Chimeric receptors of the Arabidopsis thaliana pattern recognition receptors EFR and FLS2

FLS2 and EFR are pattern recognition receptors in Arabidopsis thaliana perceiving the bacterial proteins flagellin and Elongation factor Tu (EF-Tu). Both receptors belong to the >200 membered protein family of Leucine-Rich Repeat Receptor Kinases (LRR-RKs) in Arabidopsis. FLS2 and EFR are engaged in the activation of a common intracellular signal output and they belong to the same subfamily of LRR-RKs, sharing structural features like the intracellular kinase domain and the ectodomain organized in LRRs. On the amino acid sequence level, however, they are only <50% identical even in their kinase domains. In our recently published paper¹ we demonstrated that it is possible to create chimeric receptors of EFR and FLS2 that are fully functional in ligand binding and receptor activation. Chimeric receptors consisting of the complete EFR ectodomain and the FLS2 kinase domain proved to be sensitive to elf18, the minimal peptide required for EF-Tu recognition, similar to the native EFR. In chimeric receptors where parts of the FLS2 ectodomain were swapped into the EFR LRR-domain, the receptor function was strongly affected even in cases with only small fragments exchanged. In this addendum we want to address problems and limits but also possibilities and chances of studying receptor functions using a chimeric approach.Key words: pattern recognition receptors, chimeric receptors, MAMP, flagellin perception, FLS2, EFRIn the Arabidopsis genome exist >600 genes that are predicted to encode for receptor-like kinases (RLKs).^2,3 More than 200 of them have ectodomains with LRRs. Physiological functions have been attributed only to a rather small percentage of them. Examples for known receptor-ligand pairs in A. thaliana include the well studied BRI1/Brassionlide,^4,5 AtPEPR1/Pep25,⁶ HAESA/IDA⁷ or CLV1/CLV3.⁸ While these LRR-RKs detect endogenous ligands, other members of this family function as immunoreceptors that detect ligands indicative of ‘non-self,’ such as pathogen associated molecular patterns (PAMPs). Examples of such LRR-RKs include FLS2 (Flagellin Sensing 2) and EFR (EF-Tu Receptor) from Arabidopsis and XA21 from rice.^9–11 The corresponding ligands have been identified as the flg22-epitope of bacterial flagellin for FLS2, the N-terminus of bacterial EF-Tu represented by the elf18 peptide for EFR, and the sulfated Avr21 peptide from Xanthomonas for XA21, respectively. LRR-ectodomains with related function in pathogen recognition occur also in so-called receptor-like proteins that lack the cytoplasmic kinase domains. Well studied examples include several Cf-receptor proteins which confer resistance against the fungus Cladosporium fulvum (Cf) in a gene-for-gene dependent manner. Thereby, different Cf-proteins function as recognition systems with specificity for factors determined by corresponding AvrCf products of the fungal pathogen.^12,13Receptor activation of the well studied receptor BRI1 by its ligand brassinolide involves interaction with a further receptor kinase, BAK1 (BRI1-associated receptor kinase 1).^5,14 Most interestingly, BAK1, or one of the four BAK1-related receptor kinases of the SERK protein family, also acts as a co-receptor for the ligand-dependent activation of FLS2, AtPEPR1 and EFR.^15–17 It seems that the co-receptor BAK1 plays an important role in activation of receptor kinases, serving different intracellular signaling pathways and output programs.¹⁸Up to now, little is known about the molecular details of ligand binding by the ectodomain in the apoplast and how this process leads to activation of the output signaling by the kinase moiety in the cytoplasm. The interaction with the co-receptor BAK1 suggests an activation process involving a ligand-induced intramolecular conformational change of the LRR-RK that then allows heterodimerization with the co-receptor BAK1. An initial task in elucidation of this activation process consists in defining the exact sites in the ectodomains of the receptors that interact with their corresponding ligands. So far, the clearest results for mapping ligand binding sites on LRR-receptor proteins were obtained with directed point mutations within the LRR domains as performed with the tomato receptor-like protein Cf-9,^19,20 and the Arabidopsis FLS2. There, a series of directed point mutations helped to map the LRRs 9–15 as a subdomain essential for interaction with the ligand flg22.²¹ Another interesting and promising approach consists in swaps of receptor sub-domains or exchanges of LRRs. In a remarkable, pioneering experiment this approach was used to produce chimeric receptors with the ectodomain of the brassinosteroid receptor BRI1 from Arabidopsis and the kinase domain of the immunoreceptor XA21 from rice.²² This chimera was reported to recognize the “developmental signal” brassinolide but to trigger characteristic cellular defense responses. In a recent publication²³ a domain swap between the ectodomain of the Wall Associated Kinase 1 (WAK1) and EFR was used to gain evidence for a function of the WAK1 ectodomain as a pectin receptor. Chimeric forms of the Cf receptor-like protein were used to identify subdomains carrying the specificity for the corresponding effectors from the C. fulvum pathogens.²⁴ However, as a limitation of this analysis, for none of these tomato resistance proteins a direct interaction with the corresponding effector proteins of the pathogen could be demonstrated so far.²⁵In our work, recently published in the Journal of Biochemistry,¹ we used the Arabidopsis thaliana receptors FLS2 and EFR to generate receptor chimeras. The main goal was to study the elf18 binding site in the EFR LRR-domain. In initial attempts we used EFR-constructs lacking some of the LRRs to narrow down the interaction site on the ectodomain. However, all of these truncated ectodomain versions lacking the transmembrane domain or more turned out to be unable in binding elf18 and triggering responses. In a second approach, we used the replacement of receptor parts with fragments from the structurally related receptor AtFLS2. These chimeras were tested for proper expression, localization, functionality in several plant defence related assays and affinity for the ligand elf18 in binding assays. The chimera with the complete EFR ectodomain swapped to the Kinase of FLS2 was fully functional as EF-Tu receptor. Since both receptors are known to trigger the same set of defense responses this might be not unexpected. Nevertheless, it is noteworthy that the two receptors show ∼45% sequence identity in their kinase domain, a degree of identity also shared with the kinase domains of receptors involved in other output programs, like BRI1. The 21 LRRs of EFR are sufficient for specifying full affinity for the elf18 as a ligand (

