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1.
Sergey Shabala Lone B?kgaard Lana Shabala Anja T Fuglsang Tracey A Cuin Lev G Nemchinov Michael G Palmgren 《Plant signaling & behavior》2011,6(7):1053-1056
Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper,17 we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.Key words: cytosolic calcium, reactive oxygen species, cross-tolerance, calcium pumpThe phenomenon of cross-tolerance to a variety of biotic and abiotic stresses is well-known.1,2 Some of the demonstrated examples include the correlation between oxidative stress tolerance and pathogen resistance.3–5 At the mechanistic level, changes in cytosolic Ca2+ levels [Ca2+]cyt, have long been implicated as a quintessential component of this process.6 The rise in [Ca2+]cyt is proven to be essential for the development of the oxidative burst required for triggering the activation of several plant defense reactions.7,8 The observed elevation in H2O2 level is believed to result from Ca2+-dependent activation of the NADPH oxidase,8 which then causes a further increase in [Ca2+]cyt via a positive feedback mechanism. This process is further accomplished by defense gene activation, phytoalexin synthesis and eventual cell death.9 Downstream from the stimulus-induced [Ca2+]cyt elevation, cells possess an array of proteins that can respond to a message. Such proteins include calmodulin (CaM),10 Ca2+-dependent protein kinases11 and CaM binding proteins.12 Of note is that when Ca2+ channels are blocked, biosynthesis of ROS is prevented.13While the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the involvement of active Ca2+ efflux systems in this process. In contrast, in animal systems the essential role of re-establishing [Ca2+]cyt to resting levels is widely reported. A sustained increase in [Ca2+]cyt in the alveolar macrophage is thought to be the consequence of membrane Ca2+-ATPase dysfunction.14 In endothelial cells, inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria was named as one of the reasons for [Ca2+]cyt increases.15 A significant loss of the plasma membrane Ca2+-ATPase (PMCA) activity was reported in brain synapses in response to oxidative stress,16 suggesting that PMCA may be a downstream target of oxidative stress.In our recently published paper17 we reported the phenomenon of Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana plants and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. Nonetheless, questions remain, is this transporter the only active Ca2+ efflux system involved in this process?In addition to Ca2+/H+ exchangers, active Ca2+ extrusion could also be achieved by Ca2+-ATPases. Two major types of Ca2+-ATPases that differ substantially in their pharmacology and sensitivity to CaM are known.18 Type P2A pumps (also called ER-type or ECA19,20) are predominantly ER-localized,19 although they are also present at other endomembranes (e.g., tonoplast and Golgi). Four members of this group have been identified in the Arabidopsis genome (named AtECAs 1 to 4).18,21 These pumps lack an N-terminal autoregulatory domain, are insensitive to CaM and suppressed by cyclopropiazonic acid (CPA).19 P2B (or ACA) pumps contain an autoinhibitory N-terminal domain that possesses a binding site for Ca2+-CaM.18 Ten members are known in Arabidopsis (termed AtACA1, 2, 4 and 7 to 13).21 Plant P2B pumps are located at the plasma membrane20 as well as in inner membranes such as tonoplast (e.g., ACA4), ER (e.g., ACA2) and plastids.18,19 These pumps probably constitute the basis for precise cytosolic Ca2+ regulation; as the Ca2+ concentration increases, CaM is activated and binds to the autoinhibitory domain of the Ca2+ pump. This results in the activation of the pump.In our recent study,17 we found no significant difference between the purified plasma membranes fractions isolated from control and UV-treated tobacco plants (with or without PVX inoculation) either in the Ca2+-ATPase activity or in the Ca2+-ATPase expression level and its ability to bind CaM. This suggests that the plasma membrane P2B type pumps (the only pump type known to be expressed at the plasma membrane) play no major role in removing excess Ca2+ from the cytosol under oxidative stress conditions. This led to an obvious question: what about endomembrane Ca2+-ATPases?To address this issue, microsomal membrane fractions were isolated from tobacco leaves in a manner previously described for plasma membrane fractions17 (Fig. 1A). Western blot and CaM overlay assays were then made to investigate the role of endomembrane P2B Ca2+-ATPases in our reported phenomena of acquired resistance. The results show that the expression of the P2B Ca2+ pumps in PVX-inoculated plants is significantly higher than in control plants (Fig. 1B), correlating well with the CaM overlay assay (Fig. 1C). As no difference was observed for the P2B Ca2+-ATPase expression levels in the plasma membranes,17 the observed difference in the microsomal fractions of PVX-infected plants must be due to an increased expression of endomembrane P2B Ca2+-ATPases. Given the fact that Ca2+ pumps have a high affinity for calcium, the observed increase in endomembrane P2B-type Ca2+-ATPases expression in PVX-inoculated plants may be advantageous for more efficient Ca2+ removal from the cytosol into internal organelles.Open in a separate windowFigure 1Expression of P2B Ca2+ in purified microsomal fractions from tobacco leaves. Measurements were undertaken C = mock controls; C-UV = mock controls treated with UV-light; PVX = PVX infected plants; PVX-UV = PVX inoculated plants treated with UV-light. (A) Coomassie Brilliant Blue-stained gel; (B) Protein blot immunostained with a non isoform-specific polyclonal antibody for P2B Ca2+-ATPases; (C) CaM overlay assay.To decipher the possible role of P2A Ca2+-ATPases in acquired resistance, a series of electrophysiological experiments were conducted using inhibitors of P2A-type Ca2+-ATPases, such as thapsigargin (TG)22 and cyclopiazonic acid (CPA).23 Ion-selective Ca2+ microelectrodes were prepared as described elsewhere in reference 24 and 25, and net Ca2+ fluxes were measured from tobacco mesophyll tissue following previously described protocols.17 Leaf pre-treatment for 2 h in either of these inhibitors dramatically suppressed the net Ca2+ efflux measured from tobacco mesophyll cells 2 h after UV light exposure (Fig. 2). Given the specificity of TG and CPA inhibitors for P2A-type Ca2+-ATPases, these results strongly support a hypothesis that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in plant adaptive responses to oxidative stress. This is achieved by removing excess Ca2+ from the cytosol.Open in a separate windowFigure 2Effect of known Ca2+-ATPase blockers on light-induced Ca2+ flux kinetics after 20 min of UV-C treatment. Leaf mesophyll segments were pre-treated in either 5 µM TG (thapsigargin) or 50 µM CPA (cyclopiazonic acid) for 1–1.5 h prior to exposure to UV-C light. Net Ca2+ fluxes were measured 2 h after the end of UV treatment. These were compared with two controls: (1) no pre-treatment/no UV exposure (closed circles) and (2) no pre-treatment/20 min UV exposure (open squares). Mean ± SE (n = 4 to 7).Combining these results with our previously reported observations in reference 17, the following model is proposed (Fig. 3). Oxidative stress (such as UV) causes increased ROS production in leaf chloroplasts, leading to the elevated [Ca2+]cyt. Several Ca2+ efflux systems are involved in restoring basal cytosolic Ca2+ levels. Two of these, the plasma membrane Ca2+/H+ exchanger17 and endomembrane P2A and P2B Ca2+-ATPases (as reported in this study) are upregulated in PVX inoculated plants and contribute to the improved tolerance to oxidative stress. Overall, these findings highlight the potential role of Ca2+ efflux systems in virus-induced tolerance to oxidative stress in plants. This is consistent with our previous reports on the important role of Ca2+ efflux systems in biotic stress tolerance26 and brings forth possibilities for genetic engineering of more tolerant plants by targeting expression and regulation of active Ca2+ efflux systems at either the plasma or endomembranes.Open in a separate windowFigure 3The proposed model of oxidative stress signaling and the role of Ca2+-efflux systems in acquired resistance and plant adaptation to oxidative stress.Overall, a better adaptation of virus-infected plants to a short wave UV irradiation as compared to uninfected controls may suggest that infection triggers common defense mechanisms that could be efficient against secondary unrelated stresses. This observation may lead to the development of novel strategies to protect plants against complex environmental stress conditions. 相似文献
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Huda Abdel-Hamid Kimberley Chin Dea Shahinas Wolfgang Moeder Keiko Yoshioka 《Plant signaling & behavior》2010,5(9):1147-1149
Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.Key words: calmodulin, CaM, cyclic nucleotide gated ion channels, CNGC, environmental effect, defense responses, temperatureWe recently reported about the role of the αC-helix in the cyclic nucleotide binding domain (CNBD) for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis.1 CNGCs were first discovered in vertebrate retinal photoreceptors and olfactory sensory neurons.2,3 They are composed of a cytoplasmic N-terminus, six membrane spanning regions (S1–S6), a pore domain located between S5 and S6 and a cytoplasmic C-terminus and share similarities with the voltage-gated outward rectifying K+-selective ion channel (Shaker) proteins.2 However, CNGCs are ligand gated and opened by the direct binding of cyclic nucleotides monophosphates (cNMPs), such as cAMP and cGMP (cNMPs).4 The cytoplasmic C-terminus contains a CNBD that is connected to the pore domain by a C-linker region.5 The function and regulation of CNGCs has been extensively studied in retinal photoreceptor and olfactory sensory neurons and it has been reported that their channel activity is moderated by Ca2+/calmodulin (CaM).6 A variety of Ca2+ permeable channels are known to be regulated by Ca2+/CaM. In many cases, CaM downregulates their activity, thereby creating negative feedback regulation for Ca2+ entry.7 CNGCs are also considered to follow this mode of regulation. Some animal CNGCs possess a CaM binding domain in the cytoplasmic N-terminus. For example, it has been reported that CaM binds to a short segment in the N-terminal region of the A2 subunit of CNGCs of olfactory sensory neurons in a Ca2+ dependent manner.8,9The first plant CNGC, HvCBT1 (Hordeum vulgare calmodulin (CaM)-binding transporter), was identified as a CaM-binding protein in barley.10 Interestingly, in contrast to animal CNGCs, the CaM binding domain in HvCBT1 was shown to be located at the cytoplasmic C-terminal region.10 Subsequently, several CNGCs were identified from Arabidopsis and Nicotiana tabacum11,12 and some of these CNGCs have also been shown to possess the CaM binding domain in the C-terminal region that partially overlaps with the CNBD.11–14 This difference in the location of the CaM binding domain between animal and plant CNGCs implies that different mechanisms for CNGC regulation may have evolved in animals and plants.The Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22), which contains a novel chimeric CNGC, AtCNGC11/12, shows environmentally sensitive constitutive defense responses, such as heightened salicylic acid accumulation and hypersensitive response-like spontaneous programmed cell death.15,16 It has been reported that the expression of AtCNGC11/12 and its channel activity is attributable for the cpr22 phenotype.17,18 A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12.1,18 These intragenic mutants are excellent tools to study the structure-function relationship of plant CNGCs. One of these mutants, suppressor S58, possesses a single amino acid substitution, arginine 557 to cysteine (R557C), in the αC-helix of the CNBD. The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation, under ambient temperature conditions. However, these phenotypes were recovered at 16°C, suggesting that the stability of channel function is affected by temperature.1 Interestingly, this temperature sensitivity was also observed in the original mutant, cpr22.19 All salicylic acid-dependent phenotypes of cpr22 are enhanced under low temperature and low humidity conditions. Furthermore, this type of environmental sensitivity has been reported not only for cpr22, but also for various other pathogen resistance mutants as well as for defense responses in wild type plants.16 Therefore, it is possible that the basis of the temperature sensitivity