At low micromolar concentrations, polyunsaturated fatty acids (PUFAs) alter the function of many membrane proteins. PUFAs exert their effects on unrelated proteins at similar concentrations, suggesting a common mode of action. Because lipid bilayers serve as the common ''solvent'' for membrane proteins, the common mechanism could be that PUFAs adsorb to the bilayer/solution interface to promote a negative-going change in lipid intrinsic curvature and, like other reversibly adsorbing amphiphiles, increase bilayer elasticity. PUFA adsorption thus would alter the bilayer deformation energy associated with protein conformational changes involving the protein/bilayer boundary, which would alter protein function. To explore the feasibility of such a mechanism, we used gramicidin (gA) analogues of different lengths together with bilayers of different thicknesses to assess whether docosahexaenoic acid (DHA) could exert its effects through a bilayer-mediated mechanism. Indeed, DHA increases gA channel appearance rates and lifetimes and decreases the free energy of channel formation. The appearance rate and lifetime changes increase with increasing channel-bilayer hydrophobic mismatch and are not related to differing DHA bilayer absorption coefficients. DHA thus alters bilayer elastic properties, not just lipid intrinsic curvature; the elasticity changes are important for DHA's bilayer-modifying actions. Oleic acid (OA), which has little effect on membrane protein function, exerts no such effects despite OA's adsorption coefficient being an order of magnitude greater than DHA's. These results suggest that DHA (and other PUFAs) may modulate membrane protein function by bilayer-mediated mechanisms that do not involve specific protein binding but rather changes in bilayer material properties.bilayer material properties ͉ bilayer stiffness ͉ gramicidin channels ͉ hydrophobic mismatch ͉ polyunsaturated fatty acid

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 (β-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.

Membrane fusion, essential to eukaryotic life, is broadly envisioned as a three-step process proceeding from contacting bilayers through two semistable, nonlamellar lipidic intermediate states to a fusion pore. Here, we introduced a new, to our knowledge, experimental approach to gain insight into the nature of the transition states between initial, intermediate, and final states. Recorded time courses of lipid-mixing, content-mixing, and content-leakage associated with fusion of 23 nm vesicles in the presence of poly(ethylene glycol) at multiple temperatures were fitted globally to a three-step sequential model to yield rate constants and thereby activation thermodynamics for each step of the process, as well as probabilities of occurrence of lipid-mixing, content-mixing, or content-leakage in each state. Experiments with membranes containing hexadecane, known to reduce interstice energy in nonlamellar structures, provided additional insight into the nature of fusion intermediates and transition states. The results support a hypothesis for the mechanism of stalk formation (step-1) that involves acyl chain protrusions into the interbilayer contact region, a hypothesis for a step-2 mechanism involving continuous interconversion of semistable nonlamellar intermediates, and a hypothesis for step-3 (pore formation) mechanism involving correlated movement of whole lipid molecules into hydrophobic spaces created by geometry mismatch between intermediate structures.

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