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Originally published In Press as doi:10.1074/jbc.M703895200 on July 27, 2007

J. Biol. Chem., Vol. 282, Issue 38, 27944-27952, September 21, 2007
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The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels*Formula

Alexandra Raybaud1, Ebru-Eylem Baspinar1, François Dionne, Yolaine Dodier, Rémy Sauvé, and Lucie Parent2

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

Received for publication, May 11, 2007 , and in revised form, July 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in CaV1 and CaV2 families. Herein we show that glycine substitution of the VAVIM motif in CaV2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G {approx} V1720G), similar (V1718G), or faster (I1721G {approx} M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a {approx}+10 mV shift in its voltage dependence of activation (E0.5,act). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of {approx}-20 mV in their E0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val 349 in IS6, Ile701 in IIS6, and Leu1420 in IIIS6 at positions predicted to face Val1720 in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of CaV2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val1720 in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in CaV2.3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-dependent Ca2+ channels (VDCC)3 are membrane proteins that play a key role in promoting Ca2+ influx in response to membrane depolarization in excitable cells. VDCCs arise from the multimerization of distinct subunits: CaV{alpha}1, CaVbeta, and CaV{alpha}2{delta}, and sometimes CaV{gamma} (1). To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel CaV{alpha}1 subunits (2–8) that are classified into three main subfamilies according to their gating properties (CaV1, CaV2, and CaV3). The CaV{alpha}1 subunit is the main pore-forming subunit that carries the channel activation gating among other functions. The CaV{alpha}1 subunits of VDCCs are evolutionarily related to the {alpha} subunit of Kv channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1–S6) (9). Although the overall identity at the primary sequence level is very low between CaV and Kv channels, it goes up to 10–25% when comparing the S6 transmembrane segments. As in Kv channels, the S6 transmembrane segments of CaV{alpha}1 are believed to line the channel pore and form the channel inner vestibule. It was inferred from the three-dimensional structures of KcsA, MthK, KvAP, KirBac, and Kv1.2 channels that the M2/S6 transmembrane segments include the activation gate that controls channel opening (10–14). In the Shaker K+ channels, the residue hydrophobicity in this region could alter the channel closed-open equilibrium (15, 16).

To study the functional importance of S6 residues in the gating of CaV2.3, we searched for conserved motifs of hydrophobic residues. The VAVIM motif is conserved in the S6 transmembrane segment of domain IV (IVS6) of high voltage-activated CaV1 and CaV2 families (Fig. 1A). Numerous algorithms align the PVPVIV activation locus in Shaker Kv channels with the FVAVIM (50% identity) (Fig. 1B) suggesting that this locus could play a role in the activation gating of HVA VDCCs. Precious clues regarding the role of the VAVIM motif in channel function came from genetic diseases. Mutation of the conserved Ile to Leu in CaV2.1 was identified in patients suffering from familial hemiplegic migraine (17, 18). I1811L altered the channel gating properties by shifting the voltage dependence of activation by -5 to -12 mV, depending upon the expression system (17, 19–21). Altogether, these experiments suggested that Ile could be strictly required for normal gating of HVA VDCC. Herein we show that substitutions of the Ile1721 residue in the human CaV2.3, corresponding to Ile1811 in the human CaV2.1, by residues of different structural properties (hydrophobicity, charge, polarity, and size) yielded whole cell currents with altered gating properties. Mutations of Ile1721 (Ala, Glu, Gly, His, Leu, Pro, Thr, and Val) including the conservative I1721L mutation, shifted the voltage dependence of inactivation to more negative potentials (up to -30 mV shifts) compared with the wild-type CaV2.3 channel. Glycine mutants A1719G, V1720G, I1721G, and M1722G within the VAVIM motif significantly altered the channel whole cell kinetics as well as the voltage dependence of channel activation and inactivation. The equilibrium between the channel closed and open states was modified in favor of the closed state in I1721G and M1722G. In contrast, A1719G and V1720G shifted the open/closed equilibrium toward the channel open state. Glycine mutations introduced at positions equivalent to Val1720 in S6 of Domains I (Val349), II (Ile701), and III (Leu1420) yielded similar results prompting the suggestion that the open state was stabilized in these mutants. Altogether our findings identify S6 hydrophobic residues in Domains I to IV that play a unique role in controlling the relative stability of the open and closes states in CaV2.3.


Figure 1
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  		FIGURE 1.
A, predicted secondary structure for the CaV{alpha}1 subunit in VDCCs. Primary sequences of IVS6 segments from the CaV{alpha}1 subunit of the CaV2 channel family: CaV2.1 (GenBank X57477), CaV2.2 (GenBank D14157), and CaV2.3 (GenBank L27745). The last row sums up the residues conserved in the 3 groups. Hydrophobic residues are shown in normal typeface and polar residues are shown in bold letters. The conserved isoleucine residue identified in familial hemiplegic migraine is shown with an asterisk. B, predicted alignment of S6 segments of CaV2.3 with the S6 from Shaker/Kv1.2. The top row shows the primary sequence of the S6 segments from the PDB 2A79 file for the atomic coordinates of Kv1.2. The primary sequences of the IS6, IIS6, IIIS6, and IVS6 segments of the CaV2.3 channel were aligned with S6 of Kv1.2 using the LALIGN program (see "Experimental Procedures"). This alignment was exploited to build the three-dimensional model of the S6 segments of CaV2.3. In this alignment, the mid-glycine residues in IS6 (Gly338) and IIS6 (Gly690) are not aligned with the universal glycine of K+ channels. The alignment of IIS6 was chosen based upon its overall homology with the other S6 segments of CaV2.3 (IS6, IIIS6, and IVS6) but is not the best possible alignment of IIS6 with S6 of Kv1.2. Indeed, CLUSTALW predicts the FLAIAVD locus of IIS6 to be aligned with ALPVPVI of Kv1.2. The residues of the putative activation gate in Kv channels (PVPVIV) are underlined and shown to be aligned with FVAVIM in IVS6 of CaV2.3.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant DNA Techniques—The human CaV2.3 (GenBankTM L27745 [GenBank] ) (22), the rat CaVbeta3 (GenBank M88751 [GenBank] ) (23), and the rat brain CaV{alpha}2b{delta} (GenBank NM_000722 [GenBank] ) (24) were used. Point mutations were produced with the QuikChange XL-mutagenesis kit (Stratagene) using 39-mer primers on the full-length CaV2.3 clone as described elsewhere (25). Constructs were screened by automated double-stranded sequence analysis (BioST, Lachine, Quebec, Canada). Run-off transcripts were prepared using the T7 RNA polymerase mMessage mMachine® transcription kit (Ambion, Austin, TX) and stored at -20 °C before use.

Functional Expression of CaV2.3—Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (25–27). Oocytes were injected with 46 nl of a solution containing cRNA coding for the CaV{alpha}1, CaV{alpha}2b{delta}, and CaVbeta3 subunits in a 3:1:2 weight ratio. Co-expression with the ancillary CaVbeta3, which is predominant in brain tissues like Cav2.3 (28, 29), promotes strong functional expression (30, 31), and emphasizes closed-state inactivation (32) of CaV2.3. Functional expression of mutants was deemed significant with whole cell Ba2+ currents >300 nA.

Electrophysiological Recordings in Oocytes—Wild-type and mutant channels were screened at room temperature for macroscopic Ba2+ currents, 2–4 days after RNA injection, using a two-electrode voltage-clamp amplifier (OC-725C, Warner Instruments) as described earlier (25–27, 33). Oocytes were routinely injected with 23 nl of a 50 mM EGTA (Sigma) 0.5–2 h before the experiments.

Data Acquisition and Analysis—PClamp software 8.2 (Molecular Devices, Sunnyvale, CA) was used for on-line data acquisition and analysis. A series of 450-ms voltage pulses (5 mV steps) were applied at a frequency of 0.2 Hz from a holding potential of -120 mV to take into account the negative shift of the Ile1721 mutants in the inactivation curve. Activation parameters were estimated from the peak I-V curves obtained for each channel combination and are reported as the mean of individual measurements ± S.E. Briefly, the I-V relationships were normalized to the maximum amplitude and were fitted to a Boltzmann equation with E0.5,act being the mid-potential of activation as described elsewhere (25–27). The estimation of E0.5,act using non-stationary measurements rests upon the assumption that the transition to the open state is much faster than the transition to the inactivated state. The measure of E0.5,act yields an estimation of the free energy differences between closed (C) and open (O) states as explained previously (16). Changes in E0.5,act can thus be interpreted as a modification of the ratio between the open and closed states. Time constants of whole cell current traces were estimated with the predefined Equation 1 in Clampfit 8.2 that uses the Chebyshev routine and a 4-point smoothing filter with n = 1 for deactivation time constants and n = 3 for inactivation time constants. Under the latter, the inactivation time course requires two time constants {tau}inact,fast and {tau}inact,slow.

Formula 1(Eq. 1)
As the number of exponential functions needed to account for the inactivation process varied between mutants, inactivation kinetics were quantified using r50 values. The r50 ratio is defined as the ratio of peak whole cell currents remaining 50 ms later (I50 ms/IPeak). The voltage dependence of inactivation was obtained after a series of 5-s prepulses (h5000 for h measured at 5000 ms) that varied from -120 to +30 mV at a frequency of 0.02 Hz with E0.5,inact being the mid-potential of inactivation (25–27). Most mutations inactivated completely within the 5-s pulse with less than 2% of the peak currents remaining at the end of the inactivating pulse. However, the phrase "isochronal inactivation" is more accurate when estimating the voltage dependence of inactivation of the I701G mutant that displayed 15% residual currents. Statistical analyses were performed using the one-way analysis of variance fitting routine for two independent populations included in Origin 7.0. Data were considered statistically significant at p < 0.01.


