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Originally published In Press as doi:10.1074/jbc.M607405200 on October 11, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39424-39436, December 22, 2006
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The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels*

Alexandra Raybaud, Yolaine Dodier, Pierre Bissonnette, Manuel Simoes, Daniel G. Bichet, Rémy Sauvé, and Lucie Parent1

From the Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Received for publication, August 3, 2006 , and in revised form, September 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) CaV channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in CaV1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly432 and Gly436 in CaV1.2) to form a triglycine motif unique to HVA CaV channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca2+ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In CaV1.2, {alpha}-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with CaV1.2 wild type > G770A > G422A {cong} G436A >> G432A (from the fastest to the slowest). Mutations at position Gly432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX3G residues blunted Ca2+-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In CaV2.3, mutation of the distal glycine Gly352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX3G motif in the voltage-dependent activation and inactivation gating of HVA CaV channels with the distal glycine residue being mostly involved in the inactivation gating.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-dependent calcium channels are membrane-bound proteins that form large aqueous pores for the selective diffusion of Ca2+ ions across the plasma membrane (1, 2). Native Ca2+ channels are composed of the pore-forming CaV{alpha}1, the disulfur-linked dimer CaV{alpha}2{delta}, the intracellular CaVbeta subunits (beta1-beta4), and in some cases the CaV{gamma} subunit (3). To date, molecular cloning has identified the primary structures for 10 distinct calcium channel CaV{alpha}1 subunits (1, 4-9) that are classified into three main subfamilies according to their gating properties (CaV1, CaV2, and CaV3). Whereas all voltage-gated Ca2+ channel {alpha}1 subunits activate and inactivate in response to membrane depolarization, the high voltage-activated (HVA)2 CaV1 and CaV2 {alpha}1 subunits operate at markedly more positive membrane potentials than low voltage-activated CaV3 channel {alpha}1 subunits.

In the absence of a crystal structure for these proteins, details regarding the structural determinants of the channel inner pore as well as the molecular mechanism underlying the activation of CaV{alpha}1 subunits remain sketchy. Structural studies have revealed that the architecture of the ion-selective pore is conserved in the homologous {alpha} subunit of different K+ channels (10-15) with the S6/TM2 helices lining the channel inner pore. Activation of voltage-gated K channels appears to involve the PXP locus (where X represents a hydrophobic residue) motif that is highly conserved in the lower part of S6 (16) in the family of the KV1-KV4 channels (15-18). Prolines tend to destabilize {alpha}-helices by the lack of a backbone hydrogen bond, normally formed by the amide nitrogen and by steric constraints (19, 20). Due to their typical {phi}-{Psi} bond angles, proline residues bend by at least 20° the axis of the {alpha}-helix. Based on the molecular properties of the residues, the PXP motif was thus proposed to form a "hinge" structure in voltage-gated K+ channels (16, 17, 21, 22).

In contrast, the crystal structure of a Ca2+-gated K+ channel (MthK) obtained in the presence of bound Ca2+ showed that the pore-lining helices are bent about 30° near Gly83 such that the helical bundle is splayed open (11). The closed KcsA structure, where the inner helices adopt an "inverted teepee" conformation crossing over near the intracellular surface at the helix bundle, would represent the closed state (10). Based on these static crystal structures, it was proposed that K+ channels could open by bending the pore-lining {alpha}-helix at this nearly universal glycine residue located in the middle of the pore lining helix (23). The comparison of backbone torsions was, however, limited by the poor resolution of the x-ray structure of MthK. Furthermore, the significance of this proposed gating mechanism (23) remains unclear, especially for eukaryotic ion channels.

HVA CaV1 and CaV2 channels possess four distinct S6 segments, but none possesses the tandem PXP motif that has been implicated in the activation gating of KV channels. In addition, the "universal" glycine residue is conserved only in two of the four inner helices in HVA CaV1 and CaV2 channels (Fig. 1B). Instead, a triglycine motif, GX9GX3G, has been identified in IS6 of all HVA CaV1 and CaV2 channels, whereas a lone but strictly conserved Gly residue can be found in mid-IIS6. The distal GX3G motif is reminiscent of the biglycine GXXXG or GX3G motif that provides a framework for specific transmembrane helix-helix and dimerization interactions (24-28). The proximal GX9G motif is similar to the biglycine GX8G motif conserved in TM2 of most Kir channels (13). In this regard, the second proline of the PXP motif is located at the position that corresponds approximately to the second glycine (Fig. 1B), suggesting that this glycine could be pivotal in channel gating. Finally, mutations of the distal glycine GX3G residues to polar serine and arginine residues in the human CaV1.2 contributes to the Timothy syndrome, characterized by lethal arrhythmias (29, 30). Herein, we show that relatively conserved alanine mutations of any glycine residue in IS6 significantly decreased macroscopic kinetics, with stronger effects observed with mutations of the second glycine in GX9GX3G of CaV1.2. In CaV2.3, mutation of the distal GX3G residue impacted significantly on the inactivation gating. It is proposed that the GX3G residues play a pivotal role in the gating of HVA CaV channels, with the distal glycine residue being mostly involved in the inactivation gating.


Figure 1
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  		FIGURE 1.
A, helical wheel representation of IS6 between Pro411 and Phe438 in the rabbit CaV1.2 channel. This representation suggests that the three glycine residues (empty circles) could be located on the same side of the helix. The five hydrophilic (black circles) residues appear to be facing away from the glycine residues. When two residues are shown at the same position, the inner number corresponds to the inner residue, and vice versa. Helical wheel projection was carried out with the ANTHEPROT 2000 version 5.2 software. B, primary sequences of S6/TM2 segments from K+ channels: Shaker/KV1.2, KV2.1, KvAP, KcsA, MthK, and KirBac obtained by aligning the conserved Gly residue (47) proposed to serve as the gating hinge in MthK channels (Gly83). The PVP gating hinge of Shaker channels is underlined. The bottom alignments show the primary sequences of the IS6 and IIS6 segments of the rabbit L-type CaV1.2 (GenBankTM accession number X15539) and the human CaV2.3 (GenBankTM accession number L27745) channels used in this study. The midglycine residues in IS6 (Gly422 or Gly338) and IIS6 (Gly770 or Gly690) are aligned with the glycine hinge of K+ channels. IS6 in CaV1.2 and CaV2.3 contains two additional glycine residues that are also highlighted.

