Structural Determinants of L-type Channel Activation in Segment IIS6 Revealed by a Retinal Disorder*

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.

The entry of Ca 2ϩ through voltage-gated Ca 2ϩ channels has direct effects on muscle contraction, release of hormones and neurotransmitters, hearing, vision, gene expression, and other important physiological functions (2). The pore-forming ␣ 1 -subunits of voltage-gated Ca 2ϩ channels are composed of four homologous domains formed by six transmembrane segments (S1-S6) that are linked together on a single polypeptide (3). A membrane depolarization initiates channel openings (activation) and clo-sures (inactivation). These events can be considered a multistep process consisting of a conformational change in the voltage sensor, a transmission of the signal to the pore region, the opening of the pore, and channel closure due to inactivation. The voltage-sensing machinery is formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (4). A large number of amino acids involved in Ca 2ϩ channel inactivation have been identified and several molecular mechanisms for this process have been proposed (for reviews see Refs. [5][6][7]. The molecular mechanism of the voltage-dependent pore opening of Ca 2ϩ channels, however, is less studied and largely unknown. The first attempt to localize the structural elements in Ca 2ϩ channel ␣ 1 -subunits that are involved in channel activation was made by Tanabe et al. (8) who constructed chimeric channels in which sequence stretches of a slow activating ("skeletal muscle-like") Ca V 1.1 ␣ 1 -subunit were replaced by sequences from a fast activating ("cardiac-like") Ca V 1.2 ␣ 1 -subunit. The chimeras activated slowly if repeat I of the Ca V 1.2 ␣ 1 -subunit was replaced by the Ca V 1.1 ␣ 1 -sequence. In a later study, replacement of domains I, II, and III of the low voltage and fast activating Ca V 3.1 ␣ 1 -subunit with the corresponding domains of the high voltage-activated Ca V 1.2 ␣ 1 -subunit resulted in a high voltage-activated channel (9). An important role of domains I and III but not II and IV on midpoint voltage and time constants of activation was reported by Garcia et al. (10) who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of voltage-gated Ca 2ϩ channels resulted in shortening of channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time (11).
Our present study was initiated by the recent finding that a novel retinal disorder is caused by a point mutation (I745T) in segment IIS6 of the Ca V 1.4 ␣ 1 -subunit that shifts the voltage dependence of Ca V 1.4 channel activation by approximately Ϫ30 mV (1,12). As Ca V 1.4 channels express only at low density in mammalian cell lines (13) we have decided to study the functional roles of this residue and neighboring residues in segment IIS6 by introducing and characterizing mutations in the homologous Ca V 1.2 channel. Our findings demonstrate that residue Ile-781 and three neighboring residues (Leu-779, Ala-780, and Ala-782) play a key role in gating of the Ca V 1.2 channel.

EXPERIMENTAL PROCEDURES
Mutagenesis-The Ca V 1.2 ␣ 1 -subunit coding sequence (GenBank TM X15539) in-frame 3Ј to the coding region of a modified green fluorescent protein (GFP) 4 was kindly donated by Dr. M. Grabner (14). For electrophysiological studies we used the plasmid lacking the GFP tag. Substitutions in segment IIS6 of the Ca V 1.2 ␣ 1 -subunit were introduced by the "gene SOEing" technique (15). In particular, an isoleucine to threonine mutation was introduced in position 781, and further substitutions were made at this position to proline, leucine, alanine, asparagine, glutamine, and arginine. The mutated fragments were cloned into a BamHI-AflII-cassette (nucleotides 1265 and 2689, numbering according to the Ca V 1.2 ␣ 1 -subunit coding sequence). This cassette was also used in creation of C769P, G770P, N771P, Y772P, I773P, L774P, L775P, N776P, V777P, F778P, L779P, A780P, A782P, and V783P. Mutations that did not lead to functional channels were recloned into the GFPtagged vector (14) to study membrane targeting. All constructs were checked by restriction site mapping and sequencing.
Ionic Current Recordings and Data Acquisition-Barium currents (I Ba ) through voltage-gated Ca 2ϩ channels were recorded at 22-25°C using the patch clamp technique (19) by means of an Axopatch 200A patch clamp amplifier (Axon Instruments) 36 -48 h after transfection. The extracellular bath solution contained BaCl 2 5 mM, MgCl 2 1 mM, HEPES 10 mM, and choline-Cl 140 mM, titrated to pH 7.4 with methanesulfonic acid. Patch pipettes with resistances of 1-4 megohms were made from borosilicate glass (Clark Electromedical Instruments) and filled with pipette solution containing CsCl 145 mM, MgCl 2 3 mM, HEPES 10 mM, and EGTA 10 mM, titrated to pH 7.25 with CsOH. All data were digitized using a DIGIDATA 1200 interface (Axon Instruments), smoothed by means of a four-pole Bessel filter and stored on computer hard disc. 300-and 100-ms current traces were sampled at 10 kHz and filtered at 5 kHz, for the steady state inactivation protocol, currents were sampled at 1 kHz and filtered at 0.5 kHz, and tail currents were sampled at 50 kHz and filtered at 10 kHz. Leak currents were subtracted digitally using average values of scaled leakage currents elicited by a 10 mV hyperpolarizing pulse or electronically by means of an Axopatch 200 amplifier. Series resistance and offset voltage were routinely compensated. The pClamp software package (Version 7.0 Axon Instruments, Inc.) was used for data acquisition and preliminary analysis. Microcal Origin 5.0 was employed for analysis and curve fitting.
The voltage dependence of activation was determined from currentvoltage (I-V) curves that were fitted according to the following modified Boltzmann term: where V reV is extrapolated reversal potential, V is membrane potential, I is peak current, G max is maximum membrane conductance, V 0.5,act is voltage for half-maximal activation, and k act is slope factor. The time course of current activation was fitted to a mono-exponential function: where I(t) is current at time t, A is the amplitude coefficient, is a time constant, and C is steady state current. The voltage dependence of I Ba inactivation (inactivation curve) was measured using a multistep protocol to account for run-down (see Ref. 1). The pulse sequence was applied every 40 s from a holding potential of Ϫ100 mV. Inactivation curves were drawn according to a Boltzmann equation: is membrane potential, V 0.5,inact is midpoint voltage, k is the slope factor, and I ss is the fraction of non-inactivating current. Data are given as mean Ϯ S.E. Statistical significance was assessed with the Student's unpaired t test.
Confocal Imaging-The confocal images were obtained ϳ30 h after transfection. Data illustrated are representative for 15-20 tsA-201 cells from three independent experiments. Confocal images were acquired with a Zeiss LSM-510 confocal laser scanning microscope, using a 63ϫ (1.4 NA) oil immersion objective. The plasma membrane was stained with 1 M FM4-64 (amphiphilic styryl dye, Molecular Probes). Images were acquired using an argon laser (excitation, 488 nm; emission BP505-530 nm emission filter) for the GFP-tagged Ca V 1.2 ␣ 1 -subunits and a He-Ne laser (excitation, 543 nm; emission filter, LP650 nm) for FM4-64.

