Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.
The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) 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 Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).
Kang
Kyeong-Jin
KJ
Department of Physiology & Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Kinjo
Tashi G
TG
Szerencsei
Robert T
RT
Schnetkamp
Paul P M
PP
D019943:Amino Acid Substitution; D000596:Amino Acids; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D005470:Fluorometry; D006801:Humans; D007313:Insecta; D007700:Kinetics; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
D000596:Amino Acids; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D011188:Potassium; D002118:Calcium