Structural determinants of the high affinity extracellular zinc binding site on Cav3.2 T-type calcium channels

Ca(v)3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca(v)3.2 and modulation by redox agents. His(191) is found on the extracellular face of the Ca(v)3.2 channel on the IS3-S4 linker and is not conserved in other Ca(v)3 channels. Mutation of the corresponding residue in Ca(v)3.1 to histidine, Gln(172), significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca(v)3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.