Lipid-Dependent Activation and Desensitization Mechanism of MthK

Abstract

MthK is a prokaryotic Ca2+-activated K+ channel with several high-resolution structures, and serves as a model for investigating gating mechanisms of K+ channels. Previous investigations using proteoliposome-based Tl+ flux assay indicated that MthK underwent desensitization in seconds after Ca2+ activation. In contrast, MthK studied with electrophysiology in a planar lipid bilayer system show no evidence of desensitization. We hypothesized that this is caused by the different bilayer properties between the two preparations. The lipid composition of the bilayers in both preparations is the same (DOPC:POPG, 3:1). However, the differences in curvatures (liposomes vs. planar bilayers) and thicknesses (decane, used for planar bilayer formation, leads to increased bilayer thickness) may affect channel gating. Since MthK does not appear to desensitize in the thicker, decane-containing planar bilayers, we expect that increasing the bilayer thickness in liposomes will cause slower or no desensitization in the flux assays. Conversely, decreasing the bilayer thickness in the planar bilayers will lead to desensitization in the electrophysiology assays. We performed planar bilayer and Tl+ flux experiments using MthK reconstituted in liposomes made with lipids with different lipid acyl chain lengths (PC, from C16:1 to C22:1) while maintaining the POPG mole fraction. Changing the acyl chain length did not affect the voltage-dependent gating behavior of MthK when tested in planar bilayers. In addition, MthK did not undergo desensitization in any lipid compositions tested. On the contrary, varying the acyl chain length affected both MthK activation and inactivation in flux assays. We found that MthK inactivates more slowly in liposomes with longer acyl chain lengths (C20:1 and C22:1), and faster in liposomes with shorter acyl chain lengths (C16:1). Additionally, MthK activation was markedly slower in liposomes with longer acyl-chain lipids. The mechanism for lipid-dependent activation and desensitization of MthK is under investigation.