title: Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers.
abstract: We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) 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 alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.
authors.affiliation: Department of Physiology, School of Medicine, Fukuoka University, 45-1, 7-chrome Nana-kuma, Jonan-ku, Fukuoka 814-0180, Japan. ueharaak@fukuoka-u.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Uehara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwamoto
authors.forename: Satomi
authors.initials: S
authors.lastname: Kita
authors.forename: Takao
authors.initials: T
authors.lastname: Shioya
authors.forename: Midori
authors.initials: M
authors.lastname: Yasukochi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Issei
authors.initials: I
authors.lastname: Imanaga
mesh_terms: D020816:Amino Acid Motifs / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D002412:Cations / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D005110:Extracellular Space / Q000737:chemistry; D007811:Lanthanum / Q000378:metabolism / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002412:Cations; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002104:Cadmium; C074530:potassium-dependent sodium-calcium exchanger; D003035:Cobalt; D008345:Manganese; D007811:Lanthanum; D009532:Nickel; D008274:Magnesium; D011188:Potassium