Differential effect of cholesterol on membrane interaction of charged versus uncharged 1,4-dihydropyridine calcium channel antagonists: A biophysical analysis

Abstract

Second-generation 1,4-dihydropyridine (DHP) calcium channel blockers are characterized by improved pharmacologic properties (e.g., long duration of activity and/or enhanced vasoselectivity), which are mediated, in part, by complex physicochemical interactions between the drug molecule and plasma membrane of certain contractile cells. The lipid bilayer assists in subsequent DHP receptor site recognition by optimizing drug location and concentration in equilibrium with an intrabilayer binding site. To understand these critical drug-membrane interactions, we have examined the equilibrium and kinetic binding properties of various DHP analogues, as well as verapamil and diltiazem, in reconstituted lipid bilayer vesicles. Of particular interest was the effect of cholesterol enrichment, as observed during dietary atherosclerosis, on drug binding to the lipid bilayer. Between a 0:1 and 0.3:1 cholesterol-to-phospholipid (C:PL) mole ratio, the membrane partition coefficient (KP[mem]) for uncharged DHPs (i.e., isradipine, nimodipine) decreased by over 50%, while the KP[mem] for the charged DHP amlodipine decreased by 10%. To understand the molecular basis for these results, small-angle x-ray diffraction approaches were used to generate structure profiles for membranes of varying cholesterol content to mimic normal and atherosclerotic-like conditions. One-dimensional electron density profiles (8 A resolution) demonstrated that the time-averaged membrane location for cholesterol's steroid nucleus overlaps the membrane area occupied by these DHPs. Thus, increases in cholesterol appear to reduce the molecular volume available for DHP partitioning into this region of the membrane. Despite its water solubility, amlodipine had the highest membrane partition coefficient (KP[mem]>104), the slowest rate of membrane dissociation, and was least affected by membrane cholesterol content changes. At a C-to-PL molar ratio of 0.6:1, amlodipine's KP[mem] was more than an order of magnitude greater than that of nimodipine, verapamil, or diltiazem. The results from this analysis demonstrate that there are mutual physicochemical factors that govern the interaction of lipophilic drugs with membranes of varying cholesterol composition. This study may have broad implications for the pharmacologic activity of drug molecules that bind to the membrane lipid bilayer as part of their overall mechanism of action under both normal and pathophysiologic conditions in which membrane cholesterol content is altered (e.g., atherosclerosis).