Localization of the Na/Ca exchange-dependent Ca compartment in cultured neonatal rat heart cells

It has been previously established, in both adult and cultured neonatal cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarcoplasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemmal leaflet represent together the subcellular locus of the compartment. The present study examines this proposal. The amount of Ca in the total compartment is measured isotopically in intact functional cells (using the on-line "scintillation disk" technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated from another set of intact cells by "gas dissection," and the amount of Ca bound to the membranes is measured. Probes that specifically decrease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrease total cell content and sarcolemmal binding proportionally. High-dose ryanodine (producing closure of SR channels) markedly reduces sarcolemmal binding relative to total content of the compartment. The sarcolemmal sites saturate between 1 and 2 mM extracellular Ca ([Ca]o), whereas the total compartment saturates between 4 and 6 mM [Ca]o. Below 1 mM [Ca]o, sarcolemmal binding is maintained relative to total compartment content. Finally, the total compartment increases after reversal of the intracellular Na to extracellular Na ([Na]i/[Na]o) gradient with sarcolemmal content-to-total content ratio dependent on the method used to reverse the [Na]i/[Na]o ratio. The results are consistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region ("cleft") where JSR Ca is in equilibrium with anionic inner sarcolemmal leaflet Ca binding sites.