Figure 2
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  		FIGURE 2.
Mutational analysis of the I1721 position. A, whole cell currents were recorded in 10 mM Ba2+ using 450-ms voltage pulses from -40 mV to +50 mV. Traces are shown from left to right for CaV2.3 wild-type, I1721L, I1721A, and I1721T co-expressed with CaVbeta3. Traces for I1721G are shown in Fig. 3. B, the r50 values (the fraction of peak whole cell currents remaining after a 50 ms pulse) are shown ± S.E. from 0 to +30 mV for CaV2.3 wild-type (32), I1721L (10), I1721A (9), I1721T (12), and I1721G (26) (from left to right on the bar graph). At +10 mV, the values of I1721T and I1721G were significantly different at p < 0.0001, whereas r50 of I1721L and I1721A were not significantly different (p = 0.9) as compared with the wild-type. C, the differences in the mid-points of inactivation and activation of mutants were obtained by subtracting the E0.5 values measured for the wild-type from the values of the mutants measured under the same conditions such that a {Delta} value = 0 indicates no difference from the wild-type channel. All mutants were modulated by CaVbeta3 in a typical fashion (data not shown). The dotted lines drawn at ±5 mV around 0 mV indicates a value significantly different from the wild-type channel by p < 0.01. The mid-points of inactivation E0.5,inact (striped columns) of all mutants were significantly shifted at p < 0.01 to negative potentials as compared with the wild-type channel. As compared with the wild-type channel, the mid-points of activation E0.5,act (plain gray columns) of I1721L and I1721G were significantly shifted at p < 0.001 in the negative and positive directions, respectively.

 
Homology Modeling of S6 Regions in CaV2.3—An analysis based on the SAMT02 algorithm confirmed Kv1.2 as a suitable template to model the S5 and S6 segments in each of the 4 domains of CaV2.3. The identity of Kv1.2 with CaV2.3 at the primary sequence varies from 11% for IIIS6 to up to 25% for IVS6 with values of 17% for IS6 and 22% for IIS6. The primary sequence of each of the four S6 segments of CaV2.3 was aligned with the S6 segment of Kv1.2 by using LALIGN without any gap in the structure. Introducing gaps in the alignment could in certain cases (such as in IIS6) improve local alignments while decreasing the overall homology. For each S6, the score for the alignment of the distal S6 was systematically higher than for the beginning of the S6 segment. A Kv1.2-based model of the selectivity filter and pore helix could not, however, be generated as the linkers were generally longer in CaV than in Kv1.2. Hence, the computer-based molecular model of these regions without the selectivity filter and the pore helix was achieved with Modeler 9v1 (salilab.org/modeler) using the molecular coordinates of Kv1.2 (Protein Data Bank 2A79 [PDB] ). 100 models were built and the models with the lowest objective function were further checked for internal consistency using Procheck (biotech.ebi.ac.uk/cgi-bin/sendquery). The precision of the models decreased significantly for the residues located at the C-terminal end in the absence of structural constraints. The global score of the final model was 0.10, whereas values higher than -0.50 are generally considered to be acceptable. The model was minimized with the DISCOVER module of INSIGHTII (Accelrys, San Diego, CA) with a dielectric constant of 80 to simulate the presence of solvent. The three-dimensional representations were generated with the INSIGHTII software as described elsewhere (34, 35).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Role of IVS6 in the Gating of CaV2.3—The I1811L mutation is one of the four original mutations of the CaV{alpha}1 subunit of the human CaV2.1 that is associated with familial hemiplegic migraine (17, 36). As the Ile residue is conserved in HVA VDCCs, we hypothesized that it might be important for the normal gating properties of HVA channels such as CaV2.3. Most Ile1721 mutants yielded high voltage-activated inward Ba2+ currents (Fig. 2, supplemental Fig. S1 and Table S1) with the exception of I1721D, I1721R, and I1721K that failed to express functional channels. The observation that Ile1721 tolerated a wide range of point mutations suggests a minimum of structural constraints in its local environment. Other than I1721L and I1721A, functional Ile1721 mutants produced currents with faster inactivation kinetics than the wild-type channel (supplemental Fig. S1). At +10 mV, r50 decreased from wild-type {cong}I1721L {cong} I1721A > I1721H > I1721E > I1721V {cong} I1721T {cong} I1721P > I1721G. This is the first report of mutations speeding up the inactivation kinetics of CaV2.3 because mutations in the I-II linker decreased the inactivation kinetics (33). Our results with I1721T in CaV2.3 contrast with reports that the equivalent mutation I1475T in IVS6 of the L-type CaV1.2 did not markedly alter the kinetics of whole cell current inactivation (37).

