Localization of the Activation Gate of a Voltage-gated Ca²⁺ Channel

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K⁺ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca²⁺ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the α1 subunit of P/Q type Ca²⁺ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K⁺ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting ∼1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the α1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.