observed in S58 may be related to a general environmental sensitivity in defense responses.Characterization of S58 and functional complementation using heterologous expression analyses suggested that R557 in the αC-helix of the CNBD is important for channel regulation, but not for basic channel function.1 To further investigate this, we aimed to elucidate the molecular mechanism by which R557C (S58 mutation) alters channel regulation to determine the functional role of R557. Since R557 is located in the CNBD, it is possible that R557C alters cNMP binding resulting in disruption of channel opening. In animal systems, it has been reported that cNMPs bind within the pocket formed by the αC-helix and the β-barrel that is composed of the eight β sheets in the CNBD.20,21 The αC-helix was suggested to function as the lid of this pocket that stabilizes cNMP binding by forming hydrophobic interactions with the bound cNMP.21 However, it is unlikely that R557 interacts directly with the bound cNMP because of its hydrophilic nature.Interestingly, a 19–20 amino acid sequence of the αC-helix was suggested to function as CaM binding domain in AtCNGC1 and AtCNGC2 by Köhler and Neuhaus using yeast two hybrid analysis.14 Arazi et al.13 biochemically demonstrated that a 23 amino acid sequence that overlaps with this 19–20 amino acids is the CaM binding domain in the tobacco CNGC, NtCBP4. Furthermore, they reported that the 4 additional amino acids (W R T/S W) which are located just outside of the 19–20 amino acid sequence are crucial for efficient binding to CaM. Sequence alignment revealed that R557 is located in this crucial sequence (W R T/S W).1 Thus, it can be hypothesized that the R557C mutation causes a modification in the binding affinity to CaM resulting in an alteration in channel regulation. Therefore, we generated in silico models of CaM binding with the αC-helix of the CNBD in AtCNGC12 (identical to AtCNGC11/12) and AtCNGC12:R557C (S58). We first modeled Arabidopsis CaM1 (AtCaM1) based on the crystal structure of a potato CaM, PCM6 (PDB# 1RFJ).22 There are seven different CaM genes in Arabidopsis that encode two sets of identical isoforms (CaM1 and CaM4; CaM2, CaM3 and CaM5) and two additional distinct isoforms (CaM6 and CaM7).23,24 All of them share high similarity with PCM6. Using the AtCaM1 model, we modeled possible interactions between AtCaM1 and the αC-helix of the CNBD in AtCNGC12 or AtCNGC12:R557C. As shown in Figure 1 (center part), a hydrophobic pocket of CaM that is necessary for binding with target proteins25 was seen in AtCaM1 creating a binding pocket for the αC-helix of the CNBD in AtCNGC12. In this model, R557 creates salt bridges with both D79 and E83 of AtCaM1 (indicated by a box) and these salt bridges appear to play a role for binding to CaM. This type of salt bridges have been reported to be crucial for CaM binding with several different target proteins.26 Interestingly, these salt bridges are no longer seen in AtCNGC12:R557C (Fig. 1, right part, indicated by a box); indicating that the mutation may cause an affinity change in CaM binding. Such an affinity change will likely cause an alteration in channel regulation. Since CaM is likely a negative regulator of CNGCs, this alteration in CaM affinity does not provide a simple mechanism for the R557C mutation. However, the change in CaM binding affinity may cause complex regulatory changes in CNGCs. Thus, we are currently conducting various biochemical analyses to validate this hypothesis.Open in a separate windowFigure 1Computational structural modeling of CaM binding with AtCNGC12 and AtCNGC12:R557C. Modeling of the tertiary structure of AtCaM1, and the αC-helix of AtCNGC12 and AtCNGC12:R557 was conducted using the crystallized structures of the potato CaM, PCM 6 (PDB# 1RFJ)22 and the cytoplasmic C-terminus of the invertebrate CNGC, SpIH (Flynn et al. 2007, PDB# 2PTM),27 respectively, as templates. The protein fold recognition server (Phyre)28 was used to model these proteins. The binding modeling was performed using an algorithm for molecular docking (PatchDock).29 All the images were generated using PyMOL.30 CaM is colored in cyan and the αC-helix is shown in magenta. Left part: overall binding model between AtCaM1 and AtCNGC12, Center part: close up of the boxed area of the left part in AtCNGC12, Right part: the same area in AtCNGC12:R557C. M73, M52 and M37 of AtCaM1 create a hydrophobic pocket together with F562 and I564 of AtCNGC12, which is a typical binding configuration between CaM and target proteins. R557 creates salt bridges with both D79 and E83 (center part). These salt bridges are no longer seen between AtCaM1 and AtCNGC12:R557C (right part).Although plants CNGCs have only recently been revealed to mediate multiple stress responses and also play important roles in some developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. Our current study will certainly contribute to a better understanding of the structure-function relationship and regulation of plant CNGCs. 相似文献
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Tianbao Yang Gul Shad Ali Lihua Yang Liqun Du ASN Reddy BW Poovaiah 《Plant signaling & behavior》2010,5(8):991-994
Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.1–4 Calcium/calmodulin (Ca2+/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.5 In plants, Ca2+/CaM-dependent protein phosphorylation was observed more than 25 years ago.6 Several calmodulin-regulated protein kinases have been identified and characterized.7,8 For example, plants have a unique chimeric Ca2+/CaM-dependent protein kinase (CCaMK), which exhibits Ca2+-dependent autophosphorylation and Ca2+/CaM-dependent substrate phosphorylation.9 CCaMK is required for bacterial and fungal symbioses in plants.10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.13 Ca2+/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.14 In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.19 MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies. 相似文献
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Calmodulin regulation (calmodulation) of the family of voltage-gated CaV1-2 channels comprises a prominent prototype for ion channel regulation, remarkable for its powerful Ca2+ sensing capabilities, deep in elegant mechanistic lessons, and rich in biological and therapeutic implications. This field thereby resides squarely at the epicenter of Ca2+ signaling biology, ion channel biophysics, and therapeutic advance. This review summarizes the historical development of ideas in this field, the scope and richly patterned organization of Ca2+ feedback behaviors encompassed by this system, and the long-standing challenges and recent developments in discerning a molecular basis for calmodulation. We conclude by highlighting the considerable synergy between mechanism, biological insight, and promising therapeutics.The ancient Ca2+ sensor protein calmodulin (CaM) has emerged as a pervasive modulator of ion channels (Saimi and Kung, 2002), manifesting remarkable Ca2+ sensing capabilities in this context (Dunlap, 2007; Tadross et al., 2008) and furnishing essential Ca2+ feedback in numerous biological settings (Alseikhan et al., 2002; Xu and Wu, 2005; Mahajan et al., 2008; Adams et al., 2010; Morotti et al., 2012). Such modulation was discovered via mutations in CaM that altered the motile behavior of Paramecium, stemming from blunted activation of Ca2+-dependent Na+ current or K+ current (Kink et al., 1990). Since then, numerous ion channels have been found to be modulated by CaM, as extensively reviewed (Budde et al., 2002; Saimi and Kung, 2002; Wei et al., 2003; Halling et al., 2006; Tan et al., 2011; Nyegaard et al., 2012). This paper focuses on the CaM regulation (calmodulation) of voltage-gated Ca2+ channels, which are extensively regulated by intracellular Ca2+ binding to CaM (Ca2+/CaM; Lee et al., 1999; Peterson et al., 1999; Zühlke et al., 1999; Haeseleer et al., 2000; DeMaria et al., 2001; Budde et al., 2002; Halling et al., 2006). Such feedback regulation by Ca2+/CaM demonstrates versatile functional capabilities, represents a prominent prototype for ion-channel modulation, and holds far-reaching biological consequences. These attributes contribute much to the standing of voltage-gated Ca2+ channels as the queen of ion channels, molecules that not only sculpt membrane electrical waveforms but also serve as gatekeepers of the ubiquitous second messenger Ca2+ (Yue, 2004).
Classic history of discovery
The earliest signs of Ca2+-dependent regulation of Ca2+ channels came from data showing that increased Ca2+ could accelerate the inactivation of Ca2+ currents in Paramecium (Brehm and Eckert, 1978), invertebrate neurons (Tillotson, 1979), and insect muscle (Ashcroft and Stanfield, 1981). These results gave birth to the concept of Ca2+-dependent inactivation (CDI), the then counterintuitive notion that entities other than transmembrane voltage could modulate the rapid gating of ion channels (Eckert and Tillotson, 1981).Methodological advances permitting routine isolation and recording of Ca2+ currents within vertebrate preparations clearly revealed the existence of CDI of cardiac L-type Ca2+ currents (carried by CaV1.2 channels) in both multicellular preparations (Kass and Sanguinetti, 1984; Mentrard et al., 1984) and isolated myocytes (Lee et al., 1985). Classic data illustrating CDI of cardiac L-type currents is shown in Fig. 1 (A–C). Fig. 1 A (Ca) shows Ca2+ current from frog cardiocytes, elicited by a test voltage pulse to +80 mV (Mentrard et al., 1984). CDI becomes apparent upon Ca2+ entry during a voltage prepulse to +80 mV (Fig. 1 B), which causes sharply attenuated Ca2+ current in a subsequent test pulse (diminished area of red shading). To exclude voltage depolarization itself as the cause of inactivation, a prepulse to +120 mV (Fig. 1 C) can be demonstrated to produce far less inactivation within a subsequent test-pulse current. Because this prepulse imposed strong depolarization, but diminished Ca2+ entry via decreased driving force, the reemergence of test Ca2+ current suggests that the strong inactivation evident in Fig. 1 B could be attributed to CDI. Data of this type specifies an inactivation mechanism that depends on voltage as a U-shaped function, now considered a hallmark of CDI (Eckert and Tillotson, 1981).Open in a separate windowFigure 1.Classic U-shaped signature of CDI. (A–C) Early voltage-clamp recordings from a multicellular preparation of frog atrial trabeculae cells demonstrates CDI of Ca2+ currents. All listed voltages are relative to resting potential ER (∼−80 mV) Adapted from Mentrard et al. (1984). (A) Ca2+ currents (shaded pink) evoked in response to an +80-mV depolarizing test pulse show robust inactivation. Fast capacitive transient was estimated by blocking these Ca2+ currents with 3 mM Co2+ solution (Co). Vertical bar corresponds to 0.5 µA of current; horizontal bar, 100 ms. (B) The Ca2+ current in response to the test pulse is sharply attenuated when preceded by an +80 mV prepulse. This reduction in current amplitude reflects the inactivation of Ca2+ channels. Format is as in A. (C) Further increase in the prepulse potential to +120 mV restores Ca2+ current amplitude (compare with B). Here, the diminished Ca2+ entry during the prepulse was insufficient to trigger CDI. The format is as in A. (D–F) Single channel recordings of L-type Ca2+ channels from adult rat ventricular myocytes also exhibit U-shaped dependence of Ca2+ channel inactivation. Adapted from Imredy and Yue (1994) with permission from Elsevier. (D) Depolarizing voltage pulse to +20 mV evokes representative elementary Ca2+ currents. Ensemble average currents are shown on the bottom. (E) When depolarizing pulse was preceded by +40 mV prepulse, the elementary Ca2+ currents are sparser and the first opening is delayed. This reduction in open probability parallels the sharp attenuation of macroscopic Ca2+ currents seen in B. The ensemble average is shown on the bottom. (F) Further increase in prepulse voltage to +120 mV led to a reversal of this gating pattern, once again highlighting the U-shaped dependence of CDI. Ensemble average is shown on the bottom.Still, the actual nature of effects of Ca2+ upon gating remained unclear. Fig. 1 (D–F) reproduces the profile of CDI at the single-molecule level (Yue et al., 1990; Imredy and Yue, 1992, 1994). Resolving data such as these is technically challenging, owing to the diminutive size of unitary Ca2+ currents (∼0.3 pA) and the sub-millisecond gating timescale involved. These data demonstrate the existence of U-shaped inactivation in the gating of a single cardiac L-type Ca2+ channel fluxing Ca2+, as present in an adult rat ventricular myocyte (Imredy and Yue, 1994). These results established that Ca2+ influx of a single channel suffices to trigger CDI, and demonstrated the clear correspondence of single-channel and multicellular behavior (Fig. 1). Thus, CDI emerged as a legitimate molecular-level modulatory process.Advent of Ca2+ channel calmodulation