 


Figure 2
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  		FIGURE 2.
Alanine scan in IS6 and IIS6 of CaV1.2. A-D, whole-cell current traces are shown from left to right for CaV1.2 ctrl, G422A, G432A, and G436A mutants in 10 mM Ba2+. Macroscopic activation kinetics of these mutants, estimated by the time to peak values, were estimated at 45 ± 3 ms (n = 26) for G422A, 47 ± 5 ms (n = 29) for G432A, 57 ± 3 ms (n = 21) for G436A, and 49 ± 3 ms (n = 33) for the control channel. Unless specified otherwise, mutants were expressed in Xenopus oocytes in the presence of CaV{alpha}2b{delta} and CaVbeta3 subunits, and currents were recorded using the two-electrode voltage clamp technique after a 30-min injection of a solution containing 50 mM EGTA. Oocytes were pulsed from -40 to +50 mV using 5-mV steps for 900 ms. Scale bars are 0.1µA and 100 ms for G432A and as shown for the other traces. Activation and inactivation properties are detailed in Table 1. E, the r800 values (the fraction of whole-cell currents remaining at the end of an 800-ms pulse) are shown ± S.E. for the single GA mutants from 0 to +30 mV for CaV1.2 control channel (n = 33), G422A (n = 26), G432A (n = 21), G436A (n = 21), G770A (n = 17), and G422A/G770A (n = 10) (from left to right on the bar graph). r800 values for the IS6 mutants were all statistically different from the control channel (p < 10-4). The midpotentials of inactivation (not shown) were similar for G422A, G436A, G770A, and the control channel and ranged from -21 to -23 mV. Residual currents were higher for the mutant channels with 0.69 ± 0.05 (n = 3) for G422A, 0.47 ± 0.01 (n = 12) for G436A, and 0.26 ± 0.01 (n = 8) for G770A as compared with 0.16 ± 0.01 (n = 33) for the control channel. Slow inactivation kinetics precluded the determination of the voltage dependence of inactivation for G432A.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant DNA Techniques— cDNAs coding for wild-type rabbit CaV1.2 (GenBankTM X15539 [GenBank] ), wild-type human CaV2.3 (GenBankTM L27745 [GenBank] ) (31), and rat CaVbeta3 (GenBankTM M88751 [GenBank] ) (32) were kindly donated by Drs E. Perez-Reyes and T. Schneider. The rat brain CaV {alpha}2b{delta} subunit provided by Dr. T. P. Snutch is >90% similar to GenBank NM_000722 [GenBank] (33). Point mutations were produced with the QuikChange XL mutagenesis kit (Stratagene, La Jolla, CA) using 39-bp primers as detailed before (34). CaV1.2 mutations were performed by cassette cloning using the naturally occurring SacI (956) site and the XhoI site that was engineered at nucleotide 1530 in the I-II linker of CaV1.2 (42 residues before IIS1) (34-36). This is a nonsilent mutation creating a Gly to Arg mutation (G511R). The resulting CaV1.2 (XhoI) channel (which will be referred to herein as the CaV1.2 control channel) displayed inactivation and activation kinetics similar to the wild-type CaV1.2 (35, 36). Constructs were verified by restriction mapping after religation of the mutated fragment into the SacI/XhoI sites of the wild-type CaV1.2. Recombinant clones for CaV1.2 and CaV2.3 were screened by double-stranded sequence analysis of the entire ligated cassette.


Figure 3
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  		FIGURE 3.
Western blot of nonexpressing proline and double mutants. Membrane expression of nonfunctional mutants G422P, G432P, and G422A/G432A was assessed from Western blots. Enriched plasma (lanes 1-5) and total membranes (lanes 6-10) isolated from Xenopus oocytes were separated on a 7.5% polyacrylamide gel (SDS-PAGE) as described under "Experimental Procedures." Following transfer onto a nitrocellulose membrane, proteins were revealed using the primary antibody against CaV1.2 (dilution 1:200; Alomone) and the secondary anti-rabbit horseradish peroxidase antibody (1:10,000; Jackson Immunoresearch). Lanes 1 and 6, noninjected oocytes; lanes 2 and 7,CaV1.2 control; lanes 3 and 8, G422P; lanes 4 and 9, G432P; lanes 5 and 10, G422A/G432A. Membrane preparations were isolated 3-4 days after cRNA injection to mimic the experimental conditions used for electrophysiological measurements.

 
The cDNA coding for the human camodulin (CaM) clone (GenBankTM M27319 [GenBank] ) was obtained by reverse-transcribing human placenta poly(A)+ RNA (Clontech). The cDNA was then amplified using the SMART PCR cDNA synthesis kit (Clontech) using specific primers designed to subclone into the restriction sites of the pT7TS vector (generously provided by Dr. Paul A. Krieg, University of Arizona). The pT7TS vector contains the 5'- and 3'-untranslated regions of Xenopus laevis beta-globin mRNA inserted into pGEM-4Z. The CaM1234 mutant (D20A/D56A/D93A/D129A) was obtained with the QuikChange XL mutagenesis kit according to the manufacturer's instructions.

cDNA constructs for the CaV1.2 and CaV2.3 subunits (wild type and mutants) were linearized at the 3'-end by HindIII digestion. The rat brain CaVbeta3 subunit, the CaV{alpha}2b{delta}, and the human CaM were digested by NotI, EcoRI, and BamHI, respectively. Run-off transcripts were prepared using methylated cap analog m7G (5')ppp(5')G and T7 RNA polymerase with the mMessage mMachine® transcription kit (Ambion, Austin, TX). The final cRNA products were resuspended in diethyl pyrocarbonate-treated H2O and stored at -20 °C. The integrity of the final product and the absence of degraded RNA were determined by a denaturing agarose gel stained with ethidium bromide.

Functional Expression of Wild-type and Mutant Channels— Oocytes were obtained from female Xenopus laevis clawed frog (Nasco, Fort Atkinson, WI) as described previously (34, 37). Individual oocytes free of follicular cells were obtained after a 30-40-min incubation in a calcium-free solution: 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5 mM Hepes, pH 7.6, containing 2 mg/ml collagenase (Invitrogen). 46 nl of a solution containing between 35 and 50 ng of cRNA coding for the wild-type or mutated CaV{alpha}1 subunit was injected 16 h later into stage V and VI oocytes. cRNA coding for the CaV{alpha}1 subunit was coinjected with the rat brain CaV{alpha}2b{delta} and the rat brain CaVbeta3 subunit at a 3:1:2 weight ratio, respectively. In some experiments, 27.6-55.2 ng of cRNA coding for the calmodulin wild-type or the CaM1234 mutant was co-injected with the channel subunits. For these experiments, CaM was sometimes injected 24-48 h before the CaV subunits with the same results. Oocytes were incubated at 18 °C in a Barth's solution: 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvic acid, 100 mM units/ml penicillin, 50 µg/ml gentamicin, pH 7.6. The biophysical properties of each mutant were studied in a minimum of three different oocyte batches. Furthermore, the control channel was tested periodically over time and measured in the same oocyte batch as the mutants, thus ensuring that the channel properties were recorded under the same level of endogenous CaVbeta subunits (38, 39).