Charged residues at position 1721 produced non-functional mutants save for I1721E that showed little difference in activation and inactivation potentials despite faster inactivation kinetics. I1721T displayed faster inactivation kinetics without any significant changes in E0.5,act or E0.5,inact. Both the activation and inactivation gating of I1721L and I1721A were significantly shifted by {cong}-5 to -10 mV as compared from the wild-type channel (supplemental Table S1) but inactivated with kinetics similar to the wild-type channel. In the case of I1721V, I1721P, and I1721G, the activation potentials were significantly shifted toward positive voltages, whereas inactivation potentials were shifted in the negative direction. These observations coupled with the faster inactivation kinetics of these mutants suggest that the fraction of channels in the open state at any given voltage was decreased in I1721V, I1721P, and I1721G. This conclusion holds true even when taking into account the changes in the slope factor Z. As estimated from the Formula 1 scale calculated by the group of Stephen White (38), hydrophobicity decreases in the following order: Ile {cong} Leu > Val > Ala > Thr > Gly > His > Pro > Glu, whereas volume decreases from Ile {cong} Leu > His > Val {cong} Glu > Thr > Pro > Ala > Gly. As a result, no single parameter such as hydrophobicity, charge, or volume can account for the altered channel gating of Ile1721 mutants.


Figure 3
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  		FIGURE 3.
Glycine scan of the VAVIM motif in IVS6 of CaV2.3. A, whole cell current traces are shown from left to right for A1719G, V1720G, I1721G, and M1722G co-expressed with CaVbeta3 and recorded in the presence of 10 mM Ba2+. Whole cell I/V curves of these mutants reversed between +45 and +50 mV indicating that the mutations did not affect ion selectivity. Activation and inactivation properties are detailed in supplemental Table S1. All mutants were modulated by CaVbeta3 in a typical fashion (supplemental Fig. S2B). B, the r50 values in the presence of CaVbeta3 are shown ± S.E. from 0 to +30 mV for CaV2.3 wild-type (32), V1718G (18), A1719G (20), V1720G (25), I1721G (26), and M1722G (17) (from left to right on the bar graph). C, the differences in the mid-points of inactivation and activation of mutants were obtained by subtracting the E0.5 values measured for the wild-type from the values of the mutants such that a {Delta} value = 0 indicates no difference from the wild-type channel (see supplemental Table S1 for actual values). The dotted lines drawn at ±5 mV around 0 mV indicates a value significantly different form the wild-type channel by p < 0.01. The mid-points of inactivation E0.5,inact (striped columns) of most mutants but A1719G (p < 0.01) were significantly shifted at p < 0.001 to negative potentials as compared with the wild-type channel. As compared with the wild-type channel, the mid-point of activation E0.5,act (gray columns) of I1721G was significantly shifted at p < 0.001 toward positive potentials by +10 mV, whereas E0.5,act of A1719G and V1720G was significantly shifted toward negative voltages by {cong}-17 mV (p < 0.001). In contrast, E0.5,act of V1718G was only slightly shifted at p < 0.1 by -3 mV.

 
V1720G and I1721G Yield Clues Regarding the Position of the Channel Activation Gate in IVS6—With I1721V and I1721P, I1721G produced the strongest changes in channel gating. To minimize steric and structural constraints, we chose to identify residues that are critical for channel activation gating by performing a glycine scan of the conserved VAVIM locus in the distal portion of IVS6 (Fig. 3A). Glycine mutants decreased the inactivation kinetics of neighboring A1719G and V1720G residues but sped up the kinetics of I1721G and M1722G as compared with the wild-type and V1718G channels. The r50 values thus decreased from A1719G {cong} V1720G > wild-type {cong} V1718G > I1721G {cong} M1722G (Fig. 3B). The fast inactivation kinetics of the latter were accompanied by a faster recovery from inactivation, suggesting that the inactivated state was not more stable in I1721G and M1722G (supplemental Fig. S2A). The activation potentials of the latter were either not significantly different (M1722G) or more positive (I1721G, p < 0.001) than the wild-type channel. Coupled with the fact that these 2 mutants inactivated at potentials {approx}-30 mV more negative than the wild-type channel (Fig. 3C), these observations altogether indicate that glycine mutations introduced at positions 1721 and 1722 result in a decreased channel open state. In all these mutants, activation and inactivation potentials were typically shifted toward negative voltages by CaVbeta3 (supplemental Fig. S2B). For I1721G, this means that E0.5,act = 6.9 ± 0.4 mV (n = 6) and E0.5,inact = -64 ± 3 mV (n = 3) in the absence of CaVbeta3, values that remain significantly different from E0.5,act = -1.4 ± 0.4 mV (n = 29) and E0.5,inact = -33.7 ± 0.8 mV (n = 36) for the wild-type channel under the same conditions.

