Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Histidine residues in the IS3-IS4 loop are critical for nickel-sensitive inhibition of the Cav2.3 calcium channel

We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Cav3.2 T-type Ca2+ channel. As in Cav3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Cav2.3 Ca2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Cav2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Cav2.3.     

Phosphorylation shifts the time-dependence of cardiac Ca++ channel gating currents.

A general mechanism for the physiological regulation of the activity of voltage-dependent Na+, Ca++, K+, and Cl channels by neurotransmitters in a variety of excitable cell types may involve a final common pathway of a cyclic AMP-dependent phosphorylation of the channel protein. The functional correlates of channel phosphorylation are known to involve a change in the probability of opening, and a negative or positive shift in the voltage dependence for activation of the conductance. The voltage dependence for activation appears to be governed by the properties of the charge movement of the voltage-sensing moiety of the channel. This study of the gating charge movement of cardiac Ca++ channels has revealed that isoproterenol or cAMP (via a presumed phosphorylation of the channel) speeds the kinetics of the Ca++ channel gating charge movement. These results suggest that the changes in the kinetics and voltage dependence of the cardiac calcium currents produced by beta-adrenergic stimulation are initiated, in part, by parallel changes in the gating charge movement.     

The role of the GX9GX3G motif in the gating of high voltage-activated Ca2+ channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A > G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.     

Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

The biophysical and molecular basis of TRPV1 proton gating

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.     

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1)

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.     

Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl- currents (I(ClCa)) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, I(ClCa) was evoked immediately upon membrane rupture but then exhibited marked rundown to approximately 20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, I(ClCa) was approximately 100% of initial levels. I(ClCa) recorded with AMP-PNP-containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in I(ClCa) was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater "on" rates, and voltage-dependent closing steps ("off" rates). Our model reproduced well the Ca2+ and voltage dependence of I(ClCa) as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl- channel complex influences current generation dramatically through one or more critical voltage-dependent steps.     

Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

Heme regulates allosteric activation of the Slo1 BK channel

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531-535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G-V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G-V simulated by weakening the coupling of both Ca(2+) binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca(2+)- and voltage-dependent gating as well as intrinsic stability of the open state.     

Activation of the BK (SLO1) potassium channel by mallotoxin

Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.     

Open channel block by Ca2+ underlies the voltage dependence of drosophila TRPL channel

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca(2+) blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca(2+) block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca(2+) that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca(2+) affinity are active.     

The role of distal S6 hydrophobic residues in the voltage-dependent gating of CaV2.3 channels

The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca(V)1 and Ca(V)2 families. Herein we show that glycine substitution of the VAVIM motif in Ca(V)2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G approximately V1720G), similar (V1718G), or faster (I1721G approximately M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a approximately +10 mV shift in its voltage dependence of activation (E(0.5,act)). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of approximately -20 mV in their E(0.5,act) indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val (349) in IS6, Ile(701) in IIS6, and Leu(1420) in IIIS6 at positions predicted to face Val(1720) 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 Ca(V)2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val(1720) in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in Ca(V)2.3.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.