Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude.
This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the alpha subunit of the stimulatory heterotrimeric G-protein, Galpha(s), via stimulation of the cell surface beta-adrenergic receptor. Stimulation of beta-adrenergic receptors with 10 micromol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of beta-adrenergic receptors, and thereby Galpha(s), promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.
Yarbrough
Tracy L
TL
Department of Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA.
Lu
Tong
T
Lee
Hon-Chi
HC
Shibata
Erwin F
EF
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D021941:Caveolae / Q000737:chemistry / Q000378:metabolism*; D051244:Caveolin 3; D022461:Caveolins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D015151:Immunoblotting; D066298:In Vitro Techniques; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
D000318:Adrenergic beta-Agonists; C495549:Cav3 protein, rat; D051244:Caveolin 3; D022461:Caveolins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011943:Receptors, Adrenergic, beta; D015222:Sodium Channels; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D019205:GTP-Binding Protein alpha Subunits, Gs