title: Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Nna(+)/Ca(2+) exchanger.
abstract: KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na(+)/Ca(2+) exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha-2 repeat of NCX1 identified one residue (Gly833) that caused a large (> or = 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 microM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 microM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na(+)/Ca(2+) exchange in such a way that it becomes accessible to external drug.
authors.affiliation: Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Iwamoto
authors.forename: S
authors.initials: S
authors.lastname: Kita
authors.forename: A
authors.initials: A
authors.lastname: Uehara
authors.forename: Y
authors.initials: Y
authors.lastname: Inoue
authors.forename: Y
authors.initials: Y
authors.lastname: Taniguchi
authors.forename: I
authors.initials: I
authors.lastname: Imanaga
authors.forename: M
authors.initials: M
authors.lastname: Shigekawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D004594:Electrophysiology; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D013890:Thiourea; D002118:Calcium