gramicidin-a and capsazepine

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length--as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

Topics: Algorithms; Capsaicin; Cell Line; Cell Membrane; Genistein; Gramicidin; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Isoflavones; Kinetics; Lipid Bilayers; Membrane Potentials; Membrane Proteins; Octoxynol; Phloretin; Phosphatidylcholines; Protein Conformation

At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.

Topics: Capsaicin; Gramicidin; Kinetics; Lipid Bilayers; Patch-Clamp Techniques; Scattering, Radiation; Sodium Channels