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels

Hydrogen peroxide is an important regulatory agent in plants. This study demonstrates that exogenous H2O2 application to Arabidopsis thaliana root epidermis results in dose-dependent transient increases in net Ca2+ influx. The magnitude and duration of the transients were greater in the elongation zone than in the mature epidermis. In both regions, treatment with the cation channel blocker Gd3+ prevented H2O2-induced net Ca2+ influx, consistent with application of exogenous H2O2 resulting in the activation of plasma membrane Gd3+-sensitive Ca2+-influx pathways. Application of 10 mm H2O2 to the external plasma membrane face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca2+-permeable conductance. This conductance differed from that previously characterized as being responsive to extracellular hydroxyl radicals. In contrast, in mature epidermal protoplasts a plasma membrane hyperpolarization-activated Ca2+-permeable channel was activated only when H2O2 was present at the intracellular membrane face. Channel open probability increased with intracellular [H2O2] and at hyperpolarized voltages. Unitary conductance decreased thus: Ba2+ > Ca2+ (14.5 pS) > Mg2+ > Zn2+ (20 mM external cation, 1 mM H2O2). Lanthanides and Zn2+ (but not TEA+) suppressed the open probability without affecting current amplitude. The results suggest spatial heterogeneity and differential sensitivity of Ca2+ channel activation by reactive oxygen species in the root that could underpin signalling.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane.     

