Probing Ion Binding Sites in the Na+/Ca2+ Exchanger
The membrane domain of the Na+/Ca2+ exchanger (NCX) contains two conserved internal repeat sequences, designated the ?-1 and ?-2 repeats. We have studied the topological disposition of residues in the ? repeats and a neighboring region by substituted cysteine accessibility scanning as well as the functional importance of these residues by kinetically evaluating transport activities of cysteine-substituted or other site-directed NCX1 mutants. The results suggest that the ?-1 repeat contains a reentrant loop originating from extracellular side of the membrane, while the ?-2 repeat and its neighboring region contain a complex reentrant loop structure originating from the cytoplasmic side. We identified several residues in the ?-1 repeat loop whose mutation caused significant alterations in the interaction of NCX1 with a transport substrate Ca2+o and inhibitory ions Ni2+ and Co2+. On the other hand, we found residues in the ?-2 repeat loop region whose mutation altered the interaction with an activating ion Li+ and an inhibitory drug KB-R7943 in addition to the effect on Ca2+o and Ni2+. Collectively, our data suggest that these ?-repeat regions participate in the formation of ion translocation pathway of the exchanger and that the ?-1 repeat loop plays an important role in ion selection.
ion binding sites na+/ca2+ exchanger ion translocation ncx isoforms ?-repeat loops