Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers

We examined inhibitory effects of external multivalent cations Ni2+, Co2+, Cd2+, La3+, Mg2+, and Mn2+ on reverse‐mode exchange of the K+‐dependent Na+/Ca2+ exchanger NCKX2 and the K+‐independent exchanger NCX1 expressed in CCL‐39 cells by measuring the rate of Ca2+ uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca2+ uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li+ stimulated reverse‐mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K+. Thus, both exchangers exhibited differential sensitivities to not only K+ but also many other external cations. We attempted to locate the putative binding sites within the α motifs for multivalent cations by site‐directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the α‐1 motif, but not by mutations in the α‐2 motif. These results contrast with those for NCX1 where mutations in both α‐1 and α‐2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the α‐1 and α‐2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular‐facing α‐1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both α‐1 and α‐2 motifs together form the binding sites in NCX1. © 2004 Wiley‐Liss, Inc.