The Ca2+ sensor mediating Ca2+ regulation of Ca2+ channels has long been a matter of debate, with proposals of direct binding of Ca2+ to the channel complex (Standen and Stanfield, 1982; Plant et al., 1983; Eckert and Chad, 1984), and of Ca2+-dependent phosphorylation and/or dephosphorylation of channels (Chad and Eckert, 1986; Armstrong, D., C. Erxleben, D. Kalman, Y. Lai, A. Nairn, and P. Greengard. 1988. Abstracts of papers presented at the forty-second annual meeting of the Society of General Physiologists. Abstr. 20). The road toward identification of the actual Ca2+ sensor began with the cloning and expression of recombinant Ca2+ channels, thereby enabling structure–function studies (Snutch and Reiner, 1992). This phase of discovery was initiated by a bioinformatic observation (Babitch, 1990), pointing out that the carboxy terminus of voltage-gated Ca2+ channels contains a region with weak homology to an EF hand Ca2+-binding motif (Fig. 2 A, EF1). After the recognition of EF1, the existence of a second EF hand–like motif became apparent (Fig. 2 A, EF2). This led to chimeric channel analysis (de Leon et al., 1995), wherein segments of the carboxy termini of L-type (CaV1.2) and R-type (CaV2.3) Ca2+ channels were swapped. These two types of channels exhibit strong and weak CDI, respectively, under conditions of strong intracellular Ca2+ buffering (Liang et al., 2003), making them advantageous for chimeric analysis. Results from these experiments revealed that the proximal third of the carboxy terminus (Fig. 2 A, CI region) was important for CDI, and that EF1 of CaV1.2 channels was essential for the strong CDI in these channels (de Leon et al., 1995). One possible explanation was that EF1 might be the Ca2+ binding site for CDI postulated previously (Standen and Stanfield, 1982; Plant et al., 1983; Eckert and Chad, 1984). However, mutations within the Ca2+ binding loop of EF1 failed to eliminate CDI (Zhou et al., 1997; Peterson et al., 2000), focusing the search for the Ca2+ sensor elsewhere.Open in a separate windowFigure 2.Calmodulation: the ingredients and the flavors. (A) Channel diagram illustrates overall arrangement of structural landmarks critical for CDI. The Ca2+-inactivation (CI) region, spanning ∼160 residues of the channel carboxy terminus, is highly conserved across CaV1/2 channel families and is elemental for CDI. The proximal segment (PCI) of the CI region includes the dual vestigial EF hand (EF) segments (shaded purple and blue-green). The IQ domain, a canonical CaM binding motif critical for CDI, is located just downstream of the PCI segment. The NSCaTE element in the amino terminus of CaV1.2/1.3 channels is an N-lobe Ca2+/CaM effector site. The CBD element (gray) in the carboxy terminus of CaV2 channels is a CaM binding segment thought to be critical for CDI. (B) The table outlines functional bipartition of CaM in CaV channels and the corresponding spatial Ca2+ selectivities. In CaV1.2 and CaV1.3 channels, both the C-lobe and N-lobe of CaM enable fast CDI with local Ca2+ selectivity. Latent CDI of CaV1.4 channels is revealed upon deletion of an autoinhibitory domain in the distal carboxy terminus. Throughout the CaV2 channel family, the N-lobe of CaM evokes slow CDI with global Ca2+ spatial selectivity. In CaV2.1 channels, the C-lobe of CaM supports ultra-fast facilitation (CDF) with local Ca2+ selectivity. No C-lobe triggered modulation has been reported for CaV2.2 and CaV2.3 channels.Targets of CaM binding themselves often resemble CaM, a bilobed molecule with each lobe composed of two EF hands (Jarrett and Madhavan, 1991). The dual vestigial EF hands in the carboxy terminus of channels (Fig. 2 A) may be seen as resembling a lobe of CaM, which suggests that CaM itself might be the Ca2+ sensor for CDI. Initial studies exploring this notion, however, showed that pharmacological inhibition of CaM did not eliminate CDI of L-type Ca2+ channels (Imredy and Yue, 1994; Zühlke and Reuter, 1998). Nonetheless, deletions within the CaV1.2 channel CI region identified an IQ motif (Fig. 2 A) as a critical element in CDI (Zühlke and Reuter, 1998), and mutagenesis of this IQ domain weakens CDI (Qin et al., 1999). As IQ motifs often bind Ca2+-free CaM (apoCaM; Jurado et al., 1999), the possibility that CaM acts as a CDI sensor reemerged. Definitive evidence came with studies involving a Ca2+-insensitive mutant form of CaM (CaM1234), in which point mutations within all four EF hands eliminate Ca2+ binding (Xia et al., 1998). If “preassociation” of apoCaM with target molecules were a prerequisite for subsequent regulation by Ca2+ binding to this apoCaM, then CaM1234 could act as a dominant negative to silence regulation, as seen for small-conductance Ca2+-activated K channels (Xia et al., 1998). Indeed, when CaV1.2 channels were coexpressed with CaM1234 or its analogues, CDI was ablated or strongly suppressed (Peterson et al., 1999; Zühlke et al., 1999). Additionally, apoCaM was found to bind to the CaV1.2 CI region, with critical dependence upon the IQ segment (Erickson et al., 2001; Pitt et al., 2001; Erickson et al., 2003). In retrospect, apoCaM preassociation with CaV1.2 channels would sterically protect CaM from pharmacological effects (Dasgupta et al., 1989), rationalizing the prior insensitivity of CDI to such small-molecule perturbation. In all, these data firmly substantiated CaM as the sensor for CaV1.2 channel Ca2+ regulation.Initially, it was believed that only CaV1.2 L-type channels (Fig. 2 B) were subject to CaM-mediated Ca2+ regulation (Zamponi, 2003). However, this system of calmodulation was gradually recognized to pertain to most of the CaV1 and CaV2 (but not CaV3) branches of the CaV channel superfamily (Liang et al., 2003; Fig. 2 B). P-type (CaV2.1) channels were found to be Ca2+ regulated (Lee et al., 1999), and a Ca2+/CaM binding site downstream of the IQ element (CBD; Fig. 2 A) was argued to be important. In this initial study, no role for CaM preassociation was recognized, and no role for the IQ domain was found. Moreover, Ca2+ regulation of CaV2.1 channels manifests as a facilitation of current (Ca2+-dependent facilitation; CDF), followed by a slowly developing CDI. Thus, it appeared possible that the Ca2+ regulation of CaV2.1 channels might diverge mechanistically from that of CaV1.2 channels. However, apoCaM was later found to preassociate with CaV2.1 channels (Erickson et al., 2001), Ca2+-insensitive mutant CaM molecules were found to eliminate Ca2+ regulation in CaV2.1 channels (DeMaria et al., 2001; Lee et al., 2003), and the IQ domain was determined to be structurally essential for this regulation (DeMaria et al., 2001; Lee et al., 2003; Kim et al., 2008; Mori et al., 2008). That said, the role of the CBD segment remains contentious, with some studies still arguing for this segment’s importance in CDI (Lee et al., 2003). In contrast, deletion of the entire carboxy terminus after the IQ domain (including the CBD element) completely spared CDF and CDI of CaV2.1 channels (DeMaria et al., 2001; Chaudhuri et al., 2005). Overall, CaV2.1 and CaV1.2 channels appear to be Ca2+ regulated by a largely conserved scheme.Soon thereafter, calmodulation was established for the remaining members of the CaV2 class of channels (Fig. 2 B, CaV2.2 and CaV2.3), which indicates that this modulatory system pertains throughout the CaV1-2 channel superfamily (Liang et al., 2003). Strong CaM-mediated CDI was found in CaV1.3 channels (Xu and Lipscombe, 2001; Shen et al., 2006; Yang et al., 2006); a latent capacity for CaM-mediated CDI was observed in CaV1.4 channels (Singh et al., 2006; Wahl-Schott et al., 2006); and potential indications of CaM-mediated regulation of CaV1.1 channels have been described (Stroffekova, 2008, 2011).A few more nuanced points nonetheless merit attention. First, some types of calmodulation are insensitive to strong intracellular Ca2+ buffering (e.g., CaV1.2 CDI), whereas others are not (CaV2.3 CDI; Fig. 2 B). This contrasting sensitivity enables the use of chimeric channels under increased Ca2+ buffering to identify key structural determinants (de Leon et al., 1995), despite the existence of calmodulation across the channel superfamily. Second, the mechanisms underlying CDF in CaV1.2 channels remain mysterious, as the CDF of native channels of the heart is weak to start, and attenuated by blockade of Ca2+ release from neighboring ryanodine receptor channels (RYRs; Wu et al., 2001). Additionally, CDF of CaV1.2 channels is strongly manifest only in recombinant channels containing point mutations within the IQ domain, and only when they are expressed in frog oocytes (Zühlke et al., 2000). Curiously, CDF is not observed in recombinant CaV1.2 channels expressed in mammalian cell lines (Peterson et al., 1999). In contrast, CDI of CaV1.2 channels is strong and universally observed across experimental platforms. Third, Ca2+/CaM-dependent dephosphorylation by calcineurin was initially suggested as a mechanism for CDI of Ca2+ channels (Chad and Eckert, 1986); however, this proposition has remained controversial as previously reviewed (Budde et al., 2002). Recently, calcineurin was found to preassemble with the CaV1.2 channel complex through the scaffolding protein AKAP79/150 bound to the distal carboxy terminus of the channel (Oliveria et al., 2007). Furthermore, two additional maneuvers were reported to impede CDI of these channels (Oliveria et al., 2012): the overexpression of a mutant AKAP79/150 incapable of binding calcineurin, and inhibition of calcineurin activity. However, others have found that direct inhibition of calcineurin has no effect on the CDI of L-type channels within multiple neuronal preparations (Branchaw et al., 1997; Victor et al., 1997; Zeilhofer et al., 1999). Moreover, deletion of the entire distal carboxy terminus (including the AKAP79/150 binding segment) of CaV1.2 channels preserves strong CDI (de Leon et al., 1995; Erickson et al., 2001; Crump et al., 2013). Similarly, short variants of CaV1.3 channels lacking the AKAP harboring distal carboxy termini (Marshall et al., 2011) also fully support CDI (Xu and Lipscombe, 2001; Shen et al., 2006; Yang et al., 2006; Bock et al., 2011). Altogether, it may be that calcineurin and AKAP79/150 are indirect and context-dependent modulators of CDI, rather than direct effector molecules (Budde et al., 2002).Functional bipartition of CaM and selectivity for local/global Ca2+ sources
The calmodulatory mechanism for Ca2+ channels supports remarkable forms of Ca2+ decoding. This feature echoes earlier discoveries of “functional bipartition” of CaM in Paramecium (Kink et al., 1990; Saimi and Kung, 2002). Here, “under-excitable” behavioral strains had mutations only in the N-lobe of CaM, whereas “over-excitable” strains had mutations only in the C-lobe. Thus, one lobe of CaM can mediate signaling to one set of functions, whereas the other lobe signals to an alternative set of operations. It was unclear, however, whether this bipartition extended to mammals. The requirement that Ca2+ regulation of mammalian Ca2+ channels requires apoCaM preassociation permitted direct exploration of this question. Coexpressing channels with “half mutant” CaM molecules (CaM12 and CaM34, where only C- or N-terminal lobes, respectively, bind Ca2+) revealed that the individual lobes of CaM can evoke distinct components of channel regulation (Fig. 2 B). In most CaV1 channels, the C-lobe induces a kinetically rapid phase of CDI, whereas the N-lobe yields a slower component (Peterson et al., 1999; Yang et al., 2006; Dick et al., 2008). For CaV1.2 channels, early studies that utilized high intracellular Ca2+ buffering found only minimal N-lobe–mediated CDI (Peterson et al., 1999). However, when interrogated under low intracellular Ca2+ buffering, slow but recognizable N-lobe–mediated CDI of CaV1.2 channels emerges (Dick et al., 2008; Simms et al., 2013). Most strikingly, in CaV2.1 channels, the lobes of CaM produce opposing polarities of regulation: the C-lobe of CaM triggers a kinetically rapid CDF, whereas the N-lobe evokes a slower CDI (DeMaria et al., 2001; Lee et al., 2003). CaV2.2 and CaV2.3 channels manifest CDI triggered mainly by the N-lobe of CaM (Liang et al., 2003). Whether this bipartition orchestrates divergent classes of behavior in higher-order animals remains an intriguing and open question (Wei et al., 2003).Beyond simply splitting the Ca2+ signal, however, the calmodulation of mammalian Ca2+ channels revealed that the lobes of CaM can selectively decode Ca2+ in different ways. Hints as to this capability came from the differential sensitivity of calmodulation in various channels to Ca2+ buffering. Processes evoked by the C-lobe of CaM are invariably insensitive to introduction of strong Ca2+ buffering, whereas N-lobe–mediated processes in CaV2 channels can be eliminated by the same maneuver. Strong buffering would only spare Ca2+ signals near the cytoplasmic mouth of channels where strong point-source Ca2+ influx would overwhelm buffer capacity (Neher, 1986; Stern, 1992). This argues that local Ca2+ influx through individual channels triggers C-lobe signaling; in other words, the C-lobe of CaM exhibits a “local Ca2+ selectivity.” In contrast, a spatially global elevation of Ca2+ (present in the absence of strong Ca2+ buffering) is required for N-lobe signaling of CaV2 channels (DeMaria et al., 2001; Lee et al., 2003; Liang et al., 2003); thus, the N-lobe in this context exhibits a “global selectivity.” The existence of global selectivity is notable, given that local Ca2+ influx yields far larger Ca2+ increases near a channel than does globally sourced Ca2+ (Tay et al., 2012). One exception to this pattern is the local Ca2+ selectivity of the N-lobe component of CDI in CaV1.2/1.3 channels (see two paragraphs below). Corroboration of spatial Ca2+ selectivity is provided by the ability of single CaV1.2 channels to undergo CDI (Fig. 1, D–F). By definition, only a local Ca2+ source is present in the single-channel configuration; thus, the presence of CDI in this context indicates local Ca2+ selectivity. Additionally, single-channel records of CaV2.1 channels exhibit CDF (driven by C-lobe), but not CDI (triggered by N-lobe; Chaudhuri et al., 2007), which is consistent with the proposed differential selectivities for this channel. Fig. 2 B summarizes the arrangement of spatial Ca2+ selectivities according to the lobes of CaM.The mechanisms underlying these contrasting spatial Ca2+ selectivities can be interpreted as emergent behaviors of a system in which a lobe of apoCaM must transiently detach from a preassociation site before binding Ca2+, and where a Ca2+-bound lobe of CaM must associate with a channel effector site to mediate regulation (Tadross et al., 2008). When the slow Ca2+-unbinding kinetics of the C-lobe of CaM are imposed on this scenario, local Ca2+ sensitivity invariably results. In contrast, when the rapid Ca2+-unbinding kinetics of the N-lobe are interfaced with this architecture, global Ca2+ selectivity arises if the channel preferentially binds apoCaM versus Ca2+/CaM, as in the case of CaV2 channels (Fig. 2 B). When the channel preferentially interacts with Ca2+/CaM, local Ca2+ selectivity emerges, as in the context of CaV1 channels (Fig. 2 B). The relative roles of the affinities and kinetics of Ca2+ binding to the two lobes of CaM in mediating local and global Ca2+ selectivities have been considered at length elsewhere (Tadross et al., 2008).As an explicit example of how spatial Ca2+ selectivity of N-lobe regulation is specified, we consider the effects of an N-terminal spatial Ca2+-transforming element (NSCaTE) present only on the amino terminus of CaV1.2/1.3 channels (Fig. 2 A). NSCaTE is a Ca2+/CaM binding site whose presence enhances the aggregate channel affinity for Ca2+/CaM over apoCaM (Ivanina et al., 2000; Dick et al., 2008), endowing the N-lobe component of CDI of these channels with a largely local Ca2+ selectivity of N-lobe CDI in these channels. Elimination of the NSCaTE site in these channels, which tilts channels toward a preference for apoCaM, then switches their N-lobe CDI to a global profile. Conversely, donation of NSCaTE to CaV2 channels, which causes channels to favor Ca2+/CaM binding, then endows their N-lobe CDI with local Ca2+ selectivity (Dick et al., 2008). In this manner, the presence of NSCaTE can tune the spatial Ca2+ selectivity of N-lobe–mediated channel regulation. This overall arrangement of CaM interactions with a target molecule could endow numerous other regulatory systems with spatial Ca2+ selectivity.Molecular basis of calmodulation
Elucidating the arrangement of apoCaM and Ca2+/CaM on Ca2+ channels is fundamental for the field, given the biological influence of calmodulation, and the importance of this system as an ion-channel regulatory prototype (Dunlap, 2007). This critical task, however, remains an ongoing challenge, given the >2,000 amino acids comprising the main pore-forming α1 subunit alone, the ability of multiple peptide segments of the channel to bind CaM in vitro, and the formidable nature of obtaining atomic structures for these channels.The stoichiometry of CaM interaction with channels has been debated. Crystal structures of Ca2+/CaM complexed with portions of the carboxy tail CI region of CaV1.2 channels (Fallon et al., 2009; Kim et al., 2010) suggest that multiple CaM molecules may interact to produce the full spectrum of Ca2+ regulatory functions. Moreover, CaM can interact with multiple peptide segments of the channel (Ivanina et al., 2000; Tang et al., 2003; Zhou et al., 2005; Dick et al., 2008; Ben Johny et al., 2013). In contrast, covalent fusion of single CaM molecules to CaV1.2 channels suggests that only one CaM per channel suffices to elicit CDI (Mori et al., 2004). Moreover, live-cell FRET studies of the interactions between CaM and holochannels (CaV1.2) also point to a 1:1 CaM/channel ratio (Ben Johny et al., 2012). In the holochannel, the various CaM binding segments may be arranged in close proximity so as to allow the two lobes of CaM to preferentially interact with the distinct channel segments during Ca2+ regulation (Dick et al., 2008; Ben Johny et al., 2013). Recent biochemical studies of the NSCaTE element show that this segment preferentially interacts with a single lobe of CaM (N-lobe) at a time (Liu and Vogel, 2012). Furthermore, the NSCaTE element can interact also with Ca2+/CaM prebound to an IQ domain peptide (Taiakina et al., 2013). Thus, we favor the interpretation that only one CaM interacts within holochannels, although peptide fragments of Ca2+ channels may bind to multiple CaM molecules.The IQ domain (Fig. 3 A, blue circle) is critical for calmodulation. Mutations within this domain markedly modulate the Ca2+ regulation of CaV1.2 channels (Zühlke and Reuter, 1998; Qin et al., 1999; Zühlke et al., 1999, 2000; Erickson et al., 2003), CaV1.3 channels (Yang et al., 2006; Bazzazi et al., 2013; Ben Johny et al., 2013), CaV2.1 channels (DeMaria et al., 2001; Lee et al., 2003; Kim et al., 2008; Mori et al., 2008), and CaV2.2 and CaV2.3 channels (Liang et al., 2003). Furthermore, apoCaM preassociation with CaV1.2/1.3 channels requires the IQ domain (Erickson et al., 2001; Pitt et al., 2001; Erickson et al., 2003; Bazzazi et al., 2013; Ben Johny et al., 2013). Finally, Ca2+/CaM binds well to IQ-domain peptides of many CaV channels (Peterson et al., 1999; Zühlke et al., 1999; DeMaria et al., 2001; Pitt et al., 2001; Liang et al., 2003; Bazzazi et al., 2013; Ben Johny et al., 2013). All these results gave rise to the IQ-centric hypothesis shown in Fig. 3 A. Here, the IQ domain is important for both apoCaM preassociation (left) and Ca2+/CaM binding (effector configuration shown on the right). This IQ-centric paradigm has motivated efforts to resolve crystal structures of Ca2+/CaM complexed with IQ-domain peptides of various CaV1-2 channels (Fallon et al., 2005; Van Petegem et al., 2005; Kim et al., 2008; Mori et al., 2008), as shown in Fig. 3 (B and C). Throughout, the IQ peptide segment appears as an α-helical entity with N and C termini as labeled.Open in a separate windowFigure 3.Toward an atomic-level understanding of CDI. (A) IQ-centric view of calmodulation of CaV channels. In this mechanistic scheme, ApoCaM is preassociated with the IQ domain (blue). Upon Ca2+ binding, CaM rebinds the same IQ domain with a higher affinity, and subtle conformational rearrangements are presumed to trigger CaM-mediated channel regulation. (B, left) Crystal structure of CaV1.2 IQ domain peptide in complex with Ca2+/CaM (Protein Data Bank accession no. 2BE6). The IQ domain is colored blue. CaM is show in cyan (N-lobe, pale cyan; C-lobe, cyan). Ca2+ ions are depicted as yellow spheres. The key isoleucine residue (red) serves as a hydrophobic anchor for CaM. Ca2+/CaM adopts a parallel arrangement with the IQ domain in which the N-lobe binds closer to the amino terminus of the IQ domain and the C-lobe binds further downstream. (B, right) Crystal structure of Ca2+/CaM bound to mutant CaV1.2 IQ domain with alanines substituted for the key isoleucine-glutamine residues (accession no. 2F3Z). This double mutation abolishes CDI. Structurally, however, Ca2+/CaM hugs the mutant IQ domain in a similar conformation as to its interaction with the wild-type (left). (C) Crystal structure of Ca2+/CaM bound to CaV2.1 IQ domain (left, accession no. 3BXK; right, accession no. 3DVM). The IQ domain is shaded green, CaM in cyan. Ca2+/CaM was reported to bind to the CaV2.1 IQ domain in both parallel (left) and antiparallel (right) arrangements. The antiparallel arrangement in which the C-lobe of CaM binds upstream of IQ domain has led to speculation that CDF may result from this inverted polarity of Ca2+/CaM association. (D) Simplified configurations of the CaM/channel complex relevant for calmodulation (E, A, IC, IN, and ICN). In configuration E, channels lack preassociated CaM and therefore cannot undergo CDI. Configuration A corresponds to channels bound to apoCaM, and thus capable of undergoing robust CDI. Ca2+ binding to the C-lobe and N-lobe of CaM leads to the inactivated configurations IC and IN, respectively. Ca2+ binding to both lobes of CaM then yields configuration ICN, with both C and N lobes of CaM engaged toward CDI. This final transition may exhibit positive cooperativity as specified by the constant λ >> 1. Under endogenous levels of CaM, channels may reside in any of the five configurations. Strong coexpression of wild-type or mutant CaM restricts accessibility to various states. Reproduced with permission from Ben Johny et al., 2013.However, this viewpoint remains problematic in three regards. (1) The atomic structures of Ca2+/CaM bound to wild-type and mutant IQ peptides of CaV1.2 (Fig. 3 B) show that a central isoleucine (side chains explicitly shown) in the IQ element is deeply buried within the C-lobe of Ca2+/CaM (Van Petegem et al., 2005), and that alanine substitution at this site hardly changes structure (Fallon et al., 2005). Additionally, Ca2+/CaM dissociation constants for corresponding wild-type and mutant IQ peptides are nearly the same (Zühlke et al., 2000; Bazzazi et al., 2013). Why then would alanine substitution at this well-ensconced site influence the rest of the channel to blunt regulation (Fallon et al., 2005)? (2) For CaV1.2/1.3 channels, the N-lobe of Ca2+/CaM effector site appears to be an NSCaTE element (Fig. 3 A, oval) of the channel amino terminus (Dick et al., 2008; Tadross et al., 2008; Liu and Vogel, 2012), separate from the IQ element. (3) Crystal structures of Ca2+/CaM in complex with the IQ peptide of CaV2.1 channels show that CaM can adopt both a parallel (Mori et al., 2008) and an antiparallel configuration (Kim et al., 2008; Fig. 3 C, left and right, respectively). The apparent inversion in configuration of CaM binding to IQ domain between CaV1.2 and CaV2.1 has been proposed as a mechanism for the opposing polarity of Ca2+ regulation observed in the two channels (Kim et al., 2008; Minor and Findeisen, 2010). However, detailed structural analysis along with functional systematic alanine scanning mutagenesis and chimeric channel analysis argues that the C-lobe effector site resides at a site beyond the IQ module (Mori et al., 2008).A major concern with older IQ domain analyses is that the regulatory system was not considered conceptually as a whole, as shown in Fig. 3 D (drawn with specific reference to CaV1.3 channels; Ben Johny et al., 2013). Configuration E portrays channels lacking apoCaM. Such channels can open normally, but do not manifest CDI because Ca2+/CaM from bulk solution cannot efficiently access a channel in configuration E to induce CDI (Mori et al., 2004; Yang, P.S., M.X. Mori, E.A. Antony, M.R. Tadross, and D.T. Yue. 2007. Biophysical Journal abstracts issue. 