Voltage-dependent inhibition of N- and P-type calcium channels by the peptide toxin ω-grammotoxin-SIA

We studied the mechanism by which the peptide omega-grammotoxin-SIA inhibits voltage-dependent calcium channels. Grammotoxin at concentrations of >50 nM completely inhibited inward current carried by 2 mM barium through P-type channels in rat cerebellar Purkinje neurons when current was elicited by depolarizations up to +40 mV. However, outward current (carried by internal cesium) elicited by depolarizations to >+100 mV was either unaffected or enhanced in the presence of toxin. Tail current activation curves showed that grammotoxin shifted the steady state voltage dependence of channel activation by approximate to+40 mV. Activation in the presence of toxin was far slower in addition to having altered voltage dependence. Grammotoxin also inhibited N-type calcium channels in rat and frog sympathetic neurons, with changes in channel voltage dependence and kinetics nearly identical to those of P-type channels. Experiments with monovalent ions as the only charge carriers showed that toxin effects on channel activation and kinetics depended on voltage, not on direction of current flow or on the current-carrying ion. Repeated trains of large depolarizations relieved toxin inhibition, as if toxin affinity for activated channels were low. The effects of grammotoxin on gating of P-type channels are very similar to those of omega-Aga-IVA, but combined application of the two toxins showed that grammotoxin binding is not prevented by saturating binding of omega-Aga-IVA. We conclude that grammotoxin potently inhibits both P-type and N-type channels by impeding channel gating and that grammotoxin binds to distinct or additional sites on P-type channels compared with omega-Aga-IVA.