Membrane dipole potential is sensitive to cholesterol stereospecificity: implications for receptor function

Abstract

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, an essential lipid in higher eukaryotic membranes, has previously been shown to increase membrane dipole potential. In this work, we explored the effect of stereoisomers of cholesterol, ent-cholesterol and epi-cholesterol, on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that cholesterol and ent-cholesterol share comparable ability in increasing membrane dipole potential. In contrast, epi-cholesterol displays a slight reduction in membrane dipole potential. Our results constitute the first report on the effect of stereoisomers of cholesterol on membrane dipole potential, and imply that an extremely subtle change in sterol structure can significantly alter the dipolar field at the membrane interface. These results assume relevance in the context of differential abilities of these stereoisomers of cholesterol in supporting the activity of the serotonin1A receptor, a representative G protein-coupled receptor. The close correlation between membrane dipole potential and receptor activity provides new insight in receptor-cholesterol interaction in terms of stereospecificity. We envision that membrane dipole potential could prove to be a sensitive indicator of lipid-protein interactions in biological membranes.

Keywords: Cholesterol; Dipole potential; Ent-cholesterol; Epi-cholesterol; Serotonin(1A) receptor; di-8-ANEPPS.

Fig. 1. Chemical structures of sterols used: (a) cholesterol, (b) ent-cholesterol and (c) epi-cholesterol

Both ent-cholesterol and epi-cholesterol are stereoisomers of cholesterol. ent-Cholesterol is the enantiomer of cholesterol. Enantiomers are non-superimposable mirror images of one another. epi-Cholesterol, on the other hand, is a diastereomer and is not a mirror image of cholesterol. ent-Cholesterol (but not epi-cholesterol) shares identical physicochemical properties with cholesterol. See text (section 1) for more details.

Fig. 2. Effect of stereoisomers of cholesterol on dipole potential of membranes

Dipole potential in POPC membranes plotted with increasing concentrations of cholesterol (■), ent-cholesterol (▲), and epi-cholesterol (●). Data points shown are means ± S.E. of at least three independent measurements. The ratio of di-8-ANEPPS to total lipid was 1:100 (mol/mol) and total lipid concentration was 0.43 mM. Measurements were carried out at room temperature (~23°C). Lines joining the data points are provided merely as viewing guides. The structure of voltage-sensitive probe di-8-ANEPPS is shown in the upper left side. See section 2 for details.

Fig. 3. Correlation of receptor activity with membrane dipole potential

(a) Effect of replenishment of cholesterol, epi-cholesterol (epi) and ent-cholesterol (ent) into solubilized membranes (SM) on specific binding of the agonist [³H]8-OH-DPAT to the serotonin_1A receptor. Solubilized hippocampal membranes were replenished with cholesterol, epi-cholesterol or ent-cholesterol using sterol:MβCD complex. Values are expressed as percentages of specific binding obtained in native hippocampal membranes (HM). Data shown are means ± S.E. of at least four independent experiments (taken from Jafurulla et al., 2014). (b) Correlation of membrane dipole potential with activity of serotonin_1A receptors. Specific [³H]8-OH-DPAT binding to serotonin_1A receptors (values taken from Fig. 3a) and corresponding values of membrane dipole potential containing 40 mol% sterol (from Fig. 2) are shown. Linear regression analysis yielded a correlation coefficient (r) ~0.99. The tight correlation between membrane dipole potential and receptor activity is noteworthy. See sections 2 and 3 for more details.