Figure 4
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  		FIGURE 4.
Double and multiple alanine mutants of conserved glycine residues. A-D, whole-cell current traces are shown from left to right for G422A/G436A, G432A/G436A, G422A/G432A/G436A (GGGAAA), and G422A/G770A mutants in 10 mM Ba2+. Scale bars, 0.1 µA and 100 ms for G432A/G436A and GGGAAA and as shown for the other traces. Activation and inactivation properties are shown in Table 1. E, time to peak values for the alanine mutants and the control channel are shown on a semilogarithmic bar graph between 0 and +30 mV. The time to peak values increased 4-fold from 49 ± 3 ms (n = 33) for the control channel to 202 ± 57 ms (n = 12) for the G422A/G436A mutant (p < 10-4), to 717 ± 42 ms (n = 8) for G432A/G436A, and to 631 ± 27 ms (n = 11) for the triple GGGAAA mutant. The double G422A/G770A mutant peaked at 55 ± 3 ms (n = 10) within the same time frame as the control channel. From left to right on the bar graph,CaV1.2 control channel (n = 33), G422A/G432A (n = 12), G422A/G770A (n = 10), G432A/G436A (n = 8), and G422A/G436A/G436A (GGGAAA) (n = 11) with the number of experiments shown in parentheses.

 
Western Blots—Western blots were performed on both total membranes and purified plasma membranes isolated from X. laevis oocytes as detailed elsewhere (40). Briefly, homogenates prepared from six oocytes were centrifuged at 250 x g at 4 °C to discard cell debris. Supernatants were centrifuged at 16,000 x g for 20 min to pellet down total membranes. Total membranes were resuspended in a saline solution and frozen until use. For preparations enriched in plasma membranes, 40 oocytes were rinsed in MBSS (80 mM NaCl, 20 mM MES, pH 6.0) and incubated at room temperature with 0.005% subtilisin A (Sigma) to partially digest the vitelline membrane. Polymerization of plasma membranes was performed by two sequential 60-min incubations in MBSS first with 1% Ludox and then with 0.1% polyacrylic acid (Sigma). The oocytes were then homogenized in cold HbA (5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose, and 20 mM Tris, pH 7.4, with a protease inhibitor mixture. The homogenates centrifuged twice at 16 x g for 30 s at 4 °C and then at 25 x g and finally at 35 x g. A final centrifugation step at 16,000 x g for 20 min is used to pellet the purified plasma membranes, which are resuspended in 10 µl of HbA and frozen until use. Proteins were analyzed using the primary antibody against CaV1.2 (dilution 1:200; Alomone Laboratories, Jerusalem, Israel) and the secondary anti-rabbit horseradish peroxidase antibody. The quality of the overall procedure was monitored by Ponceau red staining.

Electrophysiological Recordings in Oocytes—Wild-type and mutant channels were screened at room temperature for macroscopic Ba2+ currents 2-6 days after RNA injection using a two-electrode voltage clamp amplifier (OC-725C; Warner Instruments) as described earlier (34, 35, 37). Voltage and current electrodes were filled with 3 M KCl, 1 mM EGTA, 10 mM HEPES (pH 7.4). Whole-cell currents were measured in a 10 mM Ba2+ solution (10 mM Ba(OH)2, 110 mM NaOH, 1 mM KOH, 20 mM Hepes titrated to pH 7.3 with methane sulfonic acid or occasionally in a 10 mM Ca2+ solution, where Ca(OH)2 replaced Ba(OH)2. To minimize kinetic contamination by the endogenous Ca2+-activated Cl- current, oocytes were injected with 18.4 nl of a 50 mM EGTA (Sigma) 0.5-2 h before the experiments (41). Oocytes were superfused by gravity flow at a rate of 2 ml/min that was fast enough to allow complete chamber fluid exchange within 30 s. Experiments were performed at room temperature (20-22 °C).

Data Acquisition and Analysis—PClamp software 8.2 (Molecular Devices, Axon instruments, Foster City, CA) was used for on-line data acquisition and analysis. Unless stated otherwise, data were sampled at 10 kHz and low pass-filtered at 5 kHz using the amplifier built-in filter. For CaV1.2, a series of 900-ms voltage pulses were applied from a holding potential of -100 mV at a frequency of 0.2 Hz from -40 to +50 mV, whereas the voltage pulses were 450 ms long for CaV2.3. Activation parameters were estimated from the mean I-V curves obtained for each channel combination. The I-V relationships were normalized to the maximum amplitude and were fitted to the Boltzmann Equation 1,

Formula 1(Eq. 1)
where E0.5 represents the potential for 50% activation, z is the slope parameter, Vm is the test potential, and RT/F have their usual meanings. The fitting process generated an estimation of the error associated with the fit of each of the adjustable parameters. For statistical analysis, however, the experimental errors were estimated by calculating the individual E0.5 act for each I-V curve and extracting the S.E. from the mean of individual E0.5 act. For CaV1.2 that is slowly inactivating, inactivation kinetics were quantified using r800 values (i.e. the ratio of the whole-cell current remaining at the end of an 800-ms pulse). For CaV2.3 that is inactivating slightly faster, inactivation kinetics were quantified using r300 values (i.e. the ratio of the whole-cell current remaining at the end of a 300-ms pulse). For the CaV1.2 mutant channels that did not display significant inactivation during the depolarizing pulse (r800 > 0.98), macroscopic kinetics were characterized by the "time to peak" parameter that was extracted directly from the work-sheet generated with Clampfit 8.2. Unless specified otherwise, r800 and time to peak values were calculated at -10 < Vm < 30 mV but reported in the text at Vm = +10 mV. Capacitive transients were erased for clarity in the final figures.


Figure 5
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  		FIGURE 5.
Mutational analysis of the G436 position. A-D, whole-cell current traces are shown from left to right and from top to bottom for G436P, G436Y, G436E, and G436H mutants in 10 mM Ba2+. Peak currents were noticeably smaller than for the GA mutants. Of all of the mutants tested, the macroscopic kinetics G436A came closer to the control channel (See Fig. 2). Activation and inactivation properties are shown in Table 1. E, time to peak values for the single Gly436 mutants and the control channel were computed between 0 and 30 mV. From left to right on the bar graph, CaV1.2 control channel (n = 33), G436A (n = 21), G436P (n = 8), G436Y (n = 14), G436R (n = 7), G436K (n = 7), G436H (n = 9), G436D (n = 9), and G436E (n = 9) with the number of experiments shown in parentheses. At +10 mV, time to peak values were 57 ± 3 ms (n = 21) for G436A, 124 ± 29 ms (n = 8) for G436P (p < 0.01), 201 ± 28 ms (n = 14) for G436Y (p < 0.001), 327 ± 54 ms (n = 9) for G436E (p < 0.001), 445 ± 39 ms (n = 7) for G436R (p < 0.001), 628 ± 57 ms (n = 9) for G436H (p < 0.001), 707 ± 44 ms (n = 7) for G436K (p < 0.001), and 765 ± 26 ms (n = 9) for G436D (p < 0.001) as compared with 49 ± 3 ms (n = 33) for the control channel. The mutations to polar and charged residues had the strongest effect.

