Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states

摘要

class="enumerated" style="list-style-type:decimal">Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K⁺ channels induced by the removal of extracellular potassium, Ko+.In the absence of Ko+, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of Ko+ had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour.To characterize the nature of the interaction between Kv1.3 and Ko+ concentration ([K⁺]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of Ko+, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mm Ko+, possibly by making the channel unstable in the absence of Ko+. Mutation at a neighbouring position (400) had a similar effect.The effect of removing Ko+ on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K⁺]o dependence as the wild-type Kv1.3 channel.CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of Ko+ unless the channels were inactivated through depolarizing pulses.We conclude that removal of Ko+ induces the Kv1.3 channel to transition to a non-conducting ‘closed’ state which can switch into a non-conducting ‘inactivated’ state upon depolarization.