Regulation of Sodium Channel Function by Bilayer Elasticity: The Importance of Hydrophobic Coupling. Effects of Micelle-forming Amphiphiles and Cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid–protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel–bilayer hydrophobic interactions link a “conformational” change (the monomer ↔ dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles ( -octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less “stiff”, as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer–protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function. key words: gramicidin A • bilayer material properties • bilayer deformation energy • hydrophobic coupling • lipid–protein interactions I N T R O D U C T I O N Regulation of membrane protein function by the host bilayer lipid composition has long been enigmatic. There are numerous examples of such regulation (e.g., Bienvenüe and Marie, 1994; Dowhan, 1997; Killian, 1998; Lee, 2003), but the underlying mechanisms remain unclear, and specific lipid–protein interactions have been identified only in a few cases (e.g., Awasthi et al., 1971; Robinson, 1982; Hilgemann and Ball, 1996; Balla et al., 2000; Hilgemann et al., 2001; Hla et al., 2001). At 100 K, lipids can bind so tightly to membrane proteins that they are identified in crystal structures (Iwata et al., 1995; Luecke et al., 1999b; McAuley et al., 1999; Valiyaveetil et al., 2002). At 300 K, however, the residence time of most lipids in the annulus surrounding a protein is 10 7 to 10 6 s, and the specificity of lipid–protein interactions tends to be modest (Marsh and Horváth, 1998). Consistent with this modest specificity, accumulating evidence show that protein function may be regulated by the bilayer-collective The online version of this article contains supplemental material. Address correspondence to Jens A. Lundbæk, Institute of Biological Psychiatry, St. Hans Hospital, Boserupvej 2, DK-4000 Roskilde, Denmark. Fax: (45) 46 33 43 67. email: [email protected] Abbreviations used in this paper: OG, -octyl-glucoside; CMC, critical micellar concentration; DHA, docosahexaenoic acid; DOPC, dioleoylphosphatidylcholine; gA, gramicidin A; GX100, Genapol X-100; HH, Hodgkin-Huxley; M CD, methylated -cyclodextrin; TX100, Triton X-100; rTX100, reduced TX100. on July 1, 2017 jgp.rress.org D ow nladed fom http://doi.org/1 .1085/jgp.200308996 Supplemental material can be found at: on July 1, 2017 jgp.rress.org D ow nladed fom on July 1, 2017 jgp.rress.org D ow nladed fom on July 1, 2017 jgp.rress.org D ow nladed fom