 
In a few experiments, the voltage dependence of inactivation (isochronal inactivation) were obtained from normalized currents measured at 0 or +10 mV after a series of 5-s prepulses that varied from -100 to +30 mV at a frequency of 0.02 Hz (34). For the isochronal inactivation figures, data points represent the mean of n ≥ 4 and were fitted to a Boltzmann equation as described elsewhere (37).

Time constants of whole-cell current traces were estimated with the predefined Equation 2 in Clampfit 8.2 that uses the Chebyshev routine and a 4-point smoothing filter with n = 2 for Ba2+ current traces and n = 3 for Ca2+ current traces. Under the latter, time constant 1 ({tau}1) was defined as the slow inactivation time constant, and time constant 2 ({tau}2) was the fast inactivation time constant. The activation time constant is {tau}3.

Formula 2(Eq. 2)
Statistical analyses were performed using the one-way analysis of variance (Tukey test) for two independent populations fitting routines provided by Origin 7.0. Data were considered statistically significant for p < 0.01.

Computer-predicted Structure and Homology Modeling of IS6 in CaV1.2 and CaV2.3—The sequence alignments of the CaV channels with the KV1.2 channel and the MthK channel were based upon the universal glycine located on the S6 segment. Automated homology modeling was performed with INSIGHTII module Modeler version 8.2 (42) and involved the generation of 150 monomer models for >120 residues from S5 to S6 in both CaV1.2 and CaV2.3 channels using either KV1.2 (Protein Data Bank 2A79 [PDB] ) (15), KcsA (Protein Data Bank 1K4C [PDB] ) (14), or MthK (Protein Data Bank 1LNQ [PDB] ) channel as templates. SAMT02 (43) was used to determine the CaV residues composing the pore helix, the selectivity filter, and S6. The external loop between the selectivity filter and S6 is slightly longer in CaV than in K+ channels, thus justifying the introduction of gaps in the templates. In the resulting model, the high affinity Ca2+ binding site (Glu393 in CaV1.2) is equivalent to the high affinity ion binding site S4 in the crystallized KcsA channel (44). The models with the lowest objective function (roughly related to the energy of the model) and the lowest root mean square deviation for S6 between the template and the model were kept. The precision of the models decreased significantly for the residues located at the C-terminal end in the absence of structural constraints. Energy minimization was carried out with CHARMM as described elsewhere (45, 46).


Figure 6
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  		FIGURE 6.
G436R prevents the increased inactivation of E462R. A-C, whole-cell current traces are shown from left to right for G436R, E462R, and G436R/E462R mutants in 10 mM Ba2+. Activation and inactivation properties are detailed in Table 1. The activation potential and the reversal potential were slightly shifted to the left when compared with the single mutants (Table 1). The time to peak values of the G436R/E462R were estimated at 194 ± 14 ms (n = 13) as compared with 445 ± 39 ms (n = 7) for G436R (p < 0.001) at Vm = 10 mV, suggesting that the presence of E462R increased the macroscopic activation kinetics. D, the r800 values are shown ± S.E. from 0 to +30 mV for CaV1.2 control channel (n = 33), G436R (n = 7), E462R (n = 21), and G436R/E462R (n = 13) (from left to right on the bar graph). The double mutant did not appreciably inactivate during the 900-ms depolarizing pulse, and r800 values were 0.87 ± 0.01 (n = 13) at +10 mV and higher than 0.85 at all voltages tested (p < 0.001). At +10 mV, inactivation was slower for G436R {approx} G436R/E462R < control channel < E462R. r800 values for the mutants were all statistically different from the control channel (p < 0.001).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Three Glycine Residues in IS6 Are Involved in Voltage-dependent Gating of CaV1.2—High voltage-activated CaV1.2 and CaV2.3 channels possess three Gly residues in IS6 (GX9GX3G) and one Gly residue in mid-IIS6. Glycine residues are unique in their ability to adopt a wide range of main chain dihedral angles. When projected on a helicoidal wheel (Fig. 1A), Gly422, Gly432, and Gly436 are found to line the same side of the helix in CaV1.2, suggesting that they could act in concert. No Gly or Pro residues are present in IIIS6 and IVS6. Since Gly422 in IS6 and Gly770 in IIS6 could be aligned with the hinge Gly83 in MthK (23, 47) (Fig. 1B), we hypothesized that the {alpha}-helix flexibility conferred by these residues could be essential for the gating of CaV1.2. It is already known that mutations of the distal GX3G residues to polar residues significantly impaired voltage-dependent inactivation in CaV1.2 channels (29, 30), but little is known about the relative importance of each glycine residue in this process. To elucidate the role of glycine residues in the activation gating of CaV1.2, we undertook a mutational analysis of Gly422, Gly432, Gly436, and Gly770 residues in CaV1.2 channels by alanine substitution, the residue that has been consistently found to stabilize {alpha}-helices (48-50).

Control and mutant channels were expressed in Xenopus oocytes in the presence of CaVbeta3 to promote robust whole-cell currents. G422A, G432A, G436A, and G770A mutants yielded high voltage-activated inward Ba2+ currents that all displayed slower kinetics than the control channel (Fig. 2). Their macroscopic activation kinetics, as estimated by the time to peak values were mostly similar to the control channel. The slightly slower activation kinetics of G422A and G432A channels observed at 0 mV (p < 0.01) might be accounted for by a +5-mV shift in their activation potentials (Table 1). Whole-cell I/V curves of these mutants reversed between 42 and 48 mV, indicating that the alanine mutations did not affect ion selectivity. In contrast, the macroscopic inactivation kinetics of the GA mutants were all significantly decreased, with r800 values ranging from 0.47 ± 0.02 (33) for the control channel to 0.89 ± 0.01 (21) for G432A (Fig. 2E). At +10 mV, inactivation was slower for G432A < G422A {approx} G422A/G770A < G436A < G770A {approx} control channel. Furthermore, the inactivation kinetics of the IS6 mutants did not appreciably get faster with depolarization, unlike the control channel. Given that inactivation and activation gating are strongly coupled in CaV1.2 (51-53), a decrease in the macroscopic inactivation kinetics could result either from a decrease in the rate of inactivation, an increase in the rate of deactivation, or a decrease in the rate of activation. However the observation that the time to peak values were not significantly altered suggests that the rate of activation was not affected by the mutations.


