Identification of Critical Positive Charges in XIP, the Na/Ca Exchange Inhibitory Peptide

The peptides XIP (RRLLFYKYVYKRYRAGKQRG) and C28R2 (LRRGQILWFRGLNRIQTQIRVVKAFRSS) correspond to the autoinhibitory domains of the Na–Ca exchanger and the plasma membrane Ca pump, respectively. An increase of ionic strength reduced the inhibition of exchange activity by XIP and C28R2, consistent with an important role for electrostatic interactions. Sulfosuccunimidyl acetate (SNA)-modified XIP did not inhibit Na–Ca exchange. Because SNA modifies lysines, we conclude that at least one of the positive charges at the XIP lysine positions (7, 11, or 17) is important for inhibition. 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) has cysteines at 12 and 14 and only one lysine (at 19). 2CK-XIP inhibited Na–Ca exchange; thus positive charges at 12 and 14 are not essential. SNA-modified 2CK-XIP did not inhibit; thus a positive charge at 19 is important. Iodoacetic acid-modified 2CK-XIP inhibits the Na–Ca exchanger but not the PM Ca pump. These results show that the structural determinants for inhibition of the Na–Ca exchanger and the PM Ca pump are different, that positive charges at 7, 11, or 17 (or some combination) are more important than positive charges at 12 and 14 for inhibition by XIP of the Na–Ca exchanger.