The faster kinetics of I1721G and M1722G contrast with the slower kinetics produced by G352A in IS6 and R378E mutations in the I-II linker (33, 39). It was then proposed that the slow kinetics of these mutants resulted from either a disruption of the fast inactivated state or from a stabilization of the channel open state. To investigate whether the increased inactivation gating of I1721G and M1722G could antagonize the effects of the slow mutants, the double mutants G352A/M1722G and R378E/I1721G were produced. Unfortunately, neither double mutant expressed functional whole cell currents, leaving this question unanswered.

In contrast to I1721G and M1722G, A1719G and V1720G displayed slow inactivating kinetics accompanied with negative shifts of {approx}-20 mV in their activation potentials. This change in gating departs from our observations with G352A in IS6 (25) and the charged mutations of Arg378 in the I-II linker (33, 39) where the decreased inactivation kinetics occurred without any change in the channel activation potential of CaV2.3. The significant shifts in the E0.5,act toward negative voltages were accompanied by similar increases in the slope factor Z. Hence, introducing glycine residues at positions 1719 and 1720 appears to shift the open/closed equilibrium toward the open state. Mutations of Val1720 to Ala and Ile nearly restored the normal channel gating with smaller shifts in the mid-potentials of activation and inactivation than measured with V1720G (Fig. 4; supplemental Table S1) suggesting that hydrophobic residues at this position could shift the closed/open state equilibrium toward the channel closed state.


Figure 4
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  		FIGURE 4.
Gating properties of the Val1720 mutations in IVS6 of CaV2.3. A, whole cell current traces are shown from left to right for wild-type, V1720G, V1720A, and V1720I co-expressed with CaVbeta3 and recorded in the presence of 10 mM Ba2+. B, the r50 values are shown ± S.E. from 0 to +30 mV. At +10 mV, the r50 ranged from 0.47 ± 0.01 (32) for CaV2.3 wild-type, 0.72 ± 0.01 (25) for V1720G (p < 0.001), 0.60 ± 0.03 (8) for V1720A (p < 0.001), and 0.57 ± 0.02 (8) for V1720I (p < 0.001) (from left to right on the bar graph). C, the {Delta} of the mid-points of inactivation E0.5,inact (striped columns) and activation E0.5,act (gray columns) were calculated as described before in the presence of CaVbeta3 such that a {Delta} value = 0 indicates no difference from the wild-type channel (see supplementary Table S2 for actual values). The dotted lines drawn at ± 5 mV around 0 mV indicates a value significantly different form the wild-type channel by p < 0.01.

 
Glycine Mutations in Positions Equivalent to Val1720 Promote the Channel Open State—As the primary structure of VDCCs identifies 4 distinct S6 domains, domain-specific effects were investigated at positions equivalent to Val1720 (Fig. 5) and Ile1721 (L350 in IS6, Ala702 in IIS6, and Ile1421 in IIIS6) (supplemental Fig. S3). As seen for I1721G (IVS6), I1421G (IIIS6) displayed inactivation kinetics significantly faster than the wild-type channel at all voltages (supplemental Fig. S3). Unlike I1721G, however, the activation potential of I1421G was not significantly altered, which suggests that the mutation did not affect the relative stability of the open and closed states. L350G (IS6) behaves mostly like the wild-type channel, whereas the slower inactivation kinetics of A702G (IIS6) were observed in the absence of significant shifts in its activation and inactivation potentials (supplemental Fig. S3).

In contrast, glycine mutations at positions Val349 (IS6), Ile701 (IIS6), and Leu1420 (IIIS6) equivalent to Val1720 (IVS6) significantly altered the biophysical properties of the channel. In particular, all mutations slowed down the channel inactivation kinetics (Fig. 5B). At 0 mV, the r50 values decreased from wild-type {cong} V349G (IS6) < L1420G (IIIS6) {cong} V1720G (IVS6) < I701G (IIS6). The decrease in channel kinetics in these mutants was achieved through the progressive elimination of the fast inactivation time constant up to the point where the contribution of the fast time constant becomes negligible in I701G. The mutations also significantly hyperpolarized the activation potentials by -5 to -35 mV and the inactivation potentials by -18 to -24 mV (supplemental Tables S1 and S2 and Fig. S4). The most spectacular alterations in channel properties were, however, observed for I701G in IIS6. Indeed, the hyperpolarization of its activation curve was stronger than the negative shift in its mid-potential of inactivation (Fig. 5C). Finally, channel activation kinetics were also affected as the activation kinetics of I701G were remarkably voltage-dependent with slow {tau}act = 5.8 ± 0.4 ms (n = 9) at the peak voltage of -35 mV to reach {tau}act = 2.5 ± 0.1 ms (n = 9) at -5 mV, whereas the wild-type channel activated with reach {tau}act = 2.1 ± 0.3 ms (n = 17) at -20 < Vm < 25 mV. I701G significantly slowed deactivation kinetics (Fig. 6). Altogether, these changes in channel gating suggest that introducing a glycine residue at this position either destabilized the channel closed state or else stabilized its open state. Similar changes in channel gating were reported after substitutions of Ile781, the CaV1.2 residue corresponding to Ile701 in CaV2.3, suggesting a congruence in the gating mechanism between CaV1 and CaV2 channel types (40).