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  		TABLE 1
Biophysical properties of CaV1.2 mutants Shown are biophysical parameters of CaV1.2 wild-type and mutant channels expressed in Xenopus oocytes in the presence of CaV{alpha}2bd and CaVbeta3 subunits. The background channel used for the mutations was the CaV1.2 (XhoI) with a unique XhoI site at G511R. Whole-cell currents were measured in 10 mM Ba2+ throughout. Activation data (E0.5 act, z, and Vrev) were estimated from the mean I-V relationships and fitted to Boltzmann Equation 1. Inactivation properties were estimated from the fraction of noninactivating currents at 800 ms or r800 values. r800 values could not be determined for mutants with "time to peak" values of >500 ms. Peak IBa was determined from the peak I-V relationships for the corresponding experiments. The data are shown with the mean ± S.E. of the individual experiments, and the number of experiments appears in parentheses.

 
The {alpha}-helix-breaking proline single mutants G422P and G432P did not produce functional channels, although Western blots confirmed that the proteins were properly routed to the plasma membrane (Fig. 3). The nonfunctionality of the proline mutants within the proximal GX9G motif suggests that introducing a rigid artificial kink is incompatible with normal activation gating of CaV1.2. In contrast, the proline mutant of the midglycine in IIS6 with G770P did not significantly alter channel gating, confirming the specificity of the glycine residues in IS6. As discussed later, G436P expressed significant whole-cell Ba2+ currents with kinetics only slightly slower than the G436A mutant.

Macroscopic Activation of CaV1.2 Is Further Decreased in Multiple Glycine Mutants of IS6—The GX9GX3G motif is unique to IS6 but is conserved in all HVA CaV channels, suggesting that helix flexibility could play a critical role in channel function. Mutating two or three Gly residues by Ala further decreased macroscopic kinetics (Fig. 4). Activation kinetics were decreased to such an extent that mutants did not appear to inactivate within the 900-ms pulse duration. Macroscopic kinetics were thus quantified using the time it takes for whole-cell current traces to reach the maximum currents or time to peak values. Under our experimental conditions, an increase in the time to peak value could be obtained from an increase in the microscopic activation and inactivation time constants. Time to peak values were in the 200-300-ms range for the double and triple mutants in IS6. In contrast, they remained similar to the control channel for the double G422A/G770A, suggesting that glycine residues in IS6 are more critical than the glycine residue in IIS6 for the activation gating of CaV1.2 (Fig. 4E). These data confirm previous reports that Gly770 in mid-IIS6 is not likely to contribute to the activation gating hinge of CaV1.2 (54). Double and triple mutants that incorporated G432A were, however, the slowest channels, although 90% of the whole-cell currents peaked within 300 ms at all voltages. With their long winded activation kinetics, whole-cell Ba2+ currents of G432A/G436A and GGGAAA mutants did not appreciably inactivate within the 900-ms voltage pulse in our experiments. The double G422A/G432A channel did not produce functional channels, although some protein was detected in plasma membrane preparations (Fig. 3).


Figure 7
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  		FIGURE 7.
CDI kinetics are slower in G432S and G436R. Top panel, whole-cell current traces are shown from left to right for the CaV1.2 control channel, G432S, and G436R in 10 mM Ba2+. Bottom panel, whole-cell current traces are shown from left to right for the CaV1.2 control channel, G432S, and G436R in 10 mM Ca2+. Mutations retarded but did not eliminate calcium-dependent inactivation. Inactivation of G432S and G436R in the presence of Ca2+ was partially complete with r800 values = 0.37 ± 0.02 (n = 17) and 0.71 ± 0.02 (n = 13), respectively, as compared with 0.02 ± 0.01 (n = 11) for the control channel. The macroscopic activation kinetics of G436R were also significantly slower at p < 0.001, with time to peak values of 36 ± 1 ms (n = 13) for G436R as compared with 27.6 ± 0.5 ms (n = 17) for G432S and 23.6 ± 0.4 ms (n = 11) for the control channel at +10 mV. For the control channel, the fast {tau}2 = 25.1 ± 0.5 ms stands out over the slow {tau}1 = 196 ± 1 ms (n = 3) (A2/A1 ratio = 0.65 ± 0.03) at +10 mV. The fast inactivation time constant {tau}2 was seemingly conserved in G432S, but its relative importance decreased with {tau}2 = 33 ± 3 ms and {tau}1 = 193 ± 3 ms (n = 3) (A2/A1 ratio = 0.59 ± 0.07). G436R caused a more significant decrease in CDI kinetics with {tau}2 = 73 ± 6 ms and {tau}1 = 563 ± 33 ms (n = 3) (A2/A1 ratio = 0.23 ± 0.01). Biophysical parameters are shown in Table 2. Scale bars, 0.1 µA and 100 ms for G432S and G436R in Ba2+ (top panel) and as shown for the other traces.

 
Few Mutations at Position Gly432 Expressed Functional Ba2+ Currents—A mutational analysis was undertaken at position Gly432 implicated in the Timothy syndrome (29, 30) to investigate the structural requirements for voltage-dependent inactivation gating. Except for the G432A, G432S, and G432E mutants, the Gly432 mutations (Pro, His, Arg, Asp, and Lys) expressed whole-cell Ba2+ currents that were generally too small to characterize or failed to express any currents at all. Western blots of G432R, G432K, and G432D mutations confirmed that the proteins were processed and targeted to the membrane (results not shown). Whole-cell currents of G432A, G432E, and G432S were significantly slower than the control channel with time to peak values of 71 ± 15 ms (21), 116 ± 10 ms (6), and 711 ± 43 ms (22), respectively, as compared with 49 ± 3 ms (33) for the control channel. In addition, whole-cell Ba2+ currents of G432A, G432E, and G432S did not appreciably inactivate, with the fraction of noninactivating currents being larger than 90% within the 900-ms voltage pulse in our experiments.


Figure 8
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  		FIGURE 8.
G436R is resistant to CDI. A, normalized whole-cell current traces obtained in 10 mM Ca2+ are shown superimposed for the CaV1.2 control channel, G432S, and G436R at +10 mV (from top to bottom). CDI remained faster for G432S than for G436R despite similar rates of inactivation in Ba2+. The inactivation time constants were similar whether measured under endogenous conditions or after expression of calmodulin wild type. Under the latter conditions, {tau}2 = 26 ± 3 ms, {tau}1 = 154 ± 7 ms, and A2/A1 ratio = 0.69 ± 0.02 (n = 7) at +10 mV for the control channel using Eq 2. For G432S, {tau}2 = 29 ± 3 ms, {tau}1 = 204 ± 13 ms, and A2/A1 ratio = 0.60 ± 0.04 (n = 7). For G436R, {tau}2 = 76 ± 6 ms, {tau}1 = 615 ± 50 ms, and A2/A1 ratio = 0.35 ± 0.01 (n = 8). B, typical whole-cell Ca2+ current traces obtained for the control CaV1.2 channel are shown superimposed at +10 mV after co-expression with the cRNA coding for the calmodulin wild-type (CaM wt) or overexpression of the dominant negative calmodulin mutant D20A/D56A/D93A/D129A or CaM1,2,3,4 (from top to bottom). Traces were normalized to the peak current for each condition. Unlike what was previously reported in the presence of CaVbeta2a (85), CDI was not completely obliterated in the presence of CaVbeta3. C, typical whole-cell Ca2+ current traces obtained for G432S are shown superimposed at +10 mV after co-expression with either wild-type CaM or CaM1,2,3,4 (from top to bottom). Overexpression of CaM1,2,3,4 decreased the contribution from the fast inactivation time constant. D, typical whole-cell Ca2+ current traces obtained for G436R are shown superimposed at +10 mV after co-expression with either wild-type CaM or CaM1,2,3,4 (from top to bottom). In the presence of CaM1,2,3,4, the fast {tau}2 = 25 ± 2 ms, and {tau}1 increased to 412 ± 14 ms (n = 15) with A2/A1 ratio = 0.40 ± 0.03 at +10 mV for the control channel; the fast inactivation {tau}2 = 43 ± 10 ms and {tau}1 = 371 ± 52 ms (n = 7) with A2/A1 ratio = 0.25 ± 0.05 for G432S; and finally {tau}2 = 66 ± 4 ms and {tau}1 = 1540 ± 613 ms (n = 8) with A2/A1 ratio = 0.12 ± 0.02 for G436R. Scale bar, 200 ms throughout.

 
Gly436 Mutations with Polar Residues Decreased Macroscopic Inactivation Kinetics—Most Gly436 mutants, including G436P, expressed significant whole-cell Ba2+ currents (Fig. 5, A-D). Gly436 was thus able to tolerate a wider range of point mutations than Gly432, including proline substitutions, suggesting a minimum of structural constraints in its local environment. Gly436 mutations either failed to alter the activation potential (G436A, G436P, G436H, and G436Y) or else shifted the activation potential toward negative voltages (G436R, G436K, G436D, and G436E), suggesting that hydrophilic residues could promote the activated state (Table 1). The negative shift in the activation potential of G406R was reported previously (29, 30). Mutants activated in response to depolarization but peaked slowly as a function of time with control {cong} G436A < G436P < G436Y < G436E {cong} G436R < G436H {cong} G436K {cong} G436D. The r800 values followed a similar pattern, with faster activating mutants showing the faster inactivating kinetics. All mutations tested at this position decreased the inactivation gating of CaV1.2 with stronger effects with charged and polar residues, a distinction that was not made clear by a previous work (30). Nonetheless, both sets of data concur that the wild-type inactivation gating uniquely requires a glycine residue at this position in the CaV1.2 channel. In addition, the attractive suggestion that the impaired gating of G436R (G406 in the human construct) results from a hyperphosphorylation of the channel protein by CaMKII (55) is not supported by our observations, since the consensus sequence for phosphorylation (RXX(S/T)) requires an arginine residue at this position.

G436R Prevents the Fast Inactivation Kinetics Caused by E462R in the I-II Linker—Voltage-dependent inactivation (VDI) gating in HVA CaV1 and CaV2 channels is currently believed to proceed through a hinged lid type mechanism (56) carried out in part by the I-II linker folding back over the intracellular mouth of the pore (34-36, 53, 57-60). In particular, it has been shown that negatively charged residues at the fifth position of the {alpha} interacting domain in the I-II linker significantly decreased the VDI kinetics and voltage dependence of CaV2.3 (35). Furthermore, the negatively charged EED cluster (Glu461, Glu462, Asp463) could account for the typically slow VDI kinetics of CaV1.2 (34). Introducing a positive residue at the fifth position in the L-type CaV1.2 with E462R accelerated significantly VDI kinetics (34, 35, 58). This model implies that flexible regions on both sides of the I-II linker act as hinges to allow the linker to fold back and bind to its receptor. Due to its biochemical properties, Gly436 could bring flexibility to this region. To evaluate the requirement for Gly436 in the molecular determinants of inactivation, the gating properties of double mutants were evaluated. Fig. 6 shows whole-cell current recordings for G436R, E462R, and G436R/E462R mutants measured in the 10 mM Ba2+ solution. G432S/E462R and G422A/E462R failed to express functional currents. As seen, the G436R/E462R mutant displayed kinetics comparable with G436R and did appreciably inactivate during the 900-ms depolarizing pulse. Inactivation kinetics were slower for G436R {approx} G436R/E462R << control channel << E462R. The introduction of the fast inactivating E462R mutation in the I-II linker into the G436R background did not cancel out the slow VDI kinetics brought by G436R, indicating that the glycine residue at position 436 plays a pivotal role in bringing the channel into the inactivated state.

G436R Significantly Decreased Calcium-dependent Inactivation (CDI)— CaV1.2 channels inactivate through the combined effect of voltage (VDI) and Ca2+ (CDI) (61-64), although the latter mechanism is prevalent under physiological conditions. CDI proceeds in response to a localized elevation of intracellular Ca2+ (65) through apocalmodulin (CaM) constitutively tethered (66-69) to the C terminus of the channel in a region that comprises the consensus isoleucine-glutamine (IQ) motif (67, 70) as well as adjacent domains (71-75). Although controlled by complementary molecular determinants, VDI and CDI gating are strongly coupled (52), such that a change in the stability of the open state is predicted to affect both VDI and CDI gating (64). Molecular models have been proposed where CDI and VDI utilize the same molecular determinants for inactivation (59), with the I-II linker being the blocking particle in both situations (75). In this scheme, the Ca2+/CaM-mediated conformational change resulting from Ca2+ influx could accelerate the repositioning of the I-II linker underneath the channel mouth (75).


Figure 9
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  		FIGURE 9.
Mutations of the distal glycine residue in IS6 decreased inactivation of CaV2.3. A-E, whole-cell current traces are shown from left to right for CaV2.3 wild type, G338A, G348A, G452A, and G352R mutants in 10 mM Ba2+ in the presence of CaV{alpha}2b{delta} and CaVbeta3 subunits. F, the r300 values (the fraction of whole-cell currents remaining at the end of a 300-ms pulse) are shown ± S.E. from 0 to +30 mV. r300 values measured at +10 mV ranged from 0.07 ± 0.01 (n = 106) for the wild-type channel, to 0.10 ± 0.02 (n = 12) for G338A (p > 0.1), to 0.04 ± 0.01 (n = 10) for G348A (p > 0.1), to 0.16 ± 0.02 (n = 14) for G352A (p < 0.001), and 0.43 ± 0.01 (n = 11) for G352R (p < 10-10). r300 values for G352R and G352A were statistically different from the control channel (p < 10-3). Activation and inactivation properties are shown in Table 3.

