Sarcolemmal calcium binding sites in heart: I. Molecular origin in "gas-dissected" sarcolemma.
Calcium in the myocardial cell is highly compartmentalized and a fast, an intermediate, a slow and a nonexchangeable calcium pool have been described. The fast pool contains 66% of the total cell exchangeable calcium in cultured neonatal rat heart cells with a t1/2 of less than 1.5 sec. Though the cellular origin of this fast pool is unknown, its rapidity and its displacement by La3+ most likely places it at the sarcolemma or at least in rapid equilibrium with the sarcolemma. We isolated the sarcolemma of cultured neonatal rat heart cells using the gas-dissection technique, which yields a pure sarcolemmal preparation in less than a second, thereby precluding membrane changes which might occur during conventional plasma membrane isolation. We determined the calcium binding characteristics of these membranes, using an on-line technique to monitor 45Ca, which allows measurement of 45Ca binding characteristics in the presence of unbound 45Ca. Two classes of calcium binding sites were determined: (i) Kd of 13 microM, capacity 7 nmol/mg and (ii) Kd of 1.1 mM, capacity of 84 nmol/mg. To assess the molecular origin of the sarcolemmal calcium binding we treated the membranes with a variety of enzymes. Protease or neuraminidase treatment did not cause large changes in these parameters. Simultaneous treatment with two different phospholipases C or the extraction of the lipids with isopropanol resulted in a dramatic loss of the low-affinity binding sites. These results, in association with previously defined sarcolemmal phospholipid distribution, places the low-affinity binding sites at the cytoplasmic leaflet. The physiological implication of this localization as it pertains to cellular calcium exchange is discussed.
Post
J A
JA
Cardiovascular Research Laboratory, University of California, Los Angeles School of Medicine.
Langer
G A
GA
D000433:1-Propanol / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D004210:Dissection / Q000379:methods; D010450:Endopeptidases / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009439:Neuraminidase / Q000494:pharmacology; D009584:Nitrogen; D010743:Phospholipids / Q000032:analysis; D051381:Rats; D012508:Sarcolemma / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000494:pharmacology
D002132:Calcium Radioisotopes; D010743:Phospholipids; D000433:1-Propanol; D010738:Type C Phospholipases; D009439:Neuraminidase; D010450:Endopeptidases; D009584:Nitrogen; D002118:Calcium