Although the altered gating was more spectacular with I701G, glycine mutations of the LAIAV motif in IIS6, save for A702G, produced slowly inactivating channels that activated at more negative potentials than the wild-type channel (supplemental Fig. S5 and Table S2). In particular, V703G imparted significant hyperpolarizing shifts in the activation and inactivation potentials of CaV2.3. These data indicate that IIS6 plays a unique role in the channel equilibrium between the closed and open state(s).

Altogether, introducing glycine mutations in the distal portion of S6 segments in CaV2.3 produced position specific alterations in activation and inactivation gating within IIIS6 and IVS6 that were absent in IS6 and IIS6. Conservative mutations of the same residues to hydrophobic amino acids nearly restored the channel gating (supplemental Table S2). These observations confirm that S6 transmembrane segments of CaV channels are not functionally equivalent, an idea that was first brought up by the mutational analysis of the selectivity filter of CaV1.2 (41, 42) and recently substantiated by the SCAM study of CaV2.1 (43, 44).


Figure 5
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  		FIGURE 5.
Gating properties of the S6 residues facing Val1720 in CaV2.3. A, whole cell current traces are shown from left to right for V349G, I701G, L1420G, and V1720G co-expressed with CaVbeta3 and recorded in the presence of 10 mM Ba2+. For I701G, the 450-ms voltage pulses were applied from -70 to +30 mV to take into account the large negative shit of its E0.5,act. At 0 mV, the activation time constants were {tau}act = 2.9 ± 0.1 ms (n = 17) for the wild-type; {tau}act = 3.0 ± 0.1 ms (n = 11) for V349G; {tau}act = 2.7 ± 0.2 ms (n = 9) for I701G; {tau}act = 2.8 ± 0.1 ms (n = 10) for L1420G; and {tau}act = 3.0 ± 0.7 ms (n = 19) for V1720G. The inactivation time constants were {tau}inact,fast = 29 ± 2 ms (Afast = 0.43) and {tau}inact,slow = 116 ± 4 ms (Aslow = 0.57) (n = 17) for the wild-type channel; {tau}inact,fast = 31 ± 3 ms (Afast = 0.44) and {tau}inact,slow = 115 ± 4 ms (Aslow = 0.56) (n = 11) for V349G; {tau}inact,fast = 53 ± 3 ms (Afast = 0.45) and {tau}inact,slow = 202 ± 16 ms (Aslow = 0.55) (n = 10) for L1420G; and {tau}inact,fast = 39 ± 7 ms (Afast = 0.37) and {tau}inact,slow = 204 ± 20 ms (Aslow = 0.63) (n = 19) for V1702G. In contrast, I701G inactivated with a single {tau}inact = 344 ± 8 ms (n = 9). B, the r50 values are shown ± S.E. from 0 to +30 mV. At +10 mV, the r50 ranged from 0.46 ± 0.01 (40) for CaV2.3 wild-type, to 0.55 ± 0.02 (23) for V349G (p < 0.001), 0.80 ± 0.05 (19) for I701G (p < 0.001), 0.59 ± 0.05 (16) for L1420G (p < 0.001), and 0.70 ± 0.01 (31) for V1720G (p < 0.001) (from left to right on the bar graph). IS6 already contains 3 endogenous glycine residues that could account for the smaller impact of the glycine mutation at this position (25). C, the differences in the mid-points of inactivation and activation of V349G (Domain I), I701G (Domain II), L1420G (Domain III), and V1720G (Domain IV) were obtained from the difference between E0.5,wt and E0.5,mutant in the presence of CaVbeta3 such that a {Delta} value = 0 indicates no difference from the wild-type channel (see supplemental Table S2 for actual values). The dotted lines drawn at ± 5 mV around 0 mV indicates a value significantly different form the wild-type channel by p < 0.01. The mid-points of activation E0.5,act (gray columns) and inactivation E0.5,inact (striped columns) of mutants were significantly shifted at p < 0.001 to more negative potentials as compared with the wild-type channel.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In Kv channels, mutations to residues less hydrophobic than wild-type residues "at the closed bundle" was shown to shift the closed-open equilibrium in favor of the open state (15, 16) prompting the suggestion that the closed conformation is stabilized by hydrophobic interactions. Thus hydrophobic interactions or "hydrophobic seals" may prevent ion flow in the closed state (45). Three hydrophobic seals have been postulated in KcsA (Thr107, Ala111, and Val115) (45), whereas only Val474 and Val478 are present in Shaker K+ channels (46). Three-dimensional structures of the KcsA and Kv1.2 channels show that the residues postulated to form the hydrophobic seal project their side chains toward the channel pore. Of these two positions, Val478 was clearly identified as a key candidate for occlusion of the pore in the closed state (15).