 
When measured in the presence of Ba2+, both G432S and G436R displayed a similar decrease in inactivation kinetics, prompting the suggestion that their role could be equally important in controlling the repositioning of the I-II linker. To investigate whether the Ca2+/CaM-mediated conformational change resulting from Ca2+ influx could partially overcome these effects, CDI kinetics were evaluated for G432S and G436R (Fig. 7 and Table 2). As seen, the macroscopic kinetics measured in Ca2+ were significantly slower for G436R than for G432S, although they were both significantly faster than the kinetics measured in Ba2+. The Ca2+ current traces inactivated following a biexponential time course with the fast inactivation time constant {tau}2 dominating the inactivation process for the control channel, whereas the slow {tau}1 controls the inactivation process for both G432S and G436R. The fraction of noninactivating currents thus increased from the control channel << G432S < G436R, suggesting that the latter is a key determinant in controlling the inactivation process.


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  		TABLE 2
CDI properties of G432S and G436R mutants Shown are gating properties of G432S and G436R mutants in 10 mM Ca2+ solutions. Control and mutant channels were expressed in Xenopus oocytes in the presence of CaV{alpha}2bd and CaVbeta3 subunits. Whole cell currents were measured in the 10 mM Ca2+ solution. Current traces were recorded either in the presence of endogenous calmodulin (CaM endo), after the overexpression of wild-type human calmodulin (CaM wt), or after the overexpression of the dominant negative calmodulin mutant (CaM1,2,3,4). Activation data were estimated from the mean I-V relationships and fitted to Boltzmann Equation 1. The data are shown with the mean ± S.E., and the number of experiments appears in parentheses.

 
In all cases, co-expression with the dominant negative CaM1234 mutant, known to blunt CDI kinetics (68) by dislodging apocalmodulin binding to the CaV{alpha}1 subunit (76), reduced further CDI kinetics (Fig. 8). CaM1234 decreased the contribution from the fast {tau}2 and increased the slow {tau}1 for all channels with strongest effects on the CaV1.2 control channel. Again, the fraction of noninactivating currents increased from the control channel << G432S < G436R, confirming the crucial role of G436 in both VDI and CDI and suggesting that these two processes share Gly436 as a common molecular determinant.

The Distal Gly in IS6 Participates in the Voltage-dependent Inactivation Gating of CaV2.3—The GX9GX3G motif is conserved in all HVA CaV channels. The properties of the glycine positions in CaV2.3 were evaluated after expression of G338A, G348A, G352A, and G352R (Fig. 9). As seen, alanine mutants in the proximal GX9G motif yielded robust high voltage activated inward Ba2+ currents without significant changes in the macroscopic kinetics, reversal potentials, or voltage dependence of inactivation (Table 3). In contrast, mutations at the distal glycine Gly352 residue significantly slowed channel inactivation with G352R << G352A < G338A {approx} G348A {approx} CaV2.3 wild-type channel (from the slowest to the fastest). Furthermore, the voltage dependence of activation and inactivation were significantly different in G352R, with a -15-mV shift in the activation potential together with a +15-mV shift in the voltage dependence of inactivation, suggesting that G352R stabilized the open state of CaV2.3. Altogether, these data suggest that the proximal GX9G residues play a modest role in the gating of CaV2.3 in contrast to the situation in CaV1.2. Nonetheless, the distal glycine residue in the GX9GX3G motif confers typical inactivation kinetics to both CaV1.2 and CaV2.3, suggesting that it could play this role in all HVA CaV channels.


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  		TABLE 3
VDI properties of IS6 glycine mutants in CaV2.3 Shown are biophysical parameters of CaV2.3 wild-type and mutant channels expressed in Xenopus oocytes in the presence of CaV{alpha}2bd and CaVbeta3 subunits. Whole-cell currents were measured in 10 mM Ba2+ throughout. Activation data (E0.5 act) were estimated from the mean I-V relationships and fitted to Boltzmann Equation 1. Peak IBa was determined from the peak I-V relationships for the corresponding experiments. The voltage dependence of inactivation (E0.5 inact) was determined from the peak currents measured at 0 mV after 5-s pulses from -100 to +30 mV. Relative currents were fitted to a Boltzmann equation. The data are shown with the mean ± S.E. of the averaged experiments, and the number of experiments appears in parentheses.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the role of S6 glycine residues in the gating of high voltage-activated Ca2+ channels CaV1.2 and CaV2.3. In particular, we focused on the triglycine GX9GX3G motif that is unique to IS6 of HVA CaV channels. The first glycine residue in this motif is aligned with the mid-S6 glycine residue that is conserved in >80% of K+ channels and has been proposed to play a key role in the activation gating of some 2- and 6-TM K+ channels. Although the GX9GX3G motif is conserved in all high voltage-activated CaV channels, mutations of these residues led to significant differences in channel function between CaV1.2 and CaV2.3. At the macroscopic level, mutations of any of the glycine by the {alpha}-helicoidal-promoting alanine slowed the macroscopic kinetics of CaV1.2, but mutations of the second glycine residue (Gly432) produced the strongest changes in function and gating. In CaV2.3, the changes in channel gating were mostly observed after mutations of the distal glycine Gly352 residue.

To help identify the molecular determinants underlying gating in CaV channels, molecular models of CaV1.2 (Fig. 10, A and B) and CaV2.3 (not shown) were built based upon the crystal structures of KcsA (14) and KV1.2 (15). The S6 helices based upon the KscA channel suggest a narrower pore than the KV1.2-based IS6 helices. Within the resolution of the three-dimensional models, IS6 from CaV1.2 and CaV2.3 are predicted to exhibit the same structures. The excellent overlap between the CaV1.2 and the CaV2.3 models is not surprising, given that 15 of 17 residues are strictly conserved in the distal portion of IS6 and that the region surrounding the last two glycines is strictly conserved (10 of 10) (Fig. 1B). For both CaV1.2 and CaV2.3, the four molecular models show a 90° shift in the orientation of the first universal glycine as opposed to the distal GX3G residues. The inner facing orientation of the universal glycine is conserved in all molecular models and could not account for the differences in the function of the glycine mutants in CaV1.2 and CaV2.3. Whereas the mid-S6 glycine residue appears to be facing the pore, the distal glycine residues appear to be perfectly aligned and facing outwardly from the pore toward S5. This structural arrangement predicts that the second glycine in the GX9GX3G motif could remain inaccessible from the water-based pore in both CaV1.2 and CaV2.3 channels. SCAM studies of the HVA CaV2.1 channel support the inaccessibility of this residue from the cytoplasmic medium (77). Although the distal Gly436 and Gly352 are also predicted to lie away from the inner pore, their location at the S6/cytoplasm interface could facilitate their interactions with the cytoplasmic environment.

