title: Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its -adrenergic enhancement in ventricular myocytes.
abstract: 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 (MbetaCD), we studied the role of cholesterol in the modulation of L-type Ca(2+) currents (I(Ca,L)). MbetaCD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM MbetaCD 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 I(Ca,L) approximately threefold and caused a negative shift in V(0.5) in control cells, but it did not increase I(Ca,L) in MbetaCD-treated myocytes, nor did it shift V(0.5). The effect of MbetaCD (10 or 30 mM) was concentration dependent: 30 mM MbetaCD suppressed the ISO-induced increase in I(Ca,L) more effectively than 10 mM MbetaCD. MbetaCD dialysis also abolished the increase in I(Ca,L) elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of MbetaCD-cholesterol complex to rat myocytes attenuated the MbetaCD-mediated inhibition of the ISO-induced increase of I(Ca,L). Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by MbetaCD and increased by MbetaCD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal I(Ca,L) and beta-adrenergic cAMP/PKA-mediated increases in I(Ca,L). We suggest that cholesterol affects the structural coupling between L-type Ca(2+) channels and adjacent regulatory proteins.
authors.affiliation: Department of Physiology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Tsujikawa
authors.forename: Yumei
authors.initials: Y
authors.lastname: Song
authors.forename: Makino
authors.initials: M
authors.lastname: Watanabe
authors.forename: Haruko
authors.initials: H
authors.lastname: Masumiya
authors.forename: Sachin A
authors.initials: SA
authors.lastname: Gupte
authors.forename: Rikuo
authors.initials: R
authors.lastname: Ochi
authors.forename: Takao
authors.initials: T
authors.lastname: Okada
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002784:Cholesterol / Q000172:deficiency*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003563:Cyclic CMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology; D006352:Heart Ventricles / Q000378:metabolism; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000318:Adrenergic beta-Agonists; D002120:Calcium Channel Agonists; D020746:Calcium Channels, L-Type; D020536:Enzyme Activators; D011943:Receptors, Adrenergic, beta; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005576:Colforsin; D003563:Cyclic CMP; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002784:Cholesterol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D007545:Isoproterenol