K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels in Arabidopsis Pollen Tubes

Potassium (K⁺) influx into pollen tubes via K⁺ transporters is essential for pollen tube growth; however, the mechanism by which K⁺ transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca²⁺-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca²⁺-dependent regulation of the inward K⁺ (K+_in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K+_in currents of pollen tube protoplasts were inhibited by elevated [Ca²⁺]_cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca²⁺-dependent inhibition of K+_in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K+_in channel is the main contributor to pollen tube K+_in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca²⁺-dependent inhibition of K+_in channels and participate in the regulation of pollen tube growth in Arabidopsis.     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from Pea Nuclei

Almost all the Ca²⁺-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca²⁺ and reaches half-maximal activation at about 3 ×10⁻⁷ molar free Ca²⁺, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca²⁺-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca²⁺-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

Apelin-13 Inhibits Large-Conductance Ca2+-Activated K+ Channels in Cerebral Artery Smooth Muscle Cells via a PI3-Kinase Dependent Mechanism

Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM) cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK_Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM) caused concentration-dependent inhibition of BK_Ca in VSM cells. Apelin-13 (0.1 µM) significantly decreased BK_Ca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P<0.05). This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM) decreased the open-state probability (NP_o) of BK_Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1µM) did not alter the NP_o of BK_Ca channels, suggesting that the inhibitory effect of apelin-13 on BK_Ca is not mediated by a direct action on BK_Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK_Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM) significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK_Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes

We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca²⁺ (10 mM intracellular EGTA) was equal at all investigated temperatures (23°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca²⁺] to ~1.5 μM (9 mM Ca²⁺ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca²⁺ ionophore ionomycin (1μM), an effect that was not observed at 23°C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca²⁺-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.     

