Isolation of membranes enriched in "tetrodotoxin-insensitive" saxitoxin-binding sites from mammalian ventricle. Receptor solubilization.
Purification and characterization of Na+ channel protein from mammalian ventricular myocytes has heretofore been complicated by the low concentration of Na+ channels and by the finding that mammalian ventricles contain both tetrodotoxin (TTX)-sensitive channels (TSC), with high affinity for saxitoxin (STX), and TTX-insensitive channels (TIC), with low affinity for STX. Most (perhaps all) of the sodium current for myocardial cell action potentials is carried by TIC. Most, if not all, of the cardiac TSC reside in nerves innervating the heart. To isolate TIC in sufficient quantity for further study, we prepared t-tubular sarcolemmal vesicles from large (sheep) hearts with techniques designated to minimize contamination from nerve plasmalemma. Discontinuous sucrose density gradient centrifugation of these membranes produced membrane vesicles, some of which contained no detectable TSC (range 94-100% TIC, or 0-6% TSC), at a concentration of 200-1500 fmol total sites/mg protein, with yields of 4.0-25.0 mg protein/100 g starting material (ventricle). TTX-insensitive STX-binding sites were solubilized from the membranes by 1% digitonin (and with less stability by Triton X-100). The equilibrium binding constant and dissociation rate coefficient for STX binding to the digitonin-solubilized sites were similar to those of the binding sites for the unsolubilized membranes. Unlabeled TTX competed with [3H]STX for the site with 14 times less affinity than did unlabeled STX. Digitonin-solubilized sites had a half-life for STX binding of about 24 h. Binding could be further stabilized by addition of Mg2+ or Ca2+ and exogenous phospholipid.
Doyle
D D
DD
Department of Medicine, University of Chicago, Illinois 60637.
Winter
A
A
D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D002474:Cell-Free System; D004072:Digitonin / Q000494:pharmacology; D006352:Heart Ventricles / Q000032:analysis* / Q000378:metabolism; D012508:Sarcolemma / Q000032:analysis; D012530:Saxitoxin / Q000378:metabolism / Q000494:pharmacology; D012756:Sheep; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D012995:Solubility; D013779:Tetrodotoxin / Q000494:pharmacology
D002413:Cations, Divalent; D015222:Sodium Channels; D012530:Saxitoxin; D013779:Tetrodotoxin; D004072:Digitonin