The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.
We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane. We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the alpha repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca(2+) by up to 6-fold. In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the alpha repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.
Iwamoto
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Uehara
A
A
Imanaga
I
I
Shigekawa
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D017124:Conserved Sequence; D006224:Cricetinae; D003545:Cysteine; D004285:Dogs; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D001224:Aspartic Acid; D001216:Asparagine; D012964:Sodium; D003545:Cysteine; D002118:Calcium