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...

Fast ('concentration jump') applications of neurotransmitters are crucial for screening studies on ligand-gated ion channels. In this paper, we describe a method for automated fast perfusion of neurotransmitters (or drugs) during two-microelectrode voltage-clamp experiments on Xenopus oocytes. The oocytes are placed in a small bath chamber that is covered by a glass plate with two channels for the microelectrodes that are surrounded by a quartz funnel serving as a reservoir for test solutions. The oocytes are perfused in a vertical direction via the two channels in the plate. Automation of compound delivery is accomplished by means of a programmable pipetting workstation. A mean rise time for 10-90% current increase through muscle-type nACh channels of 55.0±1.3 ms (30 μM acetylcholine) was estimated. Automation, fast perfusion rates, and economical use of compounds (≈100 μl/data point) make the system suitable for screening studies on ligand-and voltage-gated ion channels.

The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Tuned calcium entry through voltage-gated calcium channels is a key requirement for many cellular functions. This is ensured by channel gates which open during membrane depolarizations and seal the pore at rest. The gating process is determined by distinct sub-processes: movement of voltage-sensing domains (charged S4 segments) as well as opening and closure of S6 gates. Neutralization of S4 charges revealed that pore opening of CaV1.2 is triggered by a “gate releasing” movement of all four S4 segments with activation of IS4 (and IIIS4) being a rate-limiting stage. Segment IS4 additionally plays a crucial role in channel inactivation. Remarkably, S4 segments carrying only a single charged residue efficiently participate in gating. However, the complete set of S4 charges is required for stabilization of the open state. Voltage clamp fluorometry, the cryo-EM structure of a mammalian calcium channel, biophysical and pharmacological studies, and mathematical simulations have all contributed to a novel interpretation of the role of voltage sensors in channel opening, closure, and inactivation. We illustrate the role of the different methodologies in gating studies and discuss the key molecular events leading CaV channels to open and to close.Electronic supplementary materialThe online version of this article (10.1007/s00424-018-2163-7) contains supplementary material, which is available to authorized users.

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...