Helix packing of functionally important regions of the cardiac Na(+)-Ca(2+) exchanger.
In a revised topological model of the cardiac Na(+)-Ca(2+) exchanger, there are nine transmembrane segments (TMSs) and two possible re-entrant loops (Nicoll, D. A., Ottolia, M., Lu, Y., Lu, L., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917; Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268). The TMSs form two clusters separated by a large intracellular loop between TMS5 and TMS6. We have combined cysteine mutagenesis and oxidative cross-linking to study proximity relationships of TMSs in the exchanger. Pairs of cysteines were reintroduced into a cysteine-less exchanger, one in a TMS in the NH(2)-terminal cluster (TMSs 1-5) and the other in a TMS in the COOH-terminal cluster (TMSs 6-9). The mutant exchanger proteins were expressed in HEK293 cells, and disulfide bond formation between introduced cysteines was analyzed by gel mobility shifts. Western blots showed that S117C/V804C, A122C/Y892C, A151C/T815C, and A151C/A821C mutant proteins migrated at 120 kDa under reducing conditions and displayed a partial mobility shift to 160 kDa under nonreducing conditions. This shift indicates the formation of a disulfide bond between these paired cysteine residues. Copper phenanthroline and the cross-linker N', N'-o-phenylenedimaleimide enhanced the mobility shift to 160 kDa. Our data suggest that TMS7 is close to TMS3 near the intracellular side of the membrane and is in the vicinity of TMS2 near the extracellular surface. Also, TMS2 must adjoin TMS8. This initial packing model of the exchanger brings two functionally important domains in the exchanger, the alpha 1 and alpha 2 repeats, close to each other.
Qiu
Z
Z
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1760, USA.
Nicoll
D A
DA
Philipson
K D
KD
D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008301:Maleimides / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D016296:Mutagenesis / Q000235:genetics; D009206:Myocardium / Q000737:chemistry*; D009865:Oocytes; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D004220:Disulfides; D008301:Maleimides; D008565:Membrane Proteins; D019831:Sodium-Calcium Exchanger; D003545:Cysteine; C023860:N,N'-2-phenylenedimaleimide