Calcium- and ATP-dependent regulation of Na/Ca exchange function in BHK cells: Comparison of NCX1 and NCX3 exchangers

Na+/Ca2+ exchangers (NCX) mediate bidirectional Ca2+ fluxes across cell membranes and contribute to Ca2+ homeostasis in many cell types. Exchangers are regulated by gating reactions that depend on Na+ and Ca2+ binding to transport and regulatory sites. A Na+i-dependent inactivation is prominent in all isoforms, whereas Ca2+i-dependent regulation varies among isoforms. Here we characterize new details of NCX operation and describe differences and similarities between NCX3 and NCX1 regulation by intracellular Ca2+ and ATP. To compare isoforms, we employed BHK cells expressing NCX3 or NCX1 constitutively and exchange activity was analysed in whole-cell and excised patch recordings under "zero-trans" conditions (i.e., with only one transported ion species on each side). Using BHK cells with low cytoplasmic Ca2+ buffering, outward (reverse) currents, reflecting Ca2+ influx, are activated by applying extracellular Ca2+ (Cao) in the presence of Na+ on the cytoplasmic side. When firstly activated, peak outward NCX3 currents rapidly decay over seconds and then typically develop a secondary transient peak with slower kinetics, until Cao removal abolishes all outward current. The delayed rise of outward current is the signature of an activating process since peak outward NCX3 currents elicited at subsequent Cao bouts remain stimulated for minutes and slower decline towards a non-zero level during continued Cao application. Secondary transient peaks and current stimulation are suppressed by increasing the intracellular Ca2+ buffer capacity or by replacing cytoplasmic ATP with the analogues AMP-PNP or ATPγS. In BHK cells expressing NCX1, outward currents activated under identical settings decay to a steady-state level during single Cao application and are significantly larger, causing strong and long-lived run down of subsequent outward currents. NCX1 current run down is not prevented by increasing cytoplasmic Ca2+ buffering but secondary transient peaks in the outward current profile can be resolved in the presence of ATP. Finally, inward currents recorded in patches excised from NCX3-expressing cells reveal a proteolysis-sensitive, Ca-dependent inactivation process that is unusual for NCX1 forward activity. Together, our results suggest that NCX function is regulated more richly than appreciated heretofore, possibly including processes that are lost in excised membrane patches.