1669-Plat; Liu et al., 2010; Findeisen et al., 2011). ApoCaM binding with configuration E gives rise to configuration A, where opening can also occur normally, but CDI can now take place. For CDI, Ca2+ binding to both lobes of CaM yields configuration ICN, which underlies a fully inactivated channel with strongly reduced opening. For intermediate configurations, Ca2+ binding only to the C-lobe brings about configuration IC, equivalent to a C-lobe inactivated channel; Ca2+ binding only to the N-lobe yields the N-lobe–inactivated arrangement (IN). Of particular importance, ensuing entry into ICN likely involves positively cooperative interactions specified by cooperativity factor λ >> 1.This scheme emphasizes several challenges for older analyses of the IQ domain, where CDI was mostly assessed with only endogenous CaM present. Results thus obtained would be ambiguous, because IQ-domain mutations could alter calmodulation via changes at multiple steps depicted in Fig. 3 D, whereas other interpretations largely attributed the effects to altered Ca2+/CaM binding with an IQ effector site. In contrast, IQ mutations could well weaken apoCaM preassociation and reduce CDI by favoring configuration E. Moreover, functional deficits caused by mutations that do attenuate interaction with one lobe of Ca2+/CaM may be masked by positively cooperative steps (λ in Fig. 3 D).To minimize these issues, CDI of CaV1.3 channels was recently characterized during strong coexpression with various mutant CaM molecules (Bazzazi et al., 2013; Ben Johny et al., 2013) as shown in Fig. 4 (A–C, top). For orientation, CDI of channels expressed with only endogenous CaM present is shown in the upper portion of Fig. 4 A. Strong CDI produces a rapid decay of whole-cell Ca2+ current (red trace), compared with the negligible decline of Ba2+ current (black trace). Because Ba2+ binds CaM poorly (Chao et al., 1984), the decline of Ca2+ versus Ba2+ current after 300 ms of depolarization quantifies CDI (CDI parameter plotted below in bar graphs). One can isolate the diamond-shaped subsystem lacking configuration E (Fig. 4 B, top) by leveraging mass action with strong coexpression of wild-type CaM (CaMWT). Further simplification of CDI was obtained by strongly coexpressing channels with CaM12 (Fig. 4 C, top), which empties configuration E by mass action, and forbids configurations IN and ICN. Thus, the isolated C-lobe component of CDI can be studied, with the signature rapid time course of current decay shown near the top of Fig. 4 C. Critically, this layout avoids interplay with cooperative λ steps in Fig. 3 D. Likewise, strongly coexpressing CaM34 focuses on the slower N-lobe form of CDI, with attendant simplifications.Open in a separate windowFigure 4.Residue-level roadmap for calmodulation of CaV1.3 channels. Systematic alanine scanning mutagenesis of the entire CI domain of CaV1.3 channels. (A) CDI of wild-type and mutant CaV1.3 channels recombinantly expressed in HEK293 cells was measured with only endogenous CaM present. CDI observed here reflects properties of the entire system (stick figure) diagrammed in Fig. 3 D. Exemplar whole-cell current shows robust CDI for wild-type (WT) CaV1.3, reflecting their high affinity for apoCaM. Here and throughout, the vertical bar pertains to 0.2 nA of Ca2+ current (black); the Ba2+ current (gray) has been scaled ∼3-fold downward to aid comparison of decay kinetics. Horizontal bar, 100 ms. CDI is measured as the fractional reduction of Ca2+ current in comparison to Ba2+ at 300 ms (Ben Johny et al., 2013). Bottom, bar graph summary of CDI for alanine substitutions for indicated residues. CDI for WT channels, blue-green vertical line. Alanine substitutions in both EF hand regions and IQ domain resulted in diminished CDI. (B) Overexpression of CaMWT isolates the behavior of the diamond-shaped subsystem. Such overexpression of CaMWT should rescue CDI for mutations that weakened apoCaM preassociation. For WT CaV1.3, exemplar current shows that CDI is unaltered, as expected for a channel with high affinity for apoCaM. Bottom, bar graph summary of CDI for various mutant channels with CaMWT overexpressed. Format is as in A. CDI is rescued for several mutations in the EF hand region (EF2) and the IQ domain, reflecting the preassociation of N-lobe and C-lobe of apoCaM. (C) Overexpression of CaM12 isolates C-lobe CDI. Exemplar currents for WT channels show the fast C-lobe form of CDI. Bottom, bar graph summary shows the footprint of C-lobe CDI. Mutants in both the first EF hand and the IQ domain disrupted the C-lobe CDI (see the LGF loci in the EF hand regions and TFL and IQD loci in the IQ domain). The format is as in A. (D) Overexpression of CaM34 isolates N-lobe CDI. N-lobe CDI was largely preserved by mutations in the CI region. Mutations that weakened N-lobe apoCaM preassociation exhibited enhanced N-lobe CDI (see EF2 segment). Reassuringly, alanine scanning mutagenesis of NSCaTE element resulted in specific disruption of N-lobe CDI of CaV1.3 channels (Tadross et al., 2008). Adapted with permission from Ben Johny et al. (2013) and Bazzazi et al. (2013).Accordingly, a new framework of CaM/channel configurations that underlie calmodulation could be deduced, as diagrammed in Fig. 5 (Ben Johny et al., 2013). For convenience, Fig. 4 compiles results from a systematic alanine scan covering the entire CI domain of the carboxyl tail of CaV1.3 channels (Bazzazi et al., 2013; Ben Johny et al., 2013), where substitution positions are denoted to the left of the bar graph in Fig. 4 A. The N-terminal end of the CI segment corresponds to the top of the graph, purple and blue-green regions indicate EF1 and EF2, and the lavender zone denotes the IQ domain. Electrophysiological characterization for each of the substitutions was performed for all the various subsystems (Fig. 4, A–D, top), and bar graphs plot the strength of CDI for the corresponding subsystems in each of the panels. The blue-green vertical lines reference the profile for wild-type channels. The results thus obtained were combined with CaM binding assays (not depicted) to identify meaningful structure–function correlations.Open in a separate windowFigure 5.Next-generation blueprint for CaM/CaBP regulation of CaV channels. (A) De novo molecular model of CaV1.3 CI region docked to apoCaM. The N-lobe of apoCaM is thought to preassociate with the PCI region (green), whereas the C-lobe binds to the IQ domain (blue). The NSCaTE segment (tan) is unoccupied. This model corresponds to configuration A in Fig. 3 D, where the channels are charged with an apoCaM and ready to undergo CDI. (B) Proposed model for the Ca2+ inactivated state of the channel. The N-lobe of Ca2+/CaM is shown binding to the NSCaTE segment based on a recent NMR structure (Protein Data Bank accession no. 2LQC). This configuration is believed to result in N-lobe CDI. The de novo molecular model shows C-lobe of Ca2+/CaM, the EF hand segment, and the IQ domain forming a tripartite complex resulting in C-lobe CDI. Overall, this model corresponds to the inactive configuration ICN in Fig. 3 D. (C) Proposed allosteric mechanism of CaBP action. CaBP and apoCaM may dually bind the CaV1 channel. However, CaBP may allosterically inhibit CDI of CaV1 channels by interacting with the NT, III-IV loop, or PCI segment, thereby preventing Ca2+/CaM from reaching its effector configuration. (A–C) Adapted with permission from Yang et al. (2014).Under the regimen in Fig. 4 A, where all configurations are potentially accessible, diminished CDI by alanine substitutions occurred not only in the IQ domain, but upstream in the EF hand regions (between LGF and TLF residues). The latter effects strongly suggested that something outside the IQ domain was essential. Some of these deficits in CDI could be attributed to weakening of apoCaM preassociation with channels, because overexpressing wild-type CaM to depopulate configuration E (in Fig. 4 B) substantially recovered many of these CDI deficits. Binding assays of apoCaM with various CI segments confirmed this interpretation (Bazzazi et al., 2013; Ben Johny et al., 2013).Turning to potential deficits relating to diminished Ca2+/CaM action through channel effector sites, Fig. 4 (C and D) separately interrogates the C-lobe and N-lobe components of CDI. Importantly, isolating the C- and N-lobe components minimizes masking of mutational effects by positive cooperativity, allowing CDI deficits to be more sensitively observed in this regimen. That said, Fig. 4 C displays C-lobe CDI deficits in the EF hand region (strongest for LGF substitution), as well as in the IQ domain (with the strongest effect upon substitution at the central isoleucine). These outcomes support a hypothesis where the Ca2+/CaM effector configuration for the C-lobe component of CDI involves both of these functional hotspots, as developed two paragraphs below. For the N-lobe component of CDI (Fig. 4 D), no appreciable deficits were observed, as would be expected if interaction with the channel NSCaTE element (in the channel amino terminus) serves as the effector element.Fig. 5 A displays a proposed configuration A for apoCaM interaction with the channel. This includes a homology model of the apoCaM C-lobe complexed with the IQ domain (blue), based on analogous NaV structures (Chagot and Chazin, 2011; Feldkamp et al., 2011). The portrayal of the apoCaM N-lobe incorporates ab initio structural prediction of the CI domain, with two vestigial EF hands (EF), and a protruding helix (preIQ subelement). The EF hand module (EF) resembles the structure of a homologous NaV segment (Chagot et al., 2009; Wang et al., 2012), and the preIQ helix resembles a helical segment observed in crystal structures of analogous CaV1.2 peptides (Fallon et al., 2009; Kim et al., 2010). The atomic structure of the apoCaM N-lobe (1CFD) was interfaced with shape-complementarity docking algorithms. Regarding the proposed interaction interface of the N-lobe with the channel EF domain, we note that alanine substitution therein produced a curious enhancement of N-lobe CDI seen with certain alanine substitutions, especially in the EF region (Fig. 4 D). This feature is interesting because weakening of channel interaction with the N-lobe of apoCaM would be predicted to strengthen CDI produced by the N-lobe of Ca2+/CaM (Tadross et al., 2008).Fig. 5 B displays a proposed arrangement for the Ca2+/CaM-bound configuration ICN. The N-lobe complex with NSCaTE is an NMR structure (Liu and Vogel, 2012). An alternative ab initio model of the PCI is computationally docked with the C-lobe of Ca2+/CaM (Protein Data Bank accession no. 3BXL) and IQ module, which together form a ternary complex. This ternary arrangement is consistent with the importance of both IQ and EF domains for C-lobe CDI (Fig. 4 C). Intriguingly the C-lobe configuration resembles a rather canonical CaM–peptide complex, where the channel EF module contributes a surrogate lobe of CaM. Importantly, assays of Ca2+/CaM binding to the IQ segment alone would correlate poorly with functional effects relating to the ternary complex, as experimental studies observe (Bazzazi et al., 2013; Ben Johny et al., 2013). Overall, the proposed framework in Fig. 5 (A and B) may aid future structural biology and structure–function work. In addition, this scheme of CaM exchange within its target molecule may generalize beyond the Ca2+ channel family.Biological consequences and prospects for new disease therapies
