Abstract
T-type (Cav3) Ca(2+) channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca(2+) channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I and a cluster of extracellular cysteines in the IS1-IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species-producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His(191) in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1-IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.