Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites.
To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.
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D029845:Amphibian Proteins; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D002352:Carrier Proteins / Q000032:analysis*; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D007473:Ion Channels / Q000378:metabolism; D008854:Microscopy, Electron; D009206:Myocardium / Q000166:cytology*; D010042:Ouabain / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000032:analysis*; D012756:Sheep; D012964:Sodium / Q000378:metabolism; D019810:Sodium Azide; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014018:Tissue Distribution
D029845:Amphibian Proteins; D001386:Azides; D015220:Calcium Channels; D002352:Carrier Proteins; D007473:Ion Channels; D011978:Receptors, Nicotinic; C027525:saxitoxin-binding protein, Rana catesbeiana; D010042:Ouabain; D011813:Quinuclidinyl Benzilate; D019810:Sodium Azide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium