Contribution of the NH2 terminus of Kv2.1 to channel activation

Opening and closing of voltage-operated channels requires theinteraction of diverse structural elements. One approach to theidentification of channel domains that participate in gating is tolocate the sites of action of modifiers. Covalent reaction of Kv2.1channels with the neutral, sulfhydryl-specificmethylmethanethiosulfonate (MMTS) caused a slowing of channel gatingwith a predominant effect on the kinetics of activation. These effectswere also obtained after intracellular, but not extracellular,application of a charged MMTS analog. Single channel analysis revealedthat MMTS acted primarily by prolonging the latency to first openingwithout substantially affecting gating transitions after the channelfirst opens and until it inactivates. To localize the channelcysteine(s) with which MMTS reacts, we generatedNH₂- and COOH-terminal deletion mutants and a construct in which all three cysteines in transmembrane regions were substituted. Only theNH₂-terminal deletion construct gave rise to currents that activated slowly and displayedMMTS-insensitive kinetics. These results show that theNH₂-terminal tail of Kv2.1 participates in transitions leading to activation through interactions involving reduced cysteine(s) that can be modulated from thecytoplasmic phase.