CONTRIBUTION OF THE NH2 TERMINUS OF KV2.1 TO CHANNEL ACTIVATION

Opening and closing of voltage-operated channels requires the interact ion of diverse structural elements. One approach to the identification of channel domains that participate in gating is to locate the sites of action of modifiers. Covalent reaction of Kv2.1 channels with the n eutral, sulfhydryl-specific methylmethanethiosulfonate (MMTS) caused a slowing of channel gating with a predominant effect on the kinetics o f activation. These effects were also obtained after intracellular, bu t not extracellular, application of a charged MMTS analog. Single chan nel analysis revealed that MMTS acted primarily by prolonging the late ncy to first opening without substantially affecting gating transition s after the channel first opens and until it inactivates. To localize the channel cysteine(s) with which MMTS reacts, we generated NH2- and COOH-tenminal deletion mutants and a construct in which all three cyst eines in transmembrane regions were substituted. Only the NH2-terminal deletion construct gave rise to currents that activated slowly and di splayed MMTS-insensitive kinetics. These results show that the NH2-ter minal tail of Kv2.1 participates in transitions leading to activation through interactions involving reduced cysteine(s) that can be modulat ed from the cytoplasmic phase.