Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.

A CACNA1C variant associated with cardiac arrhythmias provides mechanistic insights in the calmodulation of L-type Ca2+ channels.

We recently reported the identification of a de novo single nucleotide variant in exon 9 of CACNA1C associated with prolonged repolarization interval. Recombinant expression of the glycine to arginine variant at position 419 produced a gain in the function of the L-type CaV1.2 channel with increased peak current density and activation gating but without significant decrease in the inactivation kinetics. We herein reveal that these properties are replicated by overexpressing calmodulin (CaM) with CaV1.2 WT and are reversed by exposure to the CaM antagonist W-13. Phosphomimetic (T79D or S81D), but not phosphoresistant (T79A or S81A), CaM surrogates reproduced the impact of CaM WT on the function of CaV1.2 WT. The increased channel activity of CaV1.2 WT following overexpression of CaM was found to arise in part from enhanced cell surface expression. In contrast, the properties of the variant remained unaffected by any of these treatments. CaV1.2 substituted with the α-helix breaking proline residue were more reluctant to open than CaV1.2 WT but were upregulated by phosphomimetic CaM surrogates. Our results indicate that (1) CaM and its phosphomimetic analogs promote a gain in the function of CaV1.2 and (2) the structural properties of the first intracellular linker of CaV1.2 contribute to its CaM-induced modulation. We conclude that the CACNA1C clinical variant mimics the increased activity associated with the upregulation of CaV1.2 by Ca2+-CaM, thus maintaining a majority of channels in a constitutively active mode that could ultimately promote ventricular arrhythmias.

Sympathetic Stimulation Upregulates the Ca2+ Channel Subunit, CaVα2δ1, via the ß1 and ERK 1/2 Pathway in Neonatal Ventricular Cardiomyocytes.

Intracellular Ca2+ overload secondary to chronic hemodynamic stimuli promotes the recruitment of Ca2+-dependent signaling implicated in cardiomyocyte hypertrophy. The present study tested the hypothesis that sympathetic-mediated hypertrophy of neonatal rat ventricular cardiomyocytes (NRVMs) translated to an increase in calcium influx secondary to the upregulation of CaV1.2 channel subunits. Confocal imaging of norepinephrine (NE)-treated NRVMs revealed a hypertrophic response compared to untreated NRVMs. L-type CaV1.2 peak current density was increased 4-fold following a 24-h stimulation with NE. NE-treated NRVMs exhibited a significant upregulation of CaVα2δ1 and CaVß3 protein levels without significant changes of CaVα1C and CaVß2 protein levels. Pre-treatment with the ß1-blocker metoprolol failed to inhibit hypertrophy or CaVß3 upregulation whereas CaVα2δ1 protein levels were significantly reduced. NE promoted the phosphorylation of ERK 1/2, and the response was attenuated by the ß1-blocker. U0126 pre-treatment suppressed NE-induced ERK1/2 phosphorylation but failed to attenuate hypertrophy. U0126 inhibition of ERK1/2 phosphorylation prevented NE-mediated upregulation of CaVα2δ1, whereas CaVß3 protein levels remained elevated. Thus, ß1-adrenergic receptor-mediated recruitment of the ERK1/2 plays a seminal role in the upregulation of CaVα2δ1 in NRVMs independent of the concomitant hypertrophic response. However, the upregulation of CaVß3 protein levels may be directly dependent on the hypertrophic response of NRVMs.

An ancestral MAGUK protein supports the modulation of mammalian voltage-gated Ca2+ channels through a conserved CaVß-like interface.

Eukaryote voltage-gated Ca2+ channels of the CaV2 channel family are hetero-oligomers formed by the pore-forming CaVα1 protein assembled with auxiliary CaVα2δ and CaVß subunits. CaVß subunits are formed by a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain connected through a HOOK domain. The GK domain binds a conserved cytoplasmic region of the pore-forming CaVα1 subunit referred as the "AID". Herein we explored the phylogenetic and functional relationship between CaV channel subunits in distant eukaryotic organisms by investigating the function of a MAGUK protein (XM_004990081) cloned from the choanoflagellate Salpingoeca rosetta (Sro). This MAGUK protein (Sroß) features SH3 and GK structural domains with a 25% primary sequence identity to mammalian CaVß. Recombinant expression of its cDNA with mammalian high-voltage activated Ca2+ channel CaV2.3 in mammalian HEK cells produced robust voltage-gated inward Ca2+ currents with typical activation and inactivation properties. Like CaVß, Sroß prevents fast degradation of total CaV2.3 proteins in cycloheximide assays. The three-dimensional homology model predicts an interaction between the GK domain of Sroß and the AID motif of the pore-forming CaVα1 protein. Substitution of AID residues Trp (W386A) and Tyr (Y383A) significantly impaired co-immunoprecipitation of CaV2.3 with Sroß and functional upregulation of CaV2.3 currents. Likewise, a 6-residue deletion within the GK domain of Sroß, similar to the locus found in mammalian CaVß, significantly reduced peak current density. Altogether our data demonstrate that an ancestor MAGUK protein reconstitutes the biophysical and molecular features responsible for channel upregulation by mammalian CaVß through a minimally conserved molecular interface.