Mutation I781T Shifts Ca V 1.2 Channel Activation to More Hyperpolarized Voltages-Residue
Ile-745 in segment IIS6 of the Ca V 1.4 ␣ 1 -subunit corresponds to residue Ile-781 in the Ca V 1.2 ␣ 1 -subunit (Fig. 1A). The effects of I781T substitution on Ca V 1.2 channel activation were investigated after expressing wild-type or mutant Ca V 1.2 ␣ 1 -subunits together with auxiliary ␤ 1a and ␣ 2 -␦ 1 -subunits in tsA-201 cells. Families of inward Ba 2ϩ currents (Fig. 1B) and the corresponding current-voltage (I-V) curves (Fig. 1C) and voltage dependences of channel activation ( Fig. 2A) and inactivation (Fig. 2B) are shown. For both wild-type and I781T channels, the current reversed at ϳ50 mV indicating that the I781T mutation does not affect ion selectivity (Fig. 1C). The midpoint voltage of activation changed from Ϫ9.9 Ϯ 1.1 mV in the wild-type channel to Ϫ37.7 Ϯ 1.2 mV in the I781T channel ( Fig. 2A). I781T also shifted the voltage dependence of inactivation. The midpoint voltage of the inactivation curve changed from Ϫ38.7 Ϯ 1.0 mV in wild-type channels to Ϫ57.8 Ϯ 0.7 mV in I781T channels (Fig. 2, B and C). Wild-type channels were inactivated by 65 Ϯ 4%, whereas I781T channels were The Voltage Dependence of Ca V 1.2 Gating Was Also Shifted by Other Ile-781 Substitutions-The data presented in Figs. 1 and 2 demonstrate that Ile-781 plays a pivotal role in Ca V 1.2 gating. To gain insight into the structural requirements at this position, we subsequently replaced Ile-781 by residues of different size, polarity and charge. Replacement of Ile-781 by leucine, alanine, asparagine, and proline caused a shift in channel activation ranging from approximately Ϫ10 mV for I781L to approximately Ϫ37 mV for I781P (TABLE ONE). The same mutations also shifted the channel inactivation curves by values ranging from approximately Ϫ6 mV (I781L) to approximately Ϫ30 mV (I781P) (Fig.  2, B and C). Interestingly, the changes in the voltage dependences of channel activation and inactivation occurred in parallel. This is evident from Fig. 2C where the midpoint voltages of the activation curves are plotted versus the midpoint voltages of the inactivation curves. Fitting the data with a regression function revealed a correlation coefficient of 0.97.

Ile-781 Substitutions Showed Slow Activation Gating at Hyperpolarized Voltages and Slow Deactivation-
The voltage dependences of the activation time constants for the Ile-781 substitution mutants were also determined ( Fig. 3). Between Ϫ30 mV and ϩ30 mV, activation of the wild-type and mutant channels occurred with a similar time course (wild-type, m ranged between 3.2 ms (Ϫ30 mV) and 2.5 ms (30 mV); I781P, m ranged between 3.9 ms (Ϫ30 mV) and 2.1 ms (10 mV) (Fig.  3B)). At voltages negative to the threshold potential of wild-type Ca V 1.2 activation (Ϫ40 mV, Fig. 2A), mutant channels displayed decelerated activation kinetics (Fig. 3). At Ϫ50 mV, no channel activation was observed in wild-type and I781L (Fig. 3A), whereas the four remaining mutant channels activated at comparatively slow rates (see Fig. 3B for time constants). Moreover, activation of I781P at Ϫ60 mV was even slower ( m ϳ25 ms). In other words, the more the voltage dependence of channel activation was shifted (TABLE ONE), the more slowly the channels activated at hyperpolarized voltages (Fig. 3B).
To gain insight into the stability of the open channel conformation, we also analyzed the voltage dependence of deactivation for I781P and I781T, the mutants that induced the strongest effects on channel activation. The two mutant channels deactivated more slowly than the wild-type channel at all potentials with deactivation of I781P being slower than that of I781T (Fig. 4).
Proline Substitution of Gly-770-A similar kinetic phenotype to that observed for the I781T substitution (slow deactivation, slow activation at hyperpolarized voltages, and decelerated inactivation) was previously  reported for a bacterial sodium channel (NaChBac) when a highly conserved glycine (Gly-219) in the central third of S6 was substituted by proline (20). To evaluate an analogous role of Gly-770 in the corresponding position of segment IIS6 of Ca V 1.2 (Fig. 1A), we mutated this amino acid to proline. G770P had, however, neither significant effects on the current kinetics (Fig. 5A, second current family from top; Fig. 5B) nor on the voltage dependence of channel activation and inactivation ( Fig. 6). N771P adjacent to G770P was found to shift the voltage dependence of channel activation and inactivation (Fig. 6B,  Fig. 5), and channel deactivation was not decelerated (Fig. 4).
