Kv1.7 - Interactions with Protons and a blocking Conotoxin

Abstract

We have examined the interactions of protons and an inhibitory, poly-cationic conotoxin with the human voltage-gated potassium channel, hKv1.7. This channel differs from some members of the Kv1 sub family by having a titratable histidine residue near the N-terminal end of the putative pore-supporting P-helix, suggesting that intrinsic channel functions and pharmacology may depend on the pH of the external solution. Channels were expressed in HEK-293 cells and studied by whole-cell patch clamp. The voltage dependence of channel activation was evaluated using a tail-current protocol in which the voltage was stepped to -40 mV following a variable activating pre-pulse. Lowering of the pH of the external solution from 7.4 to 5.0 produced a positive shift in the half-activation of 36±3 mV (n=4). The lowering of pH also dramatically decreased the ability of the conotoxin to inhibit currents through the channels. Thus, following the largest depolarization, at pH5.0 very little inhibition was observed at a toxin concentration near IC50 for pH7.4. The tail current in the presence of the conotoxin was near the value seen in the absence of the toxin for external pH of 7.4. Our observations are consistent with a two-fold action of the conotoxin. First, they suggest that the positively charged toxin binds close enough to the S4 segment of the voltage sensor to inhibit activation following a depolarizing voltage step. Second, the observed decrease in maximal conductance at pH 7.4 following addition of the toxin is consistent with a block of current through the open channels. Toxin binding appears to be inhibited by protonation of a residue on the external surface of the channel, perhaps the histidine residue near the N-terminal end of the pore helix.