Ni 2 1 transport by the human Na 1 / Ca 2 1 exchanger expressed in Sf 9 cells

transport by the human Na ϩ /Ca 2ϩ exchanger expressed in Sf 9 cells. Am. block of the Na ϩ /Ca 2ϩ exchanger was examined in Sf 9 cells expressing the human heart Na ϩ /Ca 2ϩ exchanger (NCX1-NACA1). As predicted from the reported actions of Ni 2ϩ , its application reduced extracellular Na ϩ-dependent changes in intracellular Ca 2ϩ concentration (measured by fluo 3 fluores-cence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured 63 Ni 2ϩ entry. The 63 Ni 2ϩ entry was observed in Sf 9 cells expressing the Na ϩ /Ca 2ϩ exchanger but not in control cells. The established sequential transport mechanism of the Na ϩ /Ca 2ϩ ex-changer could be compatible with these results if one of the two ion translocation steps is blocked by Ni 2ϩ and the other permits Ni 2ϩ translocation. We conclude that, because Ni 2ϩ entry was inhibited by extracellular Ca 2ϩ and enhanced by extracellular Na ϩ , the Ca 2ϩ translocation step moved Ni 2ϩ , whereas the Na ϩ translocation step was inhibited by Ni 2ϩ. A model is presented to discuss these findings. heart; nickel ion quench; fluo 3 THE SODIUM/CALCIUM EXCHANGER is an important mechanism for removing Ca 2ϩ from diverse cells. In heart, it extrudes Ca 2ϩ that has entered through Ca 2ϩ channels to initiate contraction (3, 12). The Na ϩ /Ca 2ϩ exchanger transports three Na ϩ for every Ca 2ϩ it moves in the opposite direction, and it is able to maintain the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i) three to four orders of magnitude below the extracellular Ca 2ϩ concentration ([Ca 2ϩ ] o). Nevertheless, the direction of net Ca 2ϩ transport depends on the electrochemical gradient of Na ϩ (19, 20, 25). Simultaneous and consecutive transport models have been suggested for Na ϩ and Ca 2ϩ translocations (11, 22, 25, 26), and a bulk of evidence favors the latter. The individual reaction steps as well as the Na ϩ /Ca 2ϩ exchanger kinetics are largely unknown. One of the major problems for such studies is the absence of any specific pharmacological inhibitor of the Na ϩ /Ca 2ϩ exchanger (31, 32). In the absence of a specific inhibitor, blockade of the Na ϩ /Ca 2ϩ exchanger can be produced by heavy metal ions such as La 3ϩ , Mn 2ϩ , or Ni …