Three-dimensional structures of the {alpha}1 subunits of NaV and CaV channels are not known. Hence, models developed for symmetrical 6-transmembrane ion channels remain widely invoked with asymmetrical 24-transmembrane voltage-gated Na+ (47–50) and Ca2+ channels (51). To explore structural and functional similarities between the Kv and the CaV channels, we have built a three-dimensional model of the S6 segments of CaV2.3 using the atomic coordinates for Kv1.2 (Fig. 7). Structural predictions based on the threading SAMT02 algorithm suggested Kv1.2 as a suitable template to model the S5 and S6 segments in each of the 4 domains of CaV2.3 but ruled out Kv1.2 as a model for the selectivity filter, as well as for the S4–S5 and S5–S6 linkers. The absence of the selectivity filter, however, limits the precision in the spatial orientation of the four S6 transmembrane segments. Within the limits of the three-dimensional model, the sequence alignment predicts Val1720 and Ile1721 to be present in the bent section of the S6 helix. Indeed there is a >45% local identity in a 17-amino acid span in the distal region such that LALIGN, CLUSTALW, and TCOFFEE yielded the same residue alignment between the S6 of Kv1.2 and IVS6 of CaV2.3 (Fig. 1B). The three-dimensional model in Fig. 7 is supported by a SCAM study of CaV2.1 where the equivalent residues in IVS6, save for the middle Val, were accessible from the intracellular medium (43) suggesting the presence of a wide intracellular vestibule in the open state. The SCAM study of the CaV2.1 pore also showed the VAVIM locus to be modified by MTSET in a state-dependent manner (43, 44) indicating that it is located close to or at the activation gate. This contrasts with inwardly rectifying GIRK4 or Kir3.4 channels where rigidity below the transmembrane 2 helix bundle crossing appears to be a requirement for channel gating (52).

Transposing the results from mutational analyses in Shaker channels to CaV2.3 hence predicts that large hydrophobic residues at positions equivalent to Val478 should stabilize the closed conformation by strengthening the second hydrophobic seal (46). Conversely, small and/or hydrophilic residues should promote channel activation at more negative membrane potentials. Our mutational analysis of S6 segments in CaV2.3 showed that Gly substitutions at position 1721 (IVS6) but not at positions 350 (IS6), 702 (IIS6), and 1421 (IIIS6) shifted the open/closed equilibrium toward the closed state with activation potentials shifted toward positive potentials. In contrast, Gly mutations at positions 1720 (IVS6), 1420 (IIIS6), 701 (IIS6), and 349 (IS6) promoted the channel open state with channel activation significantly shifted toward more negative voltages. At positions 1720 and 1420, mutations to Ala, a residue that is more hydrophobic than Gly but less hydrophobic than the respective wild-type valine and leucine residues, tended to restore the channel gating. It is hence proposed that hydrophobic residues at positions 1720, 1420, 701, and 349 contribute to stabilize the closed state in CaV2.3. Val1720 in our three-dimensional model is aligned with Val476 (supplemental Fig. S6) and hence does not appear to project its side chain toward the pore region unlike Val478 in Kv1.2. Nonetheless, the V476A mutation in the Shaker Kv channels imparted a -20 mV shift in its E0.5,act value (15). The very good identity at the primary sequence level between the distal IVS6 of CaV2.3 and S6 of Kv1.2 supports our three-dimensional model in this region.


