Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its β-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 Ca2+ currents (I-Ca,I-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-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 ICa, L approximately threefold and caused a negative shift in V-0.5 in control cells, but it did not increase I-Ca,I-L in M beta CD-treated myocytes, nor did it shift V-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-Ca,I-L more effectively than 10 mM M beta CD. M beta CD dialysis also abolished the increase in I-Ca,I-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-Ca,I-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-Ca,I-L and beta-adrenergic cAMP/PKA-mediated increases in I-Ca,I-L. We suggest that cholesterol affects the structural coupling between L-type Ca2+ channels and adjacent regulatory proteins.