Amphiphile regulation of ion channel function by changes in the bilayer spring constant

Abstract

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.

Fig. 1.

Hydrophobic coupling between a bilayer-embedded protein and its host lipid bilayer. (A) A protein conformational change causes a local bilayer deformation. (B) Formation of a gA channel involves local bilayer thinning. Modified from ref. .

Fig. 2.

Effects of amphiphiles on gA channels in DPhPC/n-decane bilayers. (A) Current traces before and after addition of 3 μM TX100 to both sides of a bilayer doped with gA^-(13) and AgA(15). The red and blue lines denote the current levels for gA^-(13) and AgA(15) channels. (B) Concentration-dependent effects of TX100, capsaicin, capsazepine, daidzein, genistein, phloretin, or GsMTx4 on lifetime of channels formed by monomeric subunits having 13 or 15 residues (except for TX100, based on results from refs. , , and 22).

Fig. 3.

Effects of amphiphiles on lifetimes of channels formed by 13-residue subunits (expressed as ln{τ/τ_cntl}, left axis, or activation energy, , right axis) vs. corresponding effects on channels formed by 15-residue subunits (bottom or top axis). (A) Effects of TX100, capsaicin, capsazepine, daidzein, genistein, phloretin, or GsMTx4 in DPhPC/n-decane bilayers (except for TX100, results from refs. , and 22). A subset of the results was published previously (5). (B) Effects of TX100, curcumin, docosahexaenoic acid (DHA), 2,3-butanedione monoxime (BDM), reduced Triton X-100 (rTX100), Z-Gly-D-Phe (ZGdF), and Z-Gly-Phe (ZGF) (at pH 7 or pH 4) and Z-D-Phe-Phe-Gly (ZdFFG, at pH 7) in DOPC/n-decane bilayers (except for TX100, results from refs. –28). (C) Superimposition of results obtained using DPhPC (red) or DOPC (black). (D) Distribution of for individual amphiphiles using DPhPC (red) or DOPC (black). Mean ± SEM (n≥3) or ± range (n = 2).

Fig. 4.

Amphiphile-induced changes in inactivation of voltage-dependent sodium channels in HEK293 cells as a function of changes in H_B in DOPC/n-decane bilayers. Shift in membrane potential for 50% inactivation (V_in-V_in,cntl) plotted vs. ΔH_B (= H_B-H_B,cntl) in DOPC/n-decane bilayers. Amphiphiles: capsaicin, β-octyl puranoside (βOG), Genapol X-100 (GX100), reduced Triton X-100 (rTX100), and TX100. HEK293 cells were depolarized to +20 mV following 300-ms prepulses to potentials varying from -130 to +50 mV. Based on results from refs.  and .