Positional Specificity of I781P-Proline substitution of Ile-781 caused the most dramatic changes in channel activation (Figs. 2-4). To examine the positional specificity of I781P on Ca V 1.2 channel gating we substituted all amino acids between Cys-769 and Val-783 by prolines (Figs. 5 and 6, TABLE ONE). Substitution of the two alanine residues flanking Ile-781 (A780P and A782P) and of L779P substantially shifted the voltage dependence of activation (Fig. 6, TABLE ONE). A782P and L779P also showed decelerated channel activation at hyperpolarized voltages (Fig. 5B). Additionally channel inactivation was decelerated for A782P and not detectable for A780P and L779P during a 300-ms pulse (Fig. 5A, TABLE ONE). Two peculiarities were observed for A780P. This mutation i) shifted the voltage dependence of channel activation but not inactivation (Fig. 6B), and ii) this mutation slowed the kinetics of channel activation at all voltages (Fig. 5B, inset). Proline substitutions C769P, I773P, and F778P caused smaller or non-significant shifts in the voltage dependence of activation (Fig. 6A, TABLE ONE).
Evidence for Membrane Targeting of Non-functional Mutants-Six constructs (Y772P, L774P, L775P, N776, V777P, and V783P) did not conduct Ba 2ϩ currents. To investigate whether the lack of current observed for these mutants could be because of an impairment of plasma membrane targeting, we examined the subcellular distribution of GFP-tagged mutants (14) by confocal microscopy. Wild-type and mutant GFP-tagged Ca V 1.2 ␣ 1 -subunits were co-transfected with ␤ 1a and ␣ 2 -␦ 1 -subunits in tsA-201 cells, and the plasma membrane was visualized by staining with FM4-64 (Fig. 7). Consistent with previous reports (21)(22)(23), the wild-type Ca V 1.2 ␣ 1 -subunit localized predominantly at the plasma membrane with some intracellular labeling also evident. Similarly, images taken from cells expressing the non-functional channels (shown are two representative mutants) demonstrated that all mutant subunits were targeted to the plasma membrane, with some intracellular staining also detected. These findings demonstrate that the lack of current observed for these mutants cannot be attributed to a failure of the mutant GFP-tagged Ca V 1.2 ␣ 1 -subunits to reach the plasma membrane.
Bay K8644 and ␤-Subunit Modulation of Ile-781 Mutants-A shift of voltage dependence of activation toward more negative voltages is a hallmark of Bay K8644 action. It was therefore interesting to analyze if the shift in activation gating caused by the naturally occurring mutation I781T would affect BAY K8644 action. At potentials corresponding to the maximum of the current voltage relationships (0 mV for WT and Ϫ30 mV for I781T, Fig. 1C) 100 nM of BAY K8644 induced similar stimulation (2.3 Ϯ 0.3-fold, n ϭ 3 in WT and 2.6 Ϯ 0.3, n ϭ 3 in the I781T mutant) (Fig. 8, A and B). The voltage dependences of channel activation were shifted to comparable extents, Ϫ5.9 Ϯ 1.3 mV (n ϭ 4) in I781T and Ϫ6.7 Ϯ 1.4 mV in WT (n ϭ 3) (Fig. 8C).
It is well established that ␤-subunits modulate the gating of high voltage-activated Ca 2ϩ channels (for reviews see Refs. 24 -26). To elucidate whether the observed changes in the voltage dependence of channel activation interfere with ␤-subunit modulation, we co-expressed wild-type, I781T, and I781P Ca V 1.2 ␣ 1 -subunits with ␤ 1a -or ␤ 2a -subunits as well as the ␣ 2 -␦ 1 -subunit and analyzed the resulting activation curves. For each of these channels, co-expression of the ␤ 2a -subunit gave a small hyperpolarizing shift in the voltage dependence of channel activation and slower channel inactivation when compared with coexpression with the ␤ 1a -subunit (Fig. 9, TABLE ONE). These findings

A,
representative tail currents for wild-type, I781T, I781P, and N771P channels. Currents were activated during a 20-ms conditioning depolarization to 0 mV for wild-type, Ϫ40 mV for I781P, Ϫ30 mV for I781T, and Ϫ20 mV for N771P. Deactivation was recorded during subsequent repolarizations with 10-mV increments starting from Ϫ100 mV (inset). B, mean time constants of channel deactivation for wild-type, I781P, I781T, and N771P mutant Ca V 1.2 channels are plotted versus test potential. Time constants were estimated by fitting current deactivation to a mono-exponential function (see "Experimental Procedures").
suggest that these Ile-781 mutants do not substantially affect ␤-subunit modulation.