The Universal Glycine Residue—Alanine mutations of the conserved glycine residue yielded different results in CaV1.2 and CaV2.3 channels. In CaV1.2, G422A slowed inactivation kinetics with a small positive shift in the activation gating, whereas G338A did not significantly alter either parameter. In contrast to our results on HVA CaV channels, mutations of the universal glycine residue to alanine in voltage-gated K+ channels produced channels that were either nonfunctional (but present at the plasma membrane) (78, 79) or showed a reduced level of activity (80). Even when channels were functional, mutation of the glycine residue significantly shifted the channel activation potential toward more positive voltages, as in the case for the human calcium-activated K+ channel BK (78). The milder effects, if any, observed after mutation of the conserved glycine residue in CaV and NaV channels (81) may also result from the asymmetrical nature of the inner pore helices in NaV and CaV channels that do not bear four glycine residues on each S6.

Interestingly, the conserved glycine residue of ligand-activated 2-TM channels is probably more important in protein packing than it is in gating (10, 82). For KirBac1.1, small side-chain residues at the position of the universal glycine allow the inner helix to bend by 12° after the selectivity filter section (13). Furthermore, molecular dynamics simulations performed with the atomic coordinates of the pH-activated KcsA showed very little difference in the channel presumed activation mechanism following the in silico mutation of the conserved glycine (G99A) (83). In the molecular models of IS6 of CaV channels, the conserved mid-S6 glycine residue appears to be neighboring the selectivity filter in a relatively crowded space, suggesting that mutations of this residue could also add constraining interactions with critical residues. In CaV1.2, the glycine residue Gly422 is neighboring a phenylalanine residue (189.9 Å3) that is replaced by an isoleucine residue (166.7 Å3) in CaV2.3. One can always argue that the presence of a slightly larger residue adjacent to the mid-S6 glycine in CaV1.2 impacts more significantly on the structural packing of the S6 helix than the same mutation in CaV2.3. Unfortunately, the three-dimensional models cannot help resolve these differences, given the low identity between the structural template (KV1.2) and CaV1.2/CaV2.3 and the ensuing imprecision regarding the orientation of the side chains. It was, however, recently proposed that the minimal volume of this glycine residue is crucial to avoid constraining interactions in the Kir3.4 channel (82). It would be interesting to evaluate the effects of decreasing the volume of the residues adjacent to the universal glycine in both channels. Nonetheless, the difference in helix packing could not account for the observation that mutation of the universal glycine with a proline residue yielded nonfunctional channels in CaV1.2, whereas the same mutation (Table 3) did not alter CaV2.3 function.


Figure 10
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  		FIGURE 10.
Computer-based molecular models of IS6 in CaV1.2 and CaV2.3. A, superposition of two three-dimensional models of IS6 for CaV1.2 based upon the molecular coordinates of KV1.2 (green) or KcsA (red) with the pore positioned to the right of S6. Both molecular models show a 90° shift in the orientation of the first universal glycine as opposed to the distal GX3G residues. The inner facing orientation of the universal glycine was preserved when the three-dimensional models were built using the MthK channel as a template (results not shown) despite the fact that the MthK-based S6 helix is too short by 5 residues when built using the original Protein Data Bank file. The helices are shown in ribbons, and the three glycine residues are shown in the CPK representation with the same color code. The top of the figure is facing the extracellular medium. The pore helix and the selectivity filter at the N-terminal side of the IS6 helix are shown only for orientation. Only one subunit is shown. B, superposition of two same models of IS6 but this time looking from the back of S6 with the pore and the selectivity filter hidden from the viewer. See "Experimental Procedures" for details on modeling. The superposition of the modeled Protein Data Bank files was achieved with the Swiss-PDB viewer version 3.7 software (available on the World Wide Web at ca.expasy.org/spdbv/) using the first 5 residues of S6 with the "Improve Fit" command, and the figure was built using INSIGHT II.

 
The Distinct Role of the GX3G Motif in CaV Channel Gating— Mutations of the glycine residues within the distal GX3G motif impacted significantly the function and the gating of CaV1.2 and CaV2.3. In CaV1.2, most mutations of the second glycine residue yielded nonfunctional channels, and the alanine mutation of Gly432 produced channels with the slowest macroscopic kinetics. The observation that Gly432 was rather intolerant to most mutations suggests a role for this residue in the activation gating of CaV1.2. This conclusion was also reached from the analysis of the three-dimensional structure of the bacterial Kir-Bac1.1 channel (13), where it is actually preferred to the universal glycine. Nonetheless, similar mutations at the same positions in CaV2.3 had little impact on channel function, suggesting distinct gating models for the two classes of HVA channels.

In both HVA channels, mutations of the third glycine (Gly436 or Gly352) decreased the inactivation kinetics. The last glycine residue is likely to sit at the junction between S6 and the I-II linker. In both channels, the last glycine residue was found to tolerate a wider range of point mutations than Gly432, including proline substitutions, suggesting a minimum of structural constraints in its local environment. Inactivation gating could be controlled by the complex interaction between the flexibility of the distal GX3G residues and the intracellular domain formed by the I-II linker-CaVbeta complex. The introduction of the fast inactivating E462R mutation in the I-II linker into the G436R background did not cancel out the slow VDI kinetics brought by G436R, indicating that the glycine residue at position 436 plays a pivotal role in bringing the channel into the inactivated state. One could envision that intracellular elements impinge directly on inactivation gating in a manner partially akin to the MthK, KirBac, and GirK channels, gated by ligand binding of intracellular domains (11, 13, 80, 84). This model could also apply to the HVA CaV2.3 channel, since the distal Gly352 was also shown to be specifically critical for VDI in CaV2.3. Altogether, these results highlight the distinct role of the distal GX3G motif in HVA CaV channel gating while suggesting that the universal glycine might be more important for helix packing.


   FOOTNOTES
 
* This work was supported by Canadian Institutes of Health Research Grant MOP13390 and a grant from the Canadian Heart and Stroke Foundation (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dépt. de Physiologie, Université de Montréal, P.O. Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-6673; Fax: 514-343-7146; E-mail: lucie.parent{at}umontreal.ca.

2 The abbreviations used are: HVA, high voltage-activated; MES, 4-morpholineethanesulfonic acid; VDI, voltage-dependent inactivation; TM, transmembrane; CDI, calcium-dependent inactivation. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Ed Perez-Reyes and Toni Schneider for the CaVbeta3, CaVbeta2a, CaV1.2, and CaV2.3 clones; Gérald Bernatchez for subcloning calmodulin into the pT7TS vector and producing the CaM1,2,3,4 mutant; Julie Verner for assistance with oocyte culture; and Claude Gauthier for artwork.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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