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 closures (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. 5-7).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 neigh...

Ca2ϩ current through Ca V 1.2 channels initiates muscle contraction, release of hormones and neurotransmitters, and affects physiological processes such as vision, hearing, and gene expression (1). Their pore-forming ␣ 1 -subunit is composed of four homologous domains formed by six transmembrane segments (S1-S6) (2). The signal of the voltage-sensing machinery, consisting of multiple charged amino acids (located in segments S4 and adjacent structures of each domain), is transmitted to the pore region (3). Conformational changes in pore lining S6 and adjacent segments finally lead to pore openings (activation) and closures (inactivation).Our understanding of how Ca V 1.2 channels open and close is largely based on extrapolations of structural information from potassium channels. The crystal structures of the closed conformation of two bacterial potassium channels (KcsA and MlotiK) (4, 5) show a gate located at the intracellular channel mouth formed by tightly packed S6 helices. The crystal structure of the open conformation of Kv1.2 (6, 7) revealed a bent S6 with the highly conserved PXP motif apparently acting as a hinge (see 8). The activation mechanism proposed for MthK channels involves helix bending at a highly conserved glycine at position 83 (see Ref. 9, "glycine gating hinge" hypothesis).Compared with potassium channels, the pore of Ca V is asymmetric, and none of the four S6 segments has a putative helixbending PXP motif. Furthermore, the conserved glycine (corresponding to position 83 in MthK, see Ref. 10) is only present in segments IS6 and IIS6 (for review see Ref. 11). We have shown that substituting proline for this glycine in IIS6 of Ca V 1.2 does not significantly affect gating (12).Zhen et al. (13) investigated the pore lining S6 segments of Ca V 2.1 using the substituted cysteine accessibility method. The accessibility of cysteines was changed by opening and closing the channel, consistent with the gate being on the intracellular side. The general picture of a channel gate close to the inner channel mouth of Ca V 1.2 was recently supported by pharmacological studies (14).Substitution of hydrophilic residues in the lower third of segment IIS6 of Ca V 1.2 (LAIA motif,(779)(780)(781)(782)(783)(784) see Ref. 12) induces pronounced changes in channel gating as follows: a shift in the voltage dependence of activation accompanied by a slowing of the activation kinetics near the footstep of the m ∞ (V) curve and a slowing of deactivation at all potentials. Interestingly, these changes in channel gating resemble the effects of proline substitution of Gly-219 in the bacterial sodium channel from Bacillus halodurans ("Gly-219 gating hinge," see Ref. 15).The strongest shifts of the activation curve reported so far were observed for proline substitutions (12). As prolines in an ␣-helix cause a rigid kink with an angle of about 26°(16), we hypothesized that these mutants were causing a kink in helix IIS6 similar to a bend that would normally occur flexibly during the activation process (12).Here we extend ou...

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca v 1.2, we probed the closed and open conformations with the charged phenylalkylamine (؊)devapamil ((؊)qD888). To elucidate the access pathway of (؊)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca v 1.2 (1). The shifted channel gating of these mutants (by 30 -40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (؊)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca v 1.2 must be opening widely to enable free access of the charged (؊)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (؊)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca v 1.2 is able to accommodate a large cation such as (؊)D888.The pore-forming ␣ 1 -subunit of voltage-gated Ca 2ϩ channels (Ca v ) 2 is composed of four homologous domains (I-IV), each of which has six transmembrane segments (S1-S6) (2). Membrane depolarization initiates conformational changes leading to channel opening (activation) and subsequent closure (inactivation). Channel activation can be considered as a multistep process in which a conformational change in the voltage sensor (formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (3)) stimulates opening of a gate (formed by the four S6 segments).Three-dimensional structures of Ca v are not known. Comparisons of the crystal structures of the closed KcsA (4) and the open conformation of K v 1.2 (5) suggest that pore-forming S6 segments undergo substantial conformational changes during channel activation. Extension of this hypothesis to Ca 2ϩ channels is supported by recent kinetic studies on mutant Ca v 1.2 that suggest bending in segment IIS6 during channel activation (1).Ca v 1.2 channels are highly sensitive to phenylalkylamines (PAA) (6). Here we investigate whether the permanently charged (quaternary) (Ϫ)devapamil ((Ϫ)qD888) can freely access its binding pocket in the open inner pore of Ca v 1.2 from the intracellular side. To analyze the effect of the membrane potential on channel block, we used mutations of the conserved Ile-781 to threonine/proline in the pore-lining segment IIS6 of Ca v 1.2 (1, 7) that have previously been shown to shift channel activation and inactivation by 30/40 mV in the hyperpolarizing direction (1). The un...

Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust "all-or-non" device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-010-0885-2) contains supplementary material, which is available to authorized users.

A channelopathy mutation in segment IIS6 of Ca V 1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca V 1.2 (midpoint of activation curve (V 0.5 ) shifted to −37.7 ± 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V 0.5 of I781T/N785A (V 0.5 = −21.5 ± 0.6 mV) was shifted back towards wild-type (V 0.5 = −9.9 ± 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/ N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca V 1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.

the sustained phase is believed to be mediated via store depletion-activated Ca 2þ entry. Using patch-clamp recording and Ca 2þ imaging, we show here that Ca V channel currents, while found in spermatogenic cells, are not detectable in epididymal sperm and are not essential for the ZP-induced [Ca 2þ ] i changes. Instead, CATSPER channels localized in the distal portion of sperm (the principal piece) are required for the ZP-induced [Ca 2þ ] i changes. Furthermore, the ZP-induced [Ca 2þ ] i increase starts from the sperm tail and propagates toward the head.