A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit.

L-type CaV1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaVα1C assembled with CaVß and CaVα2δ1 subunits. A direct interaction between CaVα2δ1 and Asp-181 in the first extracellular loop of CaVα1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaVα2δ1 complexed with the voltage sensor domain of CaVα1C suggests that Ser-261 and Ser-263 residues in the metal ion-dependent adhesion site (MIDAS) motif are determinant in this interaction, but this hypothesis is untested. Here, coimmunoprecipitation assays and patch-clamp experiments of single-substitution variants revealed that CaVα2δ1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of CaV1.2 currents. In contrast, mutating the side chains of CaVα2δ1 Ser-263, Thr-331, and Asp-363 with alanine did not completely prevent channel function. Molecular dynamics simulations indicated that the carboxylate side chain of CaVα2δ1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of CaVα2δ1 Ser-261 and the carboxylate group of CaVα1C Asp-181. In return, the hydrogen atoms contributed by the side chain of Ser-261 and the main chain of Ser-263 bonded the oxygen atoms of CaV1.2 Asp-181. We propose that CaVα2δ1 Asp-259 promotes Ca2+ binding necessary to produce the conformation of the VWA domain that locks CaVα2δ1 Ser-261 and Ser-263 within atomic distance of CaVα1C Asp-181. This three-way network appears to account for the CaVα2δ1-induced modulation of CaV1.2 currents.

Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of CaVα2δ1 with CaVß/CaVα1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the CaV1.2/CaVα2δ1 interface are unknown. Here, a homology-based model of CaV1.2 identified protein interfaces between the extracellular domain of CaVα2δ1 and the extracellular loops of the CaVα1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with CaVα2δ1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with CaVα2δ1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of CaVß/CaV1.2 with CaVα2δ1 and the CaVα2δ1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in CaV1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of CaV1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the CaVα2δ1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of CaV1.2. We conclude that CaV1.2 Asp-181 anchors the physical interaction that facilitates the CaVα2δ1-mediated functional modulation of CaV1.2 currents. By stabilizing the first extracellular loop of CaV1.2, CaVα2δ1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels.

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVß, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

A helical segment makes potassium channels go-go.

More than 500 variants in the KCNH2 gene, which encodes the cardiac human ether-a-go-go (hERG) ion channel, have been associated with sudden cardiac death, but only a subset of these variants have been investigated. Matthew D. Perry and colleagues now combine NMR spectroscopy and electrophysiological experiments to explore the functional properties of mutations within an overlooked hERG helix, finding important contributions to channel function.

Inherited Ventricular Arrhythmias: The Role of the Multi-Subunit Structure of the L-Type Calcium Channel Complex.

The normal heartbeat is conditioned by transient increases in the intracellular free Ca2+ concentration. Ca2+ influx in cardiomyocytes is regulated by the activity of the heteromeric L-type voltage-activated CaV1.2 channel. A complex network of interactions between the different proteins forming the ion channel supports the kinetics and the activation gating of the Ca2+ influx. Alterations in the biophysical and biochemical properties or in the biogenesis in any of these proteins can lead to serious disturbances in the cardiac rhythm. The multi-subunit nature of the channel complex is better comprehended by examining the high-resolution three-dimensional structure of the closely related CaV1.1 channel. The architectural map identifies precise interaction loci between the different subunits and paves the way for elucidating the mechanistic basis for the regulation of Ca2+ balance in cardiac myocytes under physiological and pathological conditions.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity.

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca(2+) channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of CaVα2δ1 as well as the CaVα2δ1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca(2+) channels.

Functional characterization of CaVα2δ mutations associated with sudden cardiac death.

L-type Ca(2+) channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVß and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30-33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca(2+) currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ.

Aromatic-aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process.

The Ca(2+)-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca(2+) concentrations (Pomax) is low, typically 0.1-0.2 for KCa3.1 wild type. This observation argues for the binding of Ca(2+) to the calmodulin (CaM)-KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca(2+)-dependent gating of KCa3.1 originates from the binding of Ca(2+) to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic-aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic-aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

Cooperative activation of the T-type CaV3.2 channel: interaction between Domains II and III.

T-type CaV3 channels are important mediators of Ca(2+) entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial NaV channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG(interact) ≥ 1.5 kcal mol(-1)) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type CaV3 channel.

Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process.

The Ca(2+)-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca(2+) sensitivity of KCa3.1 is conferred by the Ca(2+)-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca(2+) to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe-KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca(2+) (Pomax). A structural homology model of the KCa3.1-CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM-KCa3.1 complex throughout gating.

A quartet of leucine residues in the guanylate kinase domain of CaVß determines the plasma membrane density of the CaV2.3 channel.

Ca(V)ß subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)ß (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, ß10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)ß3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)ß3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)ß-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.

The C-terminus of human Ca(v)2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines.

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.

Double mutant cycle analysis identified a critical leucine residue in the IIS4S5 linker for the activation of the Ca(V)2.3 calcium channel.

Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel.