The Outer Transmembrane Domain is Involved in a Slow Voltage-Dependent Gate in a K+ Channel

Abstract

Many voltage-dependent channels activate in a time-dependent manner. A lesson on the mechanism of this slow gating can be learned from the small viral K+ channel Kcv. This channel, with a monomer size of 94 amino acids, has the advantage of being truly minimal; it consists of an outer (TM1) and an inner (TM2) transmembrane domain and a pore loop with minimal N and C termini.Kcv reveals in Xenopus oocytes a time-dependent inward rectification. This slow activating component is absent when the channel is expressed in HEK293 cells. It can can be regained in the latter expression system when Pro13, the amino acid, which marks entry of TM1 into the membrane, is replaced by an alanine. Single channel recordings of Kcv-P13A reveal that the open probability is much higher than in the wild-type.A similar gain in function is obtained when TM1 is extended by insertion of alanine downstream of Pro13. The region in which an extension of TM1 promotes this gain of function shows high flexibility in molecular dynamics (MD) simulations of Kcv. The idea that flexibility is related to slow gating is supported by the temperature sensitivity of the kinetics. In mutants with an extended TM1 the time constant of activation is strongly temperature-dependent, decreasing at high temperature.Experimental and theoretical data supports a model in which the movement of the N-terminal part of TM1 is involved in time dependent gating. MD simulation shows transient salt bridge patterns between TM1 and TM2 controlling the entry of ions into the cavity. We speculate that formation and disruption of these salt bridges is part of the slow gating process and that an increased flexibility of TM1 modulates the frequency for salt bridge formation.