Domain-Specific Gating-Modifier Toxins for Voltage-Gated Calcium Channels

Abstract

Few gating-modifier toxins have been reported to specifically target T-type calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Unlike the homotetrameric Kv channels, voltage-gated calcium channels are comprised of four different domains, presenting the possibility of multiple toxin binding sites. Using chimeric constructs, we screened existing gating-modifier toxins against the putative paddle motif from each domain of T-type calcium channel, Cav3.1. This helix-turn-helix motif is a critical structure responsible for both voltage-sensing and toxin sensitivity in Kv channels. We found that the four individual paddle motifs of Cav3.1 channels display unique toxin binding capabilities, suggesting that gating-modifier toxins can bind to T-type calcium channels in a domain-specific fashion. For two known T-type gating-modifier toxins, kurtoxin and ProTx-II, we identified key acidic and hydrophobic residues necessary for toxin binding in Cav3.1/Kv2.1 channel chimeras. Mutations of these residues in the wildtype Cav3.1 channels reduce toxin sensitivity, providing critical information on the binding sites of gating-modifier toxins in T-type calcium channels.