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.