Calmodulin has the Potential to Function as a Ca2+-Dependent Adaptor Protein

Calmodulin (CaM) is a versatile Ca²⁺-binding protein that regulates the activity of numerous effector proteins in response to Ca²⁺ signals. Several CaM-dependent regulatory mechanisms have been identified, including autoinhibitory domain displacement, sequestration of a ligand-binding site, active site reorganization, and target protein dimerization. We recently showed that the N- and C-lobes of animal and plant CaM isoforms could independently and sequentially bind to target peptides derived from the CaM-binding domain of Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1), to form a 2:1 peptide:CaM complex. This suggests that CaM might facilitate the dimerization of NtMKP1, although the dimerization mechanism is distinct from the previously described simultaneous binding of other target peptides to CaM. The independent and sequential binding of the NtMKP1 peptides to CaM also suggests an alternative plausible scenario in which the C-lobe of CaM remains tethered to NtMKP1, and the N-lobe is free to recruit a second target protein to the complex, such as an NtMKP1 target. Thus, we hypothesize that CaM may be capable of functioning as a Ca²⁺-dependent adaptor or recruiter protein.Key Words: calmodulin, calcium, EF-hand, adaptor protein, mitogen-activated protein kinase phosphataseCalcium (Ca²⁺) is a dynamic secondary messenger that regulates many signaling events in both plant and animal cells. Intracellular Ca²⁺ transients and oscillations (Ca²⁺ signals) are decoded by a large superfamily of calcium-binding proteins, the most important of which is calmodulin (CaM).^1–3 The prototypical CaM protein consists of four tandem helix-loop-helix “EF-hand” Ca²⁺-binding motifs that are divided into distinct N- and C-terminal globular lobes connected by a flexible linker. CaM proteins from all species including the single mammalian CaM and the many different plant CaM isoforms each undergo similar Ca²⁺-induced conformational changes involving a rearrangement of the position of its α-helices that opens distinct hydrophobic target protein-binding patches on the surface of each lobe; known as the “open” conformation (Fig. 1B). These hydrophobic patches can interact with numerous different target proteins including protein kinases, protein phosphatases, cytoskeletal proteins and other cell signaling enzymes, to regulate their activity. The closed or semi-open conformations adopted by the N- and C-lobes of Ca²⁺-free CaM (apo-CaM) (Fig. 1A) can also interact with another subset of proteins, to target CaM to certain cellular locations or facilitate Ca²⁺-independent regulatory events.^1–3Open in a separate windowFigure 1Structures of CaM and CaM-target complexes. (A) apo-CaM (PDB:1DMo), (B) Ca²⁺-CaM (PDB:1CLL). Complexes of CaM bound to (C) CaMBD of smooth muscle myosin light chain kinase (PDB:1CDL), (D) partial CaMBD of plasma membrane Ca²⁺-pump C20W (PDB:1CFF), (E) the adenylyl cyclase protein from Bacillus anthracis (PDB:1K93), (F) 2 glutamate decarboxylase CaMBD''s (PDB:1NWD), (G) 2 CaM proteins bound to 2 small conductance Ca²⁺-activated potassium channel (SK channel) CaMBD''s (PDB:1G4Y), (H) 2 apo-CaM proteins bound to 2 tandem IQ motifs from murine myosin V (PDB:2IX7). In each panel CaM is shown in ivory, the target molecule is shown in blue and the Ca²⁺ ions bound to the N- and/or C-lobes of CaM are represented by red spheres.The CaM-dependent regulation of target proteins can occur through numerous different mechanisms. For example, Ca²⁺-CaM can relieve autoinhibition by binding to a short (20–25 residue) calmodulin-binding domain (CaMBD) sequence that is adjacent to or within an autoinhibitory region of the enzyme (Fig. 2A).³ Numerous structures of these Ca²⁺-CaM-CaMBD complexes have been reported, which reveal a characteristic “wrap-around” binding mode (Fig. 1C). Typically the CaM C-lobe binds with high affinity to a Trp residue within the N-terminal part of the target sequence, and the flexible central linker allows the N-lobe to pivot and bind to a second bulky hydrophobic “anchor” residue within the C-terminal part of the target sequence.³ Truncation of this second anchor residue can lead to binding of only one CaM domain and an extended CaM conformation (Fig. 1D).^4,5 Studies with plant CaM isoforms having mutations to non-CaMBD-coordinating residues have also suggested that a secondary binding interface exists on the opposite surface of the CaM protein which also contributes to the activation of some of these target enzymes.^6,7Open in a separate windowFigure 2Schematic model for the various mechanisms of CaM-dependent target regulation. (A) autoinhibitory domain displacement, (B) sequestering of a ligand binding site, (C) active-site reorganization, (D) CaM-induced target protein dimerization (1:2 complex), (E) CaM-induced target protein dimerization (2:2 complex), (F) hypothesized model for CaM acting as an adaptor/recruiter protein. In each panel CaM is shown as a red dumbbell shaped molecule with Ca²⁺ ions represented by yellow circles, and the target proteins are shown in various colors. See the text for details on each model.Another regulatory mechanism involving Ca²⁺-CaM-binding to a single contiguous CaMBD sequence may occur with the potato kinesin-like CaM-binding protein (KCBP)⁸ as well as some plant cyclic-nucleotide gated channels (CNGC''s).⁹ In both cases the Ca²⁺-CaM binding site on the target protein overlaps with the respective ligand binding site, and thus the binding of KCBP to microtubules or the binding of cyclic nucleotide monophosphates to CNGC''s may be prevented by interaction with Ca²⁺-CaM (Fig. 2B). In a variation on this mechanism, CaM can bind to the cytoplasmic juxtamembrane region of the human epidermal growth factor receptor and sequester a threonine residue which is a specific phosphorylation target of protein kinase C (PKC). CaM-binding inhibits PKC phosphorylation of this threonine, and PKC phosphorylation inhibits CaM-binding.¹⁰There are also several examples of CaM-target interactions where the N- and C-lobes bind to noncontiguous target protein regions, and play distinct roles in target regulation. The structures of a CaM-activated adenylyl cyclase from Bacillus anthracis with and without bound CaM shows how the N- and C-lobes of CaM can bind two distant regions of the adenylyl cyclase enzyme and induce a conformation reorganization that creates the enzyme''s active site (Figs. 1E and ​and2C2C).