Gating modification by ProTxII in voltage-gated Sodium+ and T-type Calcium2+ channels

ProtoxII (ProTxII) is peptide toxin that modifies gating in voltage dependent Na+ and T-type Ca2+ channels. The toxin inhibits activation gating in both types of channels in grossly similar ways, inducing a decrease in macroscopic conductance and a slowing of activation timecourse. 1. ProTxII has multiple effects on activation gating in the voltage-dependent Na+ channel isoform NaV1.5. Toxin induces a positive shift in the voltage range of activation, decreases maximum macroscopic conductance, slows the timecourse of activation, and speeds the timecourse of deactivation. These effects are separable suggesting multiple toxin-channel interaction sites. An electrostatic, surface charge-like effect accounts for some, but not all of the toxin's ability to shift the voltage range of activation. 2. ProTxII also inhibits activation gating in NaV1.4. In this case the toxin induced a lengthening of the Cole-Moore shift and slowed gating current kinetics, though total gating charge was unchanged. Site-directed fluorescence measurements showed perturbation of voltage sensor movements in domains II, III, and IV. The shape of the fluorescence-voltage relationship in domain II in the presence of ProTxII was dramatically affected, particularly at very positive potentials, pointing to direct toxin-channel interactions in this domain. 3. The effects of ProTxII on the T-type Ca2+ channel isoform CaV3.1 are similar to its effects on NaV1.5. The decrease in macroscopic conductance was not due to a change in single channel current amplitude or total gating charge. The toxin slowed the timecourse of current activation, lengthening both the time to peak current and the Cole-Moore shift. Ionic current simulation based on a simplified gating model for this channel suggested that inhibition of multiple points along the activation pathway could produce effects similar to that seen in the presence of ProTxII. 4. The remaining two T-type Ca2+ channel isoforms show different sensitivities to ProTxII with CaV3.2 displaying no toxin sensitivity at all. To investigate potential locations for electrostatic toxin-channel interactions between ProTxII and CaV3.1, CaV3.1/Ca V3.2 reciprocal mutations were made at three sites within the extracellular linkers at which the primary sequence of the two channels differ in their charge characteristics. Two sites in domain II were found to be important in determining the effect of ProTxII on macroscopic conductance (near the p-loop) and on the kinetics of activation (near the voltage sensor). The third site, near the voltage sensor in domain III, disrupted all of the effects of ProTxII on CaV3.1. Taken together, the results of these studies suggest a common mechanism of action of ProTxII to inhibit activation in both voltage-gated Na+ and T-type Ca2+ channels at the level of the voltage sensors. Thus, ProTxII represents a promising tool for exploring gating mechanisms these two types of channels may have in common.