Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels.

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

Identification of inactivation determinants in the domain IIS6 region of high voltage-activated calcium channels.

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (alpha(1E)) calcium channels to slowly inactivating L-type (alpha(1C)) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575-24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, alpha(1C) and alpha(1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha(1C) with the corresponding alpha(1E) residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated alpha(1C) inactivation could still be antagonized following coexpression of the rat beta(2a) subunit or by domain I-II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I-II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.

Structural determinants of fast inactivation of high voltage-activated Ca(2+) channels.

The fast inactivation of voltage-dependent Ca(2+) channels is a key mechanism that contributes to the precise control of Ca(2+) entry into excitable cells. Recent advances have revealed that multiple structural elements contribute to the intrinsic inactivation properties of the alpha(1) subunit, including its cytoplasmic and transmembrane regions. Another major determinant of Ca(2+) channel inactivation is the association with one of four types of ancillary beta subunits that differentially modulate the intrinsic inactivation properties of the alpha(1) subunit. This could occur partly via interactions with the N-terminal region of the alpha(1) subunit and through lipid modification of the beta subunit. However, the latest findings suggest a mechanism in which fast Ca(2+) channel inactivation could occur through physical occlusion of the pore of the channel in a manner reminiscent of Na(+) and K(+) channel inactivation.

Differential modulation of N-type 1B and P/Q-type 1A calcium channels by different G protein subunit isoforms.

Using transient calcium phosphate transfection into the human embryonic kidney tsa-201 cell line and subsequent whole-cell patch-clamp protocols, we examined the tonic modulation of cloned N- and P/Q-type calcium channels by five different G protein beta subunits via strong depolarizing voltage prepulses. For N- and P/Q-type channels, the magnitude of inhibition was dependent on the Gbeta subtype co-expressed. Both the absolute and relative magnitudes of Gbeta subunit-induced inhibition of P/Q-type channels differed from those observed with the N-type channel. For each calcium channel subtype, kinetics of both the prepulse-mediated recovery from inhibition and the re-inhibition following the prepulse were examined for each of the Gbeta subunits by varying either the duration between the pre- and the test pulse or the length of the prepulse. For each channel subtype, we observed a differential Gbeta subunit rank order with regard to the rates of re-inhibition and recovery from inhibition. On average, P/Q-type channels exhibited more rapid rates of recovery from inhibition than those observed with N-type channels. Different Gbeta subtypes mediated different degrees of slowing of activation kinetics. The differential modulation of P/Q- and N-type channels by various Gbeta subtypes may provide a mechanism for fine tuning the amount of calcium entering the presynaptic nerve termini.

Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.

Block of voltage-dependent calcium channel by the green mamba toxin calcicludine.

A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (alpha(1)(C)) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics, or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity with an IC(50) value of 88 nm. Three other types of high voltage activated channels tested (alpha(1)(A), alpha(1)(B), and alpha(1)(E)) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore, the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized test potentials, calcicludine actually upregulated current activity of (N-type) alpha(1)(B) channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of alpha(1)(C) and alpha(1)(E) revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall, calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.

Modulation of rod, but not cone, cGMP-gated photoreceptor channels by calcium-calmodulin.

Inside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at +/-30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was approximately 28 microM and approximately 37 microM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was approximately 2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the beta subunit of the cone channel must be different from that of the rod channel.

Block of the cGMP-gated cation channel of catfish rod and cone photoreceptors by organic cations.

Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.