Figure 6
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  		FIGURE 6.
Deactivation time constants for the S6 residues facing Val1720 in CaV2.3. A, representative tail currents for wild-type, V349G, L1420G, and V1720G channels. Currents were activated during a 8-ms conditioning depolarization to 0 mV for CaV2.3 wild–type, V349G, and L1420G; and -10 mV for V1720G. Deactivation was recorded during subsequent repolarizations with 10-mV increments starting from -120 mV (test potentials). Time constants were estimated by fitting current deactivation to a monoexponential function. B, representative tail currents for I701G channels. Currents were activated during a 15-ms conditioning depolarization to -40 mV. Deactivation was recorded during subsequent repolarizations with 10-mV increments starting from -120 to -40 mV (test potentials). Time constants were estimated by fitting current deactivation to a mono- or a biexponential function. Monoexponential functions were found to fit reasonably well the tail currents of all channels and allow for a better comparison between the wild-type and other mutants. C, mean time constants of channel deactivation (monoexponential functions) for CaV2.3 wild-type, V349G, I701G, L1420G, and V1720G are plotted versus test potential. At -40 mV, the time constants {tau}deact were: 1.7 ± 0.1 ms (n = 8) for wild-type; 2.0 ± 0.2 ms (n = 7) for V349G; 3.3 ± 0.06 ms (n = 6) for L1420G; 5 ± 1 ms (n = 6) for V1720G; and 130 ± 20 ms (n = 6) for I701G such that deactivation kinetics decreased from wild-type {cong}V349G < L1420G < V1720G << I701G. The pulse protocol is shown in the inset.

 


Figure 7
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  		FIGURE 7.
Computer-based molecular model of S6 segments in CaV2.3 using the molecular coordinates of Kv1.2. Computer-based molecular model of the pore region of CaV2.3 (IS6, IIS6, IIIS6, and IVS6 transmembrane segments without the selectivity filter and the pore helix) is shown as a ribbon representation. Kv1.2 (PDB code 2A79) is a suitable template for the S5 and S6 segments but not for the selectivity filter or the cytoplasmic S4–S5 linker. Modeling was achieved with Modeler 9v1. Although the S6 remains helical at the site of the PVP motif (putative activation gate in Kv1.2), the distal residues are located in the relaxed portion of the S6 helix. Color codes indicate IS6 (red), IIS6 (violet), IIIS6 (white), and IVS6 (blue). The residues in IS6 (orange), IIS6 (pink), and IIIS6 (gray) equivalent to the VAVIM locus in IVS6 (turquoise) are shown in the same color with a different shade. The representation shows the channel pore in a transversal cut with the top of the S6 helices facing the extracellular medium. A, putative projection in a ball-and-sticks representation of the side chains at positions Leu350 (IS6), Ala702 (IIS6), Ile1421 (IIIS6), and Ile1721 (IVS6). B, putative projection in a ball-and-sticks representation of the side chains at positions Val349 (IS6), Ile701 (IIS6), Leu1420 (IIIS6), and Val1720 (IVS6). The figure was produced using INSIGHT II (Accelrys).

 
The position of key residues in IIS6 remains, however, open to discussion. Alignment predictions vary whether or not gaps are introduced in the structure and in some alignments, Val703 would be facing with Val476 in Kv1.2 and Val1720 in IVS6. To note, mutations at position Val703 were also seen to significantly promote the channel open state. The orientation of the side chains facing directly or not the channel permeation pathway may or may not influence channel activation depending upon helix packing in the region. In this regard, it was concluded from the MTSET modification of 5 consecutive positions in IIS6 of CaV2.1, that the S6 segments of CaV channels are more loosely packed than S6 of Kv channels (43).

The observation, that residues Ile701 and Val703 located within a half-helicoidal turn of each other in IIS6 affects channel activation, raises the possibility that cytoplasmic linkers might play an active role in controlling transition(s) from the closed to the open state. Indeed, the role of the S4–S5 intracellular linker in the activation of HVA CaV channels remains to be addressed. Numerous studies have already shown that mutations in the cytoplasmic S4–S5 alter the activation kinetics of Kv2.1 (53) and HERG channels (54) among others, suggesting that interactions between the S4–S5 linker and the S6 helix seem to be crucial for the coupling of the voltage sensor movements to the activation gate. The group of MacKinnon and co-workers (55) has proposed in 2005 that the S4–S5 linker functions as a lever that exerts a mechanical force on the activation gate. Inward movement of the voltage sensor with hyperpolarization is postulated to compress the S4–S5 linker against the S6 helices, resulting in the tightening of the permeation pathway (55). It is thus possible that the distal S6 mutations in CaV2.3 influence the angle between the proximal and distal S6 such that it disrupts the coupling between the voltage sensor and the activation gate. As mentioned earlier, the S4–S5 and the S5–S6 linkers were not modeled in our study due to the limited identity between Kv1.2 and CaV2.3 in these regions. It thus remains to be seen whether the molecular mechanisms underlying the transition between the closed and open state are transposable from Kv1.2 to CaV2.3. Questions regarding the role of the S4–S5 linker go beyond the scope of this current investigation and await further structural studies.


   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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. S1–S6. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: 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.

3 The abbreviation used is: VDCC, voltage-dependent Ca2+ channel. Back


   ACKNOWLEDGMENTS
 
We thank Julie Verner for oocyte culture, Michel Brunette for computer maintenance, Claude Gauthier for artwork, and Sébastien Wall-Lacelle for some data analysis.



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 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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