Epigallocatechin‐3‐gallate induces mesothelioma cell death via H2O2−dependent T‐type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin‐3‐gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H₂O₂ release in cell cultures, and exogenous catalase (CAT) abrogated EGCG‐induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3‐loaded, EGCG‐exposed MMe cells showed significant [Ca²⁺]_i rise, prevented by CAT, dithiothreitol or the T‐type Ca²⁺ channel blockers mibefradil and NiCl₂. Cell loading with dihydrorhodamine 123 revealed EGCG‐induced ROS production, prevented by CAT, mibefradil or the Ca²⁺ chelator BAPTA‐AM. Direct exposure of cells to H₂O₂ produced similar effects on Ca²⁺ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Ca_v3.2 T‐type Ca²⁺ channels in these cells, compared to normal mesothelium. Also, Ca_v3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Ca_v3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T‐type Ca²⁺ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.     

Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca²⁺] and [Cd²⁺], or [Ca²⁺] and [Ba²⁺], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba²⁺] was added to a B cell line that had been stimulated with anti-immunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca²⁺], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba²⁺. The conductance for Ba²⁺ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca²⁺] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba²⁺.     

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana.

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.     

Capacitation suppression by mouse seminal vesicle autoantigen involves a decrease in plasma membrane Ca2+‐ATPase (PMCA)‐mediated intracellular calcium

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca²⁺‐ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca²⁺]_i and NaHCO₃‐induced [cAMP]_i. Cyclic AMP agonists bypassed the SVA's suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca²⁺]_i and intracellular pH (pH_i) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA's suppression of [Ca²⁺]_i. The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM–SVA (with a K_d of ～3 µM) which was close to the IC₅₀ of SVA's suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)‐1 expression stimulated by tumor growth factor (TGF)‐β and CD. These observations supported the membrane lipid‐raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca²⁺]_i, thereby decreasing the [cAMP]_i level and preventing sperm pre‐capacitation. J. Cell. Biochem. 111: 1188–1198, 2010. © 2010 Wiley‐Liss, Inc.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+-activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.     

Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

F‐ATP synthases convert the electrochemical energy of the H⁺ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F‐ATP synthases can also undergo a Ca²⁺‐dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca²⁺ binding site and the mechanism(s) through which Ca²⁺ can transform the energy‐conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the “Ca²⁺‐trigger site” of the PTP to the catalytic site of the F‐ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca²⁺‐ATP hydrolysis, confer resistance to Ca²⁺‐induced, PTP‐dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.     

Structures and recognition modes of toll‐like receptors

Toll‐like receptors (TLRs) recognize common structural patterns in diverse microbial molecules and play central roles in the innate immune response. The structures of extracellular domains and their ligand complexes of several TLRs have been determined by X‐ray crystallography. Here, we discuss recent advances on structures and activation mechanisms of TLRs. Despite the differences in interaction areas of ligand with TLRs, the extracellular domains of TLRs all adopt horseshoe‐shaped structures and the overall M‐shape of the TLR–ligand complexes is strikingly similar. The structural rearrangement information of TLRs sheds new light on their ligand‐recognition and ‐activation mechanisms. Proteins 2016; 85:3–9. © 2016 Wiley Periodicals, Inc.     

Effect of temperature on plasma membrane and tonoplast ion channels in Arabidopsis thaliana

The temperature dependence of the activity of ion channels was investigated, by means of the patch-clamp technique in the 'whole-cell' configuration, using protoplasts and vacuoles isolated form Arabidopsis thaliana L. cultured cells. The effect of temperature changes in the range 11–22°C was tested on the hyperpolarization and depolarization-activated K⁺ currents in the plasma membrane and on the hyperpolarization-activated K⁻ currents in the tonoplast (vacuolar membrane). All 3 kinds of currents were unaffected by increasing temperature up to 15°C and were activated between 15 and 20°C.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca²⁺-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K⁺ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na⁺ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na⁺ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca²⁺ Ba²⁺ Mn²⁺ Sr²⁺ Na⁺ K⁺ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca²⁺ Sr²⁺ Na⁺ Mn²⁺ Ba²⁺ = 19 18 14 12 10. Unitary Ca²⁺ currents display a tendency to saturate with the Ca²⁺ concentration increase with apparent dissociation constant (K _d ) of 10 mm. No block of Na⁺ permeation by extracellular Ca²⁺ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca²⁺-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.