Probing ion binding sites in the Na+/Ca2+ exchanger.
The membrane domain of the Na(+)/Ca(2+) exchanger (NCX) contains two conserved internal repeat sequences, designated the alpha-1 and alpha-2 repeats. We have studied the topological disposition of residues in the alpha repeats and a neighboring region by substituted cysteine accessibility scanning as well as the functional importance of these residues by kinetically evaluating transport activities of cysteine-substituted or other site-directed NCX1 mutants. The results suggest that the alpha-1 repeat contains a reentrant loop originating from extracellular side of the membrane, while the alpha-2 repeat and its neighboring region contain a complex reentrant loop structure originating from the cytoplasmic side. We identified several residues in the alpha-1 repeat loop whose mutation caused significant alterations in the interaction of NCX1 with a transport substrate Ca(2+)(o) and inhibitory ions Ni(2+) and Co(2+). On the other hand, we found residues in the alpha-2 repeat loop region whose mutation altered the interaction with an activating ion Li(+) and an inhibitory drug KB-R7943 in addition to the effect on Ca(2+)(o) and Ni(2+). Collectively, our data suggest that these alpha-repeat regions participate in the formation of ion translocation pathway of the exchanger and that the alpha-1 repeat loop plays an important role in ion selection.
Shigekawa
Munekazu
M
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan. shigekaw@ri.ncvc.go.jp
Iwamoto
Takahiro
T
Uehara
Akira
A
Kita
Satomi
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000378:metabolism*
D002412:Cations; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002118:Calcium