DISCUSSION
In this study we demonstrate a crucial role of a cluster of amino acids (Leu-779, Ala-780, Ile-781, Ala-782) in the pore-forming segment IIS6 in activation and inactivation of the Ca V 1.2 channel. This finding is related to a recently described channelopathy, where a mutation of the corresponding isoleucine to threonine in Ca V 1.4 (I745T) causes a severe X-linked retinal disorder (12). As previously described for the I745T Ca V 1.4 mutant (1), I781T resulted in an almost identical shift in the voltage dependence of activation (Fig. 1C) and slowed inactivation of Ca V 1.2 (TABLE ONE) suggesting a common mechanism of gating disturbances in both channel types. Additional analyses revealed that the I781T mutation also caused a negative shift in the voltage dependence of inactivation (Fig. 2B), slowed activation at hyperpolarized voltages (Fig. 3), and slowed deactivation at all potentials (Fig. 4). These changes in gating of Ca V 1. Ile-781 Substitutions May Destabilize the Closed State-The five Ile-781 substitutions that produced functional channels caused similar alterations in channel gating (Fig. 2). It seems more feasible to explain FIGURE 5. Positional specificity of the effects of proline substitution on the activation kinetics of Ca V 1.2. A, left panel shows ␣-helical representation of the amino acid sequence of segment IIS6 of the ␣ 1 -1.2 subunit. Functional proline mutants are shown in gray, non-functional mutants are shown in black. Right panel shows representative families of I Ba through wild-type and mutant channels. Barium currents were evoked during depolarizing test pulses from a holding potential of Ϫ100 mV (increments, 10-mV). Currents at threshold potentials are indicated. B, voltage dependence of channel activation time constants ( m ). Data were obtained by fitting the activation phase of currents to a mono-exponential function. The dashed line represents the voltage dependence of I781P activation from Fig. 3. The inset illustrates the voltage dependence of channel activation time constants of the slowly activating A780P mutant.  Transiently transfected tsA-201 cells expressing wild-type and two representative non-functional Ca V 1.2 channels, L755P and Y772P, are shown. The wild-type and mutant GFP-tagged (green) Ca V 1.2 ␣ 1 -subunits were co-expressed with ␤ 1a and ␣ 2 -␦ 1subunits, and the cells were stained with FM4-64 (red), a plasma membrane marker. In these merged images, GFP-tagged ␣ 1 -subunits located in the plasma membrane are seen as punctate yellow clusters. this finding by a destabilization, rather than by a stabilization, of channel conformations. A possible mechanism for destabilization of the closed state could be a reduction in hydrophobic interactions in position 781 with neighboring residues in the S6-bundle crossing region. This explanation is in line with the lower hydrophobicity and larger shifts in the activation curve of threonine when compared with alanine and leucine ( Fig. 2A). Thus, hydrophobic interactions of Ile-781 with neighboring residues in the bundle crossing region might contribute to stabilization of the closed conformation in wild-type channels. Changes in hydrophobicity alone can, however, not explain the full picture. Asparagine is less hydrophobic than threonine (27), although it causes a similar shift (Fig. 2C). As leucine differs from isoleucine by the position of only one methyl group and showed the smallest shift, we speculated that Ile-781 forms part of a tightly fitted region in the closed channel state. Proline caused the largest effect on channel activation. Its hydrophobicity is, however, closer to the wild-type isoleucine than to threonine and asparagine. Proline might disturb the interaction with neighboring amino acids in the closed state by favoring bending of the ␣-helix (28) and may thereby favor an open pore conformation.
Evidence for Stabilization of the Open State-The deceleration of deactivation observed for I781P and I781T (Fig. 4)  We speculate that closure of voltage-gated Ca 2ϩ channels requires the return of all four pore-forming elements into the resting state. If one element of the gate structure does not precisely fit the resting (closed) state, then the conducting pore may remain open for a longer period.