The biological consequences of Ca2+ regulation by CaM promise to be wide ranging and immense. In the heart, elimination of CaV1.2 CDI by means of dominant-negative CaM elicits marked prolongation of ventricular action potential duration (APD), implicating CDI as a dominant control factor in specifying APD (Alseikhan et al., 2002; Mahajan et al., 2008). As APD is one of the main determinants of electrical stability and arrhythmias in the heart, pharmacological manipulation of such regulation looms as a future antiarrhythmic strategy (Mahajan et al., 2008; Anderson and Mohler, 2009). Most recently, genome-wide linkage analysis in humans has uncovered heritable and de novo CaM mutations as the probable cause of several cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), with altered CaM-ryanodine receptor function implicated as a major contributing factor (Nyegaard et al., 2012). Whole-exome sequencing has revealed an association between three de novo missense CaM mutations and severe long-QT (LQT) syndrome with recurrent cardiac arrest (Crotti et al., 2013), quite plausibly acting via disruption of CaV1.2/1.3 channel CDI (Limpitikul et al., 2014).In brain, state-of-the-art analysis of genome-wide single-nucleotide polymorphisms (SNPs) has identified CaV channels as a major risk factor for several psychiatric disorders (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013). More specifically to calmodulation, CaV1.3 channels constitute a prominent Ca2+ entry portal into pacemaking and oscillatory neurons (Bean, 2007), owing to the more negative voltages required to open these ion channels. These channels are subject to extensive alternative splicing (Hui et al., 1991; Xu and Lipscombe, 2001; Shen et al., 2006; Bock et al., 2011; Tan et al., 2011) and RNA editing (Huang et al., 2012) in the carboxy tail, in ways that strongly modulate the strength of CDI (Shen et al., 2006; Liu et al., 2010; Huang et al., 2012). This fine tuning of CDI appears to be important for circadian rhythms (Huang et al., 2012). From the specific perspective of disease, CaV1.3 channels contribute a substantial portion of Ca2+ entry into substantia nigral neurons (Bean, 2007; Chan et al., 2007; Puopolo et al., 2007; Guzman et al., 2009), which exhibit high-frequency pacemaking (Chan et al., 2007) that drives dopamine release important for movement control. Notably, loss of these neurons is intimately related to Parkinson’s disease, and Ca2+ disturbances and overload are critical to this neurodegeneration (Bezprozvanny, 2009; Surmeier and Sulzer, 2013). Accordingly, an attractive therapeutic possibility for Parkinson’s disease involves discovery of small molecules that selectively down-regulate the opening of CaV1.3 versus other closely related Ca2+ channels (Kang et al., 2012). Thus, understanding the mechanisms underlying the modulation of CaV1.3 CDI is crucial, particularly to furnish specific molecular interfaces as targets of rational screens for small-molecule modulators.The fundamental mechanism of action of a natural modulator of CaV1.3 CDI may be especially relevant. In particular, recent discoveries identify Ca2+-binding proteins (CaBPs), a family of CaM-like brain molecules (Haeseleer et al., 2000) that may also bind CaV channels and other targets (Yang et al., 2002; Kasri et al., 2004). Like CaM, CaBPs are bilobed, with each lobe containing two EF hand Ca2+ binding motifs (Haeseleer et al., 2000), and a recent crystal structure shows overall similarity to CaM (Findeisen and Minor, 2010). However, whereas all four EF hands bind Ca2+ in CaM, one lobe is nonfunctional in CaBPs. Coexpressing CaBPs with CaV1 channels eliminates their CDI, thereby potentially influencing diverse biological processes (Zhou et al., 2004; Yang et al., 2006). Some have argued that CaBPs may simply compete for apoCaM on channels (Lee et al., 2002; Findeisen et al., 2013; Oz et al., 2013). More recent data argue for a different mechanism (Fig. 5 C), in which CaBP and apoCaM can both preassociate with the channel (Yang et al., 2014). In particular, by binding to channel regions that overlap Ca2+/CaM effector loci, CaBPs may preemptively retard the ability of Ca2+/CaM to reach its effector configuration (Fig. 5 B), allowing low concentrations of CaBP molecules in the CNS to still exert functional effects in the presence of much higher CaM levels (Yang et al., 2014). If this scheme is correct, the basic mechanisms of CaBP action may have implications for drug discovery, in that small molecules that target channel interaction surfaces for CaBP and/or Ca2+/CaM may exert potent modulatory actions.In conclusion, this overview of the calmodulation of voltage-gated Ca2+ channels highlights an impressive synergy among elegant molecular regulatory mechanisms, vital biological functions, and pathogenesis and potential therapy. As such, this field promises considerable mystery, enrichment, and enlightenment in the years ahead. 相似文献7.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
8.
Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.1,2 Actin inhibitors effectively retard the movements of mitochondria,3–6 peroxisomes,5,7–11 Golgi stacks,12,13 endoplasmic reticulum (ER),14,15 and nuclei.16–18 These organelles are co-aligned and associated with actin filaments.5,7,8,10–12,15,18 Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.19Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).22 The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,26 whereas phot2 alone is essential for the avoidance response.27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.29 CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles. 相似文献
9.
Hiroaki Ishida Mario Rainaldi Hans J. Vogel 《The Journal of biological chemistry》2009,284(41):28292-28305
The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca2+-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca2+-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel “double anchor” motif. Moreover, the orientation of the helical peptide on the surface of Ca2+-SCaM4 is distinct from other known complexes. The N-lobe of Ca2+-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca2+ are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.Calmodulin (CaM)2 is a ubiquitous intracellular Ca2+ sensor protein that plays an essential role in various Ca2+ signaling pathways. Contiguous and unique CaM-binding domain (CaMBD) regions are found widely distributed in many different types of CaM target proteins including protein phosphatases and kinases, cytoskeletal proteins, ion channels, and pumps (1, 2). Even though the CaMBD from various proteins share relatively poor amino acid sequence similarity, the majority of CaMBD become α-helical upon binding to CaM, and they can be grouped into either the 1-5-10 or the 1-8-14 motif, where the first and the last number indicate the position of two hydrophobic anchor residues that attach the CaMBD to the two binding pockets of the bilobal Ca2+-CaM. However, several noncanonical classes of CaMBD have also been identified. For example, in the CaMBD of the MARCKS protein, the two anchor residues are separated by a single amino acid residue (3). On the other hand, in the recently determined crystal structure of Ca2+-CaM complexed with the CaMBD of the skeletal muscle ryanodine receptor RYR1, they are separated by 15 residues (1–17 motif) (4). Bulky hydrophobic side chains of residues such as Trp, Leu, Phe, and Ile are most commonly utilized as anchor residues (see Fig. 1a), and these are usually deeply inserted into the hydrophobic target-binding pocket of either the N- or C-lobe of Ca2+-CaM. However, in several cases, including the N-methyl-d-aspartate receptors (NMDAR) (5) and the voltage-gated Ca2+ channels (Cav1–2) (6, 7), a polar side chain from Thr or Tyr has also been found to act as an anchor residue. In almost all Ca2+-CaM complexes studied to date, the two lobes of Ca2+-CaM become collapsed on the helical CaMBD, and they form a globular complex structure. An exception was found in the case of the Ca2+-CaM complex with an incomplete CaMBD from the plasma membrane Ca2+ pump (C20W), where only the C-lobe of Ca2+-CaM binds to the CaMBD, and the N-lobe was free in solution (8). The versatility of CaM target protein binding has been discussed in many recent reviews (for example, Refs. 2, 9–11).Open in a separate windowFIGURE 1.a, amino acid sequences of CaMBD from tobacco NtMKP1 (residues 438–449), Arabidopsis AtMKP1 (residues 451–462), and rice OsMKP1 (residues 456–467) are compared with various CaMBDs. The sequences are aligned at the position of the first hydrophobic anchor residue. The hydrophobic anchor residues are colored in red, while the other hydrophobic residues are shown in green. The basic residues and acidic residues are colored in cyan and pink, respectively. The residue numbers of the NtMKP1 sequence in SCaM4-NtMKP1/NtMKP1 protein are also indicated. b, schematic drawing of the two fusion proteins, SCaM4-NtMKP1 and SCaM4CT-NtMKP1.In plant cells, Ca2+ signals, arising from various extracellular stimuli such as abiotic stresses, hormones, or phytopathogens are mediated by multiple CaM isoforms to create specific cellular responses. In contrast, only a single CaM protein exists in animal cells. For example, the model plant Arabidopsis thaliana has nine CaM genes (CaM1–9) encoding seven different CaM isoforms (12, 13). Five distinct CaM genes (SCaM1–5) encoding four different CaM proteins have been identified so far in the soybean genome (14). Despite the relatively high amino acid sequence identity among these CaM isoforms (50–90%), each isoform is utilized to regulate different target enzymes related to specific cellular responses (15, 16). For example, the expression of two soybean CaM isoforms, SCaM4 and 5 is markedly up-regulated after pathogen infection, and these two proteins can activate the enzyme nitric-oxide synthase (NOS) (17). Production of nitric oxide is thought to be one of the early events in the plant defense reactions (18, 19). On the other hand, SCaM1 is incapable of activation of the NOS enzyme, and it does in fact act as a competitive inhibitor. Likewise, in Nicotiana tabacum (tobacco), the CaM isoforms NtCaM1 and NtCaM13 are overexpressed in tobacco leaf tissue in response to wounding and the TMV-triggered hypersensitive reaction, respectively (20). Recently, we have addressed the question as to how distinct CaM isoforms can give rise to selectivity in their target regulation by determining the solution structures of the two soybean CaM isoforms, SCaM1 and SCaM4 (21). However, there are currently no structures available for plant CaM isoforms in a complex with a target CaMBD peptide, although many such complex structures have been determined for animal CaM. Therefore, determining the structure of plant CaM-target complexes and uncovering their unique features relative to those of animal CaM or other plant CaM isoforms will undoubtedly enhance our understanding of the CaM-target regulation mechanisms in plants and mammals.The mitogen-activated protein kinase cascade (MAPK cascade) is an important signal transduction pathway in animals, yeast as well as in plants (22). The activity of MAPK is regulated via phosphorylation by its immediate upstream regulator, MAPK kinase (MAPKK). Following activation, MAPKs are dephosphorylated and inactivated by MAPK phosphatases. Recently, the MAPK phosphatase from tobacco (NtMKP1) was shown to be a novel plant-specific CaM-binding protein (23). This finding indicated a possible link between the MAPK cascade and Ca2+ signaling pathways in plant cells. The MAPK cascade is thought to play an important role in plant defense signaling, and the accumulation of Ca2+ in plant cells is also a well-known response to pathogens and other stresses (for recent reviews, see Refs. 24, 25). The putative CaMBD reported for NtMKP1 (residues 396–447) is located directly upstream of the conserved Ser-rich domain in the middle of the protein. Mutagenesis studies have revealed that Trp440 and Leu443 are indispensable for Ca2+-CaM binding. More recently, we have tested various truncated versions of the CaMBD of NtMKP1 and narrowed it down to 12 residues (residues 438–449) (Fig. 1a) that are sufficient for Ca2+-CaM binding (26). Interestingly, the NtMKP1 CaMBD does not belong to any of the typical CaM-binding motif classes. Binding assays using isothermal titration calorimetry (ITC) as well as NMR titration studies for various SCaM isoforms, and their half-lobe fragments have revealed a novel sequential target binding mechanism for the Ca2+-CaM isoforms. The first strong binding event involves the C-lobe of Ca2+-CaM and the reported binding constant for NtMKP1 peptide was 107–108 m−1. On the other hand, the binding of a second CaMBD of NtMKP1 occurs only through the N-lobe of Ca2+-CaM, and the binding constant was around 105 m−1 (26). To date, structural information about the manner in which Ca2+-CaM binds sequentially to the unusual amino acid sequence of the CaMBD of NtMKP1 is unavailable. Here, we have determined the solution NMR structure of the C-lobe fragment of SCaM4 (SCaM4CT) fused with the CaMBD of NtMKP1 (Fig. 1b). We have chosen SCaM4 over other SCaM isoforms, as the stress-induced SCaM4 would provide a more important connection between stress MAPK response and Ca2+ signaling. The interaction of the α-helical CaMBD of NtMKP1 with SCaM4CT domain is stabilized by hydrophobic interactions mainly through Trp440 and Leu443 in the NtMKP1 sequence. Moreover, the basic residue, Arg444 that is unusual at position 5 (Fig. 1a) stays outside of the hydrophobic patch, and it seems to form a unique hydrogen bond to Glu84 of the SCaM4CT domain. The resulting orientation of the α-helical CaMBD relative to the SCaM4CT domain is therefore very different from those seen in the other previously reported Ca2+-CaM-target complexes. We have also studied binding of a synthetic CaMBD peptide to intact Ca2+-SCaM4 fused at the C-terminal end with the CaMBD of NtMKP1 (Fig. 1b) providing additional information about the role of the N-lobe of Ca2+-SCaM4 in NtMKP1 binding. Furthermore, we have studied the interactions between the N-lobe of Ca2+-SCaM4 and a second potential CaMBD in the C-terminal region of NtMKP1. 相似文献
10.
