Residues Contributing to the Ca2+ and K+ Binding Pocket of the NCKX2 Na+/Ca2+-K+ Exchanger

The Na+/Ca2+-K+ exchanger (NCKX) extrudes Ca2+ from cells utilizing both the inward Na+ gradient and the outward K+ gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca2+ and K+ and alternates between inward and outward facing conforma...

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Published inThe Journal of biological chemistry Vol. 280; no. 8; pp. 6823 - 6833
Main Authors Kang, Kyeong-Jin, Kinjo, Tashi G., Szerencsei, Robert T., Schnetkamp, Paul P.M.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 25.02.2005
American Society for Biochemistry and Molecular Biology
Subjects
Amino Acid Substitution
Amino Acids
Animals
Binding Sites - genetics
Biological Transport
Calcium - metabolism
Calcium - pharmacology
Cell Line
Fluorometry
Humans
Insecta
Kinetics
Potassium - metabolism
Potassium - pharmacology
Sodium-Calcium Exchanger - chemistry
Sodium-Calcium Exchanger - genetics
Sodium-Calcium Exchanger - metabolism
Static Electricity
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Summary:The Na+/Ca2+-K+ exchanger (NCKX) extrudes Ca2+ from cells utilizing both the inward Na+ gradient and the outward K+ gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca2+ and K+ and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K+ and Ca2+ dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K+ and Ca2+ dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu188 and Asp548, whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro187 and Pro547), three additional acidic residues (Asp258, Glu265, Glu533), and two hydroxyl-containing residues (Ser185 and Ser545) showed smaller shifts, but shifts in Ca2+ dependence were invariably accompanied by shifts in K+ dependence. We conclude that Glu188 and Asp548 are the central residues of a single cation binding pocket that can accommodate both K+ and Ca2+. Furthermore, a single set of residues lines a transport pathway for both K+ and Ca2+.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M407933200