Simulation of the two parallel NCX1 inactivation reactions revealed in studies of cardiac giant membrane patches. (A) Exchange currents recorded after step changes of cytoplasmic Na and Ca. From left to right: (1) When exchange current is activated by a step increase of cytoplasmic Na (100 mM) in the presence of cytoplasmic Ca (2 µM), exchange currents decay by 10 to >90% with a tau of several seconds. (2) In the absence of cytoplasmic Ca, no current is activated by application of Na. (3) In the presence of Na, outward exchange current turns on and off with a tau of ∼8 s when cytoplasmic Ca is applied and removed. (4) When Na is applied and Ca is removed simultaneously, current decays to negligible values with a multisecond time course. (5) Na application without Ca activates no current. (6) However, simultaneous application of Na and Ca after a period of no Na/no Ca activates the peak exchange current, reflecting a fast transition from state 7 to 4 in the diagram of Fig. 2. (B) Simulation of the same exchange current function. (C) Effect of increasing solely the rates of the two Na-dependent inactivation processes in parallel (i.e., transitions 4 to 5 and 7 to 6). The changes that occur for Ca regulation include both a shift of the half-maximal Ca to higher concentrations and a decrease of the maximal exchange current observed. The rightward shifts of these curves correspond well to the observed effects of deleting ATP and/or anionic phospholipids from patches, while the leftward shifts correspond well to effects of increasing anionic phospholipids. This includes the development of high exchange activity at extremely low free Ca.