Mutations in the Cavity Affect the Rate of Slow Inactivation in Shaker K+ Channels

Abstract

It has been reported earlier that the activation and inactivation gates of Shaker channels are coupled, and this coupling might be mediated by a rotational motion of the S6 helix. We hypothese that either the electrostatics or the volume/hydrophobicity of the side chains pointing into the cavity can affect this coupling. Accordingly, we have introduced side-chains at position 470 in a T449A Shaker-IR background with different hydrophobicities according to the Wimley-White hydrophobicity scale (Ala, Cys, or Trp) and charges (Glu, Asp, Arg, Lys) and measured the inactivation kinetics. Mutant channels were transiently expressed in tsA_201 cells. All ionic current experiments were performed with excised inside-out patches. The introduction of an ionizable cysteine and the small but neutral alanine dramatically slowed entry into the inactivated state (the inactivation time constants for Shaker mutant T449A/I470C, T449A/I470A and T449A were 1.4 s, 250 ms and 50 ms, respectively). We could not detect any current on mutant T449A/I470X (X = Asp, Glu, Trp) but the transfection with a mixture of mutant and wild-type expression plasmids expressed currents. These heterotetramers had slower inactivation kinetics with respect to the T449A channels. Almost all of the channels had a midpoint voltage for activation in the range −50 mV to −20 mV. The only exception was the I470C mutant which had a large shift in voltage dependence (midpoint ∼5 mV). Mutant channels containing Arg or Lys (I470R, I470K) did not give functional expression either as homotetramers or as heterotetramers. Our results show that not the size but the physicochemical properties of the side chains (hydrophobicity and charge) at position 470 determine the inactivation kinetics of voltage-gated K+ channels, which may reflect interaction of the side chains with permeant ions.