There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.1 All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.2 The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca2+ release channels6,7 and associates with the cell cycle regulator TGF-β.8 Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,9 leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,18 the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates17 because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers15 and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel). 相似文献
11.
Aphids ingest from the sieve tubes and by doing so they are confronted with sieve-tube occlusion mechanisms, which are part of the plant defense system. Because aphids are able to feed over longer periods, they must be able to prevent occlusion of the sieve plates induced by stylet penetration. Occlusion probably depends upon Ca2+-influx into the sieve element (SE) lumen. Aphid behavior, biochemical tests and in vitro experiments demonstrated that aphid''s watery saliva, injected during initial phase of a stylet penetration into the SE lumen, contains proteins that are able to bind calcium and prevent calcium-induced SE occlusion. In this addendum, we speculate on the consequences of saliva secretion for plant resistance. (a) The release of elicitors (e.g., oligogalacturonides) due to cell wall digestion by gel saliva enzymes may increase the resistance of cortex, phloem parenchyma cells and companion cells (CC) around the puncture site. (b) Ca2+-binding by aphid watery saliva may suppress the local defense responses in the SEs. (c) Signaling cascades triggered in CCs may lead to systemic resistance.Key words: aphid saliva, calcium binding, elicitor, oligogalacturonides, local plant defense, systemic plant defense, phloem translocation, aphid/plant-interactionAfter having penetrated the sieve-element (SE) plasma membrane, aphids encounter unspecific wound-induced occlusion reactions to prevent sap leakage.1–4 Occlusion mechanisms by callose, structural P-proteins and forisomes are likely induced by a sudden calcium influx into the sieve-tube lumen.5 Calcium possibly enters the sieve-tube lumen through the stylet wounding-site in the plasma membrane and/or stretch-activated calcium-channels.6–8 After SE penetration, aphids secrete watery saliva that contains calcium-binding proteins presumed to sabotage sieve-plate occlusion.9,10We demonstrated that Megoura viciae (Buckton) is most likely able to prevent or reduce sieve-tube occlusion in Vicia faba by secretion of watery saliva. By in vitro confrontation of isolated forisomes, protein bodies responsible for sieve-tube occlusion in Fabaceaen,5 and watery saliva concentrate, we were able to show that salivary proteins convey forisomes from a dispersed (+Ca2+) into a condensed (−Ca2+) state.10 The dispersed forisome functions in vivo as a plug, leading to stoppage of mass flow.5This in vitro evidence was corroborated by aphid behavior in response to leaf tip burning, which triggers an electrical potential wave (EPW) along the sieve tubes. Such an EPW induces Ca2+-influx and corresponding SE occlusion along the pathway.11 The passage of the EPW is associated with a prolonged secretion of watery saliva of aphids. This is interpreted as an attempt to unplug the SEs by calcium binding.10 Similar behavioral changes in response to leaf-tip burning were observed in an extended set of aphid/plant species combinations, indicating that attempted sabotage of sieve-tube occlusion by aphid saliva is a widespread phenomenon (unpublished).Aphid feeding was reported to induce local (on the same leaf) and systemic (in distant leaves) reactions of the host plant. The local response led to enhanced feeding,12–14 while the systemic response showed reduced ingestion and extended periods of watery saliva secretion in sieve tubes distant from previous feeding sites.12–14 These contrasting observations were described to be independent of the aphid species.13 The question arises how aphids induce these seemingly opposite plant responses?The aphid stylet pushing forward through cortical and vascular tissue is surrounded by a sheath of gel saliva, secreted into the apoplast.15,16 Gel saliva contains cellulase and pectinase that amongst others produce oligogalacturonides (OGs) along the stylet sheath by digestion of cell wall material.17,18 Usually, OGs act as elicitors, triggering a variety of plant responses against pathogens and insects in which the activation of calcium channels is involved.19,20 This seems to conflict with a suppression of resistance as observed for the impact of watery saliva in SEs.10 We will make an attempt to explain this paradoxon.OG induced defense responses may be triggered in all cell types adjacent to the salivary sheath (Fig. 1). Because watery saliva is only secreted briefly into these cells, which are punctured for orientation purposes (Hewer et al., unpublished), it seems unlikely that OG induced defense is suppressed here by saliva-mediated calcium binding.15 The diffusion range of OGs may be restricted to the close vicinity of the stylet sheath leading to an enhanced regional defense with a limited sphere of action (Fig. 1). Because the settling distance of aphids is restricted by their body size (1–10 mm),21 aphids feeding on the same leaf are probably hardly confronted with the regional defense induced by another aphid (Fig. 1). Otherwise, they would show an increased number of test probes before first phloem activity, as described for volatile mediated plant defense in cortex cells.13 Circumstantial support in favor of our hypothesis is provided by production of hydrogen peroxide in the apoplast,22 which is most likely associated with the action of OGs.22 Observations of hydrogen peroxide production during aphid (Macrosiphum euphorbiae) infestation of tomato in a limited area along the leaf veins, the preferred feeding sites of this species, indicate a locally restricted defense response (Fig. 1 and and22).4 The question arises why the cell signals are not spread via plasmodesmata to adjacent cells to induce resistance in a more extended leaf area? Dissemination of the signals may be prevented by closure of plasmodesmata (Fig. 1) through callose deposition,23,24 which is most likely directly coupled with calcium influx induced by OGs,25 by apoplastic hydrogen peroxide and to a minor extent by stylet puncture (Fig. 2).7,26Open in a separate windowFigure 1Hypothetical model on how stylet penetration induces and suppresses plant defense. Sheath saliva (light blue) that envelopes the stylet during propagation through the apoplast contains cellulase and pectinase,17,18 enzymes producing elicitors (e.g., oligogalacturonides (oGs)) by local cell wall digestion.19 Parenchyma cells adjacent to the sheath may develop a defense response owing to signaling cascades triggered by oG-mediated Ca2+-influx.19 Together with a Ca2+-dependent transient closure of plasmodesmata by callose (black crosses),23,24 the focused production of oGs may cause a defense response with a limited sphere of action (red—strong, brown—light, green—none). This restricted domain of defense may not be perceived by other aphids, since the settling distance is limited by the aphid body size. Nearby aphids do not show any sign of defense perception in their probing and feeding behavior.14 Signaling cascade compounds may be channeled from parenchyma cells to CCs (dashed yellow arrows), where they are subsequently released into the SEs. There they may act as long-distance systemic defense components (grey arrows). In contrast to the parenchyma domain (where only minor amounts of watery saliva are secreted), Ca2+-mediated reactions such as defense cascades and sieve-plate (SP) occlusion are suppressed in SEs by large amounts of watery saliva. The left aphid penetrates an SE and injects watery saliva (red cloud; ws) that inhibits local sieve-plate occlusion and,10 most likely, is transported by mass flow (black arrow) to adjacent SEs,27 where occlusion is impeded as well. A short-distance systemic spread over a few centimeters may explain local suppression of plant defense resulting in a higher rate of colonization. Salivary proteins or their degradation products may serve as systemic defense signals as well (grey arrows), but may also diffuse via the PPUs into CCs where additional systemic signals are induced (yellow arrows).Open in a separate windowFigure 2Hypothetical involvement of Ca2+-channels in aphid-induced cell defense (detail). During probing with its stylet the aphid secretes gel saliva as a lubrication substance (light blue) into the apoplast.15 on the way to the sieve tubes, aphids briefly puncture most non-phloem cells (red) after which the puncturing sites are sealed with gel saliva.7,16 Gel saliva also most likely prevents the influx of apoplastic calcium into pierced sieve elements (green) by sealing the penetration site.7 Watery saliva (red cloud), injected into the SE lumen,9 contains proteins which bind calcium ions (marked by X) that enter the SE via e.g., mechano sensitive Ca2+-channels activated by stylet penetration (blue tons).10 In this way, aphids suppress SE occlusion and activation of local defense cascades. In the parenchyma cells around the gel saliva sheath, a small cylindrical zone of defense may be induced by oligogalacturonides (oGs; brown triangles) produced by cell wall (grey) digestion.17–19 Perceived by unknown receptor proteins (R; e.g., a receptor like protein kinase)34 and kinase mediation (black dotted and dashed arrows), oGs lead to a Ca2+-influx through kinase activated calcium channels (orange tons).25 Around the probing site, aphids apparently induce the production of superoxide by Ca2+-induced activation of the NADPH oxidase (violet box) and its following conversion to hydrogen peroxide (red spots) is mediated by superoxide dismutase (SoD).4 Hydrogen peroxide activates Ca2+-channels (violet tons) and diffuses through plasma membrane (curled arrows) therefore potentially acting as a intracellular signal.26By contrast, Ca2+-influx into SEs, induced by presence of OGs or stylet insertion (Fig. 2), is not expected to trigger local defense given the abundant excretion of Ca2+-binding watery saliva.7,10,25 Watery saliva may spread to down-stream and adjacent SEs through transverse and lateral sieve plates (Fig. 1).7,27 Aphids puncturing nearby SEs may therefore encounter less severe sieve-plate occlusion which results in facilitated settling and thus in increased population growth. Aggregation of feeding aphids would self-amplify population growth until a certain density is attained. Farther from the colonization site, this effect may be lost due to dilution. Stimulation of aphid feeding by aphid infestation was observed locally on potato by Myzus persicae and M. euphorbiae, respectively, 96 h after infestation.13 However, a similar effect was not observed for M. persicae on Arabidopsis thaliana where aphids induced premature leaf senescence and resistance 12 h after infestation,28 possibly induced by OGs.19As a speculation, OG induced Ca2+-influx into parenchyma cells adjacent to the salivary sheath activate Ca2+-induced signaling cascades via CaM,26,29 CDPKs,30,31 MAPKinases and reactive oxygen species (Fig. 2).32 Systemic resistance, induced by aphid infestation,12–14 is mediated by unknown compounds such as, e.g., salivary proteins, their degradation products, signal cascade products or volatiles.13 Compounds produced in CCs first have to pass the PPUs, while SE signaling elements can be directly transported via mass flow (Fig. 1).The question arises if aphids profit from induced resistance on local (cortex and parenchyma cells) and systemic (distant plant organs) levels as holds for suppression of defense in SEs. Possibly settling and subsequent spread of competing pathogens/herbivores (e.g., fungi or other piercing-sucking insects) are suppressed by induced defense. In this context it is intriguing to understand how aphids cope with the self-induced systemic resistance, which probably lasts over weeks.33 相似文献
12.
