Regulation and deregulation of cardiac Na⁺–Ca²⁺ exchange in giant excised sarcolemmal membrane patches

Abstract

A PLASMALEMMAL Na⁺–Ca²⁺ exchange mechanism^1,2 is an important electrogenic determinant of contractility in cardiac cells^3–5. As in other cell types^6–8, calcium influx by Na⁺–Ca²⁺" exchange is secondarily activated by cytoplasmic calcium⁴ and probably ATP⁹, but these modulatory mechanisms are either absent or altered in isolated cardiac sarcolemmal vesicles^5,12. Involvement of a calcium-dependent protein kinase in exchange regulation has been suggested^7,10 but not verified^5,11. Here I describe measurements of outward Na⁺–Ca²⁺ exchange current, corresponding to calcium influx, in giant excised sarcolemmal Patches¹¹ from guinea pig myocytes. The exchange current is stimulated by both calcium and Mg–ATP from the cytoplasmic face, evidently through separate mechanisms. Activation by cytoplasmic calcium takes place within seconds, is reversible, and does not require ATP. Stimulation by Mg–ATP reverses only slowly over > 10 min, or not at all. Unexpectedly, a substantial decrease in exchange current occurs during activation by cytoplasmic sodium, which seems to reflect an inactivation process rather than ion concentration changes or a 'first pass' exchange cycle. This apparent inactivation, and the modulations by cytoplasmic calcium and Mg–ATP, are all abolished by brief treatment of the cytoplasmic surface with chymotrypsin, leaving the exchanger in a maintained state of high activity. Therefore, limited proteolysis deregulates Na⁺–Ca²⁺ exchange and could contribute to the loss of secondary regulation of the exchange in isolated sarcolemmal vesicles.