Calcium channel gating.

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.

Correlation between human ether-a-go-go-related gene channel inhibition and action potential prolongation.

BACKGROUND AND PURPOSE: Human ether-a-go-go-related gene (hERG; Kv 11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The interference from accompanying block of Cav 1.2 and Nav 1.5 channels was investigated along with an in silico AP model. EXPERIMENTAL APPROACH: Drug-induced changes in APD were assessed in hiPSC-CMs using voltage-sensitive dyes. The IC50 values for dofetilide and 13 derivatives on hERG current were estimated in an HEK293 expression system. The relative potency of each drug on APD was estimated by calculating the dose (D150 ) required to prolong the APD at 90% (APD90 ) repolarization by 50%. KEY RESULTS: The D150 in hiPSC-CMs was linearly correlated with IC50 of hERG current. In silico simulations supported this finding. Three derivatives inhibited hERG without prolonging APD, and these compounds also inhibited Cav 1.2 and/or Nav 1.5 in a channel state-dependent manner. Adding Cav 1.2 and Nav 1.2 block to the in silico model recapitulated the direction but not the extent of the APD change. CONCLUSIONS AND IMPLICATIONS: Potency of hERG current inhibition correlates linearly with an index of APD in hiPSC-CMs. The compounds that do not correlate have additional effects including concomitant block of Cav 1.2 and/or Nav 1.5 channels. In silico simulations of hiPSC-CMs APs confirm the principle of the multiple ion channel effects.

Methods for quantification of pore-voltage sensor interaction in Ca(V)1.2.

Voltage sensors (VSs) initiate the pore opening and closure in voltage-gated ion channels. Here, we propose a technique for estimation of the equilibrium constant of the up- and downward VS movements and rate constants of pore transitions from macroscopic current kinetics. Bell-shaped voltage dependence of the activation/deactivation time constants and Bolzmann distributions of CaV1.2 activation were analyzed in terms of a circular four-state (rest, activated, open, deactivated) channel model: both dependencies uniquely constrain the model parameters. Neutralization of gating charges in IS4 or IIS4 only slightly affects the equilibrium constant of VS transition while affecting simultaneously the rate constants of pore opening and closure. The application of our technique revealed that pore mutations on IS6-IVS6 segments induce pronounced shifts of the VS equilibrium between the resting (down) and activated (up) position. Analyzing a channelopathy mutation highlighted that the leftward shift of the activation curve induced by I781T on IIS6 is only partially (35 %) caused by a destabilization of the channel pore but predominantly (65 %) by a shifted VS equilibrium towards activation. The algorithm proposed for CaV1.2 may be applicable for calculating rate constants from macroscopic current kinetics in other voltage-gated ion channels.

Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2.

BACKGROUND AND PURPOSE: Diltiazem inhibits Ca(V)1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of Ca(V)1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α1 subunits. EXPERIMENTAL APPROACH: Ca(V)1.2 composed of α1, α2-δ and ß2a subunits were expressed in tsA-201 cells and barium currents through Ca(V)1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion). KEY RESULTS: Quaternary derivative d-cis-diltiazem inhibited Ca(V)1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca(V)1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil. CONCLUSION AND IMPLICATIONS: The data show that use-dependent block of in Ca(V)1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.

Automated fast perfusion of Xenopus oocytes for drug screening.

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 muM acetylcholine) was estimated. Automation, fast perfusion rates, and economical use of compounds ( approximately 100 mul/data point) make the system suitable for screening studies on ligand- and voltage-gated ion channels.