Allosteric Regulation of Na/Ca Exchange Current by Cytosolic Ca in Intact Cardiac Myocytes

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Abstract
The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca]i such that when [Ca]i is low, NCX current (INCX) deactivates. In this study, we used membrane potential (Em) and INCX to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca]i in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca]i would be to increase inward INCX (Ca efflux) and to decrease outward INCX. On the other hand, Ca activation would increase INCX in both directions. Thus, we attributed [Ca]i-dependent increases in outward INCX to allosteric regulation. Ca activation of INCX was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Δ680–685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (KmCaAct = 125 ± 16 nM SEM ≈ resting [Ca]i). This, along with the observation that no delay was observed between measured [Ca]i and activation of INCX under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the INCX activation state may influence SR Ca load and resting [Ca]i, helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in KmCaAct from other species. Model predictions for Ca activation, on which our estimates of KmCaAct are based, confirm that Ca activation strongly influences outward INCX, explaining why it increases rather than declines with increasing [Ca]i.