Positional Specificity of Ile-781-To examine the positional specificity of I781P we substituted the residues between Cys-769 and Val-783 by prolines (see Fig. 5A). Amino acids causing prominent shifts in the voltage dependence of channel activation and inactivation were localized in a cluster near the intracellular channel mouth (I781P Ͼ L779P, A782P, A780P). The more the voltage dependence of channel activation was shifted, the slower these channels activated at hyperpolarized potentials (Figs. 5 and 6A). A782P showed moderately decreased inactivation, whereas inactivation was severely reduced for L779P and A780P suggesting that all three residues contribute to both activation and inactivation gating to different extents.
In the upper part of IIS6, N771P caused a shift (Ϫ17 mV) of the activation curve and moderately slowed inactivation, but did not shift the inactivation curve markedly and did not affect the rate of channel activation at hyperpolarized voltages, (Fig. 5B, Fig. 6B, TABLE ONE). Moreover, unlike I781P and I781T, the voltage dependence of channel deactivation was unaltered in this mutant (Fig. 4B). Together these findings suggest that mutation N771P does not stabilize the open state but is more likely to destabilize the closed state.
The correlation between shifts in the activation and inactivation curves observed for all mutants with the exception of A780P represents an interesting finding per se Figs. 2B and 6B. A model describing channel activation in terms of a voltage-sensing machinery, a discrete voltageindependent mechanism of the pore opening and inactivation events might reproduce this correlation.
Although correctly targeted to the plasma membrane, some proline mutants did not conduct barium currents (Fig. 7). These data suggest that  proline substitutions in Ca 2ϩ channel S6 segments may disrupt the functionality of the conducting pore. Similar observations were recently made for proline substitutions in the central part of NaChBac segment S6 (29). We cannot exclude, however, that other substitutions in these positions would produce functional Ca 2ϩ channels with altered gating properties.
Bay K8644 and ␤-Subunit Modulation-Slowing of deactivation and a shift of steady state activation as observed for the naturally occurring mutation I781T and other effective mutations is reminiscent of BAY K8644 action. Interestingly, mutation I781T neither prevented a further shift of channel activation nor affected the stimulation of the current (Fig. 8). These data suggest that Bay K8644 and mutation I781T affect the channel via independent mechanisms. Functional and structural studies suggest that the modulation of channel activation and inactivation by ␤-subunits occurs by a direct modulation of the movement of the pore-forming segment IS6 (26,30). The I781P and I781T mutants showed the same shift in the voltage dependence of channel activation and slowed inactivation that occurs when wild-type ␣1 subunits are coexpressed with ␤ 2a rather than ␤ 1a (Fig. 9, TABLE ONE). These findings suggest that ␤-subunit modulation of segment IS6 and the effects of residue 781 on pore stability are determined by independent mechanisms.
Significance of Ile-781 and Adjacent Residues for Activation Gating of Ca 2ϩ Channels-The crystal structure of the 2-TM helix calcium-gated potassium channel MthK revealed that a conserved glycine at position 83 is responsible for bending the pore-lining M2 helix leading to channel opening (31). Previous studies have shown that the corresponding glycine (Gly-219) in the bacterial voltage-gated sodium channel NaCh-Bac represents a "gating hinge" (20). This conclusion is based on two principle findings, i) G219P shifts the activation curve to hyperpolarized voltages, and ii) this shift is accompanied by a deceleration of deactivation kinetics. Mutating the corresponding Gly-770 in Ca V 1.2 to proline did not cause significant effects on channel gating (Fig. 6, TABLE ONE). Localization of a gating hinge at residue Gly-770 is therefore not likely. The kinetic phenotype of Ile-781 and adjacent residues (Leu-779, Ala-780, and Ala-782) observed in the present study is, however, similar to the one described for NaChBac construct G219P (20). Our data therefore suggest that helix bending is more likely to occur close to the inner channel mouth in Ca V 1.2. Residues 779 -782 are conserved in high voltage-activated Ca 2ϩ channels, and it is tempting to speculate that this sequence (LAIA) might participate in a common mechanism of gating.