Cholesterol depletion modulates basal L-type [Ca.sup.2+] current and abolishes its [beta]-adrenergic enhancement in ventricular myocytes

Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-[beta]-cyclodextrin (M[beta]CD), we studied the role of cholesterol in the modulation of L-type [Ca.sup.2+] currents ([I.sub.Ca,L]). M[beta]CD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM M[beta]CD for 10 min, a positive shift in membrane potential at half-maximal activation ([V.sub.0.5]) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased [I.sub.Ca,L] approximately threefold and caused a negative shift in [V.sub.0.5] in control cells, but it did not increase [I.sub.Ca,L]. in M[beta]CD-treated myocytes, nor did it shift [V.sub.0.5]. The effect of M[beta]CD (10 or 30 mM) was concentration dependent: 30 mM M[beta]CD suppressed the ISO-induced increase in [I.sub.Ca,L] more effectively than 10 mM M[beta]CD. M[beta]CD dialysis also abolished the increase in [I.sub.Ca,L] elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of M[beta]CD-cholesterol complex to rat myocytes attenuated the M[beta]CD-mediated inhibition of the ISO-induced increase of [I.sub.Ca,L]. Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by M[beta]CD and increased by M[beta]CD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal [I.sub.Ca,L] and [beta]-adrenergic cAMP/ PKA-mediated increases in [I.sub.Ca,L]. We suggest that cholesterol affects the structural coupling between L-type [Ca.sup.2+] channels and adjacent regulatory proteins. lipid raft; adenosine 3',5'-cyclic monophosphate; protein kinase A; phosphorylation