¹¹ An interesting feature of this interaction is that the CaM N-lobe remains Ca²⁺-free and in a closed conformation, while the C-lobe is in a canonical Ca²⁺-bound open conformation. Indeed, Ca²⁺-binding to the C-lobe but not N-lobe is required for activation of the adenylyl cyclase.¹²The N- and C-lobes of Ca²⁺-CaM can also each simultaneously bind to identical peptides derived from the petunia glutamate decarboxylase (GAD) enzyme to form a 1:2 Ca²⁺-CaM:GAD complex (Fig. 1F).^13,14 This suggests that Ca²⁺-CaM-induced target protein dimerization may be another way in which CaM can regulate target proteins (Fig. 2D). CaM-dependent dimerization has also been shown to regulate the activity of small conductance Ca²⁺-activated K⁺ channels (SK channel), although in this case a novel 2:2 CaM:SK channel complex is formed (Figs. 1G and ​and2E2E).¹⁵ This structure is also unique because Ca²⁺ is bound to the “lower affinity” N-lobe EF-hands, but not to the “higher affinity” C-lobe EF-hands of CaM.In addition to the SK channel, CaM can regulate voltage-gated sodium channels, voltage-gated calcium channels, as well as ryanodine-sensitive calcium release channels.¹⁶ With these channels CaM typically binds in complex Ca²⁺-dependent and Ca²⁺-independent ways to several noncontiguous target sequences in the same protein, and often to so-called IQ motifs (IQXXXRGXXXR). IQ motifs are generally thought to be constitutive apo-CaM binding sites which retain CaM under resting (low [Ca²⁺]) cellular conditions to ensure a rapid response to Ca²⁺-stimuli.¹⁷ However many IQ motifs can also bind specifically to Ca²⁺-CaM or to both apo-CaM and Ca²⁺-CaM. Structures of some Ca²⁺-CaM-IQ domain complexes have revealed wrap-around binding modes, albeit with differences in lobe and peptide orientation compared to other complexes.^18–20 For a discussion about the mechanisms of CaM-dependent ion channel regulation (see ref. ¹⁶). A very recent crystal structure of apo-CaM bound to an IQ domain from myosin V (Fig. 1H) has also revealed yet another variation on the wrap-around binding mode, where the apo-C-lobe of CaM adopts a semi-open conformation and forms numerous interactions with the target sequence, while the apo-N-lobe adopts a closed conformation and forms weaker interactions with the IQ domain.²¹Using several biophysical techniques we recently characterized the interaction between CaM isoforms (mammalian CaM, soybean CaM isoforms SCaM-1 and SCaM-4) and a novel CaMBD derived from the Nicotiana tabacum mitogen-activated protein kinase phosphatase (NtMKP1).²² The NtMKP1 protein was initially identified as a CaM-binding protein by Ohashi and coworkers,²³ and the same group recently showed that CaM-binding NtMKP1 homologs are also present in other plant species as well.²⁴ We found that each CaM isoform was capable of binding to the NtMKP1 CaMBD in the absence of Ca²⁺ using only the apo-C-lobe, with the primary binding site consisting of NtMKP1 residues N438 - S449, and additional C-terminal residues G450 - K460 enhancing the overall binding affinity (K_d ∼10⁻⁵ M). In the presence of Ca²⁺, a 1:1 complex could be formed with the CaM C-lobe having significantly increased affinity for the N438 - S449 region of NtMKP1 (K_d 10⁻⁷ − 10⁻¹⁰ M). However, the Ca²⁺-loaded CaM N-lobe interacted only very weakly with the C-terminal NtMKP1 sequence in this 1:1 complex, despite an abundance of seemingly suitable hydrophobic “anchor” residues in this region. Interestingly, the addition of more peptide triggered the independent binding of a second NtMKP1 peptide to the Ca²⁺-CaM N-lobe (Kd 10⁻⁵ − 10⁻⁶ M) to form a 1:2 Ca²⁺-CaM:NtMKP1 complex. As with GAD, these results suggest that CaM is capable of facilitating the dimerization of NtMKP1, although the independent and sequential NtMKP1 peptide binding to the C- and N-lobes markedly distinguishes the CaM-NtMKP1 interaction from the simultaneous high-affinity binding of 2 GAD CaMBD''s to CaM.While our NtMKP1 study was ongoing, Ohashi and coworkers reported that CaM is incapable of stimulating the phosphatase activity of the NtMKP1 enzyme, thereby implying that the CaM-NtMKP1 interaction is necessary for something other than direct enzyme regulation.²⁵ The independent and sequential binding of the NtMKP1 fragments to the Ca²⁺-saturated C- and then N-lobes of CaM observed in our study suggests a plausible situation in which the C-lobe of CaM is tightly bound to NtMKP1, leaving the N-lobe free to recruit a different target protein to the complex, for example, a NtMKP1 protein substrate. Therefore, CaM may be capable of acting as an adaptor or recruiter protein, which would add yet another mechanism of target regulation to CaM''s repertoire (Fig. 2F). In addition to NtMKP1 peptides, the isolated N-lobe of CaM is capable of binding to other CaMBD peptides^26,27 as well as intact target proteins,²⁸ increasing the likelihood that the N-lobe could serve as a recruiter domain. The pre-association of the apo-C-lobe of CaM with NtMKP1 under resting conditions would also ensure a rapid response response to Ca²⁺-stimuli, since CaM would only need to recruit one rather than both protein targets.Although the ability of CaM to act as an adaptor protein in vivo has not yet been demonstrated, there are examples of related EF-hand proteins acting as adaptor proteins, including centrin²⁹ and calcium- and integrin-binding protein 1.³⁰ With the abundance of poorly characterized CaM-binding proteins in plants, many of which have CaMBD''s with little sequence resemblance to the better characterized motifs in animals¹ it seems likely that sequences will be identified which bind preferentially to the CaM N-lobe. Considering the incredible assortment of known CaM interaction modes and regulatory mechanisms, many of which have only been identified within the last decade, it is likely only a matter of time before CaM is proven to function as an adaptor protein in vivo.     

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

Genetic Analysis of Mutants with a Reduced Ca2+-Dependent K+ Current in PARAMECIUM TETRAURELIA

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.     

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K⁺ channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K⁺ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K⁺ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.