The prion hypothesis1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.6 For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,10 Rnq1p,11 Sup35,12 Swi1,13 and Cyc8,14 from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),6,19 Rnq1p(153-405)20 and HET-s(218-289)21). The same conclusion was reached by investigating Sup35(1-254).22 All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.23 An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR24 (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.23Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p25,26 and HET-s27), as well as NMR (mammal prions15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.25It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains? 相似文献
13.
Takamitsu Kurusu Jumpei Hamada Haruyasu Hamada Shigeru Hanamata Kazuyuki Kuchitsu 《Plant signaling & behavior》2010,5(8):1045-1047
Cytosolic free Ca2+ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca2+ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa2+ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca2+ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca2+ concentration.1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca2+ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca2+ sensors of animals.3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).5 The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,8 followed by a form of programmed cell death known as hypersensitive response (HR).9,10 Because complexed spatiotemporal patterns of cytosolic free Ca2+ concentration ([Ca2+]cyt) have been suggested to play pivotal roles in defense signaling,1,9 multiple Ca2+ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)14–19 has been suggested, other Ca2+-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity. 相似文献
14.
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Jennifer C Mortimer Anuphon Laohavisit Henk Miedema Julia M Davies 《Plant signaling & behavior》2008,3(9):698-699
The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca2+ channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca2+ channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence. Activity of root NADPH oxidase was found to be stimulated by restricting Ca2+ influx, suggesting that these enzymes are involved in sensing Ca2+ entry into cells.Key words: calcium, channel, NADPH oxidase, oscillation, root hairElevation of cytosolic free Ca2+ ([Ca2+]cyt) encodes plant cell signals.1 Reactive oxygen species (ROS) are potent regulators of the PM Ca2+ channels implicated in signalling and developmental increases in [Ca2+]cyt.1,2 Plasma membrane (PM) voltage (Vm) also plays a significant part in generating specific [Ca2+]cyt elevations through the opening of voltage-gated Ca2+-permeable channels, allowing Ca2+ influx.1,3 Patch clamp electrophysiological studies on the root hair apical PM of Arabidopsis have revealed co-localisation of hyperpolarisation-activated Ca2+ channels (HACCs),4 ROS-activated HACCs5 and depolarisation-activated Ca2+ channels (DACCs).6 The DACC characterisation pointed to the presence of a Cl−-permeable conductance that was activated by moderate hyperpolarisation (−160 mV) but rapidly inactivated when the voltage was maintained at such negative values.6 This may be the R-type anion efflux conductance previously described in Arabidopsis root hair and root epidermal PM.7 Previous studies have shown that root hair PM also harbors K+ channels (mediating inward or outward flux)8–10 and a H+-ATPase.11 A key problem to address now is how these transporters interact to generate and be influenced by PM Vm, thus gating and in turn being regulated by their companion Ca2+ channels to encode developmental and environmental signals at the hair apex.A seminal study on the relationship between Vm and ionic fluxes in wheat root protoplasts not only confirmed oscillatory events but also determined that the PM can exist in three distinct states.12 In the “pump state” the H+-ATPase predominates, there is net H+ efflux and the hyperpolarized Vm is negative of the equilibrium potential for K+ (EK). In the “K state”, K+ permeability predominates but there is still net H+ efflux and Vm = EK. In the third state, there is net H+ influx and Vm > EK. In this depolarized H+-influx state, the H+-ATPase is thought to be inactive. Oscillations in PM Vm and H+ flux may be more profound in growing cells13,14 and oscillations between these states may explain the temporal changes in H+ flux recently observed at the apex of growing Arabidopsis root hairs.15 Peaks of H+ influx may reflect a depolarized Vm that could activate DACC, suggesting that DACC would play a significant role in growth regulation. The view has arisen that the HACC would be the main driver of growth, primarily because in patch clamp assays its current is greater than DACC4–6 and because resting Vm is usually found to be hyperpolarized. In a growing cell, with a Vm oscillating between a hyperpolarized and depolarized state, a DACC could just as well be a driver of growth given that the Ca2+ influx it permits could be amplified through intracellular release.The PM H+-ATPase traditionally lies at the core of models of voltage and ionic flux14,16 but in terms of [Ca2+]cyt regulation, the activity of PM NADPH oxidases must also now be considered. The Arabidopsis root hair apical PM also contains an NADPH oxidase (AtrbohC) that catalyses extracellular superoxide production.5 AtrbohC is implicated in the transition to polar growth at normal extracellular pH5 and also osmoregulation.17 NADPH oxidases catalyse the transport of electrons out of the cell and thus, in common with PM redox e− efflux systems,18 their activity would depolarize the membrane voltage unless countered by cation efflux or anion influx.19 Two H+ would also be released into the cytosol for every NADPH used. The voltage-dependence of plant NADPH oxidases is unknown but e− efflux by animal NADPH oxidases is fairly constant over negative Vm and decreases at very depolarized Vm.20 AtrbohC is implicated in generating oscillatory ROS at the root hair apex and loss of function affects magnitude and duration of apical H+ flux oscillations.15 The latter suggests that AtrbohC function does in some way affect Vm, a situation extending to other root cell types (such as the epidermis) expressing NADPH oxidases.21NADPH oxidase activity in roots is under developmental control but also responds to anoxia and nutrient deficiency22,23 to signal stress conditions. Blockade of PM Ca2+ channels by lanthanides increases superoxide production in tobacco suspension cells.24 This suggests that NADPH oxidases are involved in sensing the cell''s Ca2+ status and the prediction would be that extracellular Ca2+ chelation would increase their activity. To test this, superoxide anion production by excised Arabidopsis roots was measured using reduction of the tetrazolium dye XTT (Sodium, 3′-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulphonic acid).25,26 Lowering extracellular Ca2+ from 0.5 mM to 1.4 µM by addition of 10 mM EGTA caused a mean 95% increase in diphenyliodinium-sensitive superoxide production (Fig. 1; n = 9), implicating NADPH oxidases as the source of this ROS. Stimulation of NADPH oxidase activity by decreasing Ca2+ influx at first appears contradictory as NADPH oxidases are stimulated by increased [Ca2+]cyt27 (Fig. 1). However, reduction of Ca2+ influx should promote voltage hyperpolarisation (just as block of K+ influx causes hyperpolarisation in root hairs28) and this could feasibly cause increased NADPH oxidase activity. Production of superoxide could then result in ROS-activated HACC activity5 to increase Ca2+ influx.Open in a separate windowFigure 1Superoxide anion production by Arabidopsis roots. Assay medium comprised 10 mM phosphate buffer with 0.5 mM CaCl2, 500 µM XTT, pH 6.0. Production was linear over the 30 min incubation period. Control, mean ± standard error, n = 9. Test additions were: 20 µM of the NADPH oxidase inhibitor diphenylene iodonium (DPI; n = 6); 100 µM of the Ca2+ ionophore , A2318730 to increase [Ca2+]cyt (n = 9); 10 mM of the chelator EGTA (n = 9). Dimethyl sulphoxide [DMSO; 1% (v/v)] was used as a carrier for XTT and DPI and a separate control for this is shown (n = 9).In addition to Vm, activities of PM transporters in vivo will be subject to other levels of regulation such as phosphorylation, nitrosylation and the action of [Ca2+]cyt itself. Distinct spatial separation of transporters will undoubtedly play a significant role in governing Vm and [Ca2+]cyt dynamics, particularly in growing cells. An NADPH oxidase has already been found sequestered in a potential PM microdomain in Medicago.29 While there is still much to do on the “inventory” of PM transporters involved in Ca2+ signalling in any given cell, placing them in context not only requires knowledge of their genetic identity but also modelling of their concerted action. 相似文献
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As a second messenger, H2O2 generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H2O2 signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H2O2 generation, inhibition of inward K+ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H2O2 signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H2O2, is essential to plant growth and development in response to stresses,1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H2O2 generation.6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H2O2 signaling regardless of SA mimicking ABA in regulating stomatal closure.2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H2O2 generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,14–16 Pisum sativum,17 Medicago sativa18 and tobacco.19 The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,20 tobacco tissue21 and suspension-cultured Oryza sativa cells.22 Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H2O2 signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H2O2 generation and decreasing K+ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H2O2 signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H2O2 signaling,23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase23,24 is not always identical to that of MEK1/28,25 in ABA-induced H2O2 signaling of Vicia guard cells. For example, H2O2- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H2O2, the effects of both ABA- and H2O2-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H2O2-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H2O2-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.26 Similar to this, SB203580 improves H2O2-inhibited inward K+ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H2O2 signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,1,2,8,23,24 and the ABA and H2O2 pathways diverge further downstream in their actions on the K+ channels and, thus, on stomatal control. Other differences in action between ABA and H2O2 are known. For example, Köhler et al. (2001) reported that H2O2 inhibited the K+ outward rectifier in guard cells shows that H2O2 does not mimic ABA action on guard cell ion channels as it acts on the K+ outward rectifier in a manner entirely contrary to that of ABA.27Open in a separate windowFigure 2Effect of SB203580 on ABA- and H2O2-inhibited inward K+ currents. The experimental procedure and data analysis are according to the previous publication.24 SB203580 directs ABA- and H2O2-inactivated inward K+ currents across plasma membrane of Vicia guard cells. Here the inward K+ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H2O2 signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H2O2 signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition. 相似文献