Residues contributing to the Na(+)-binding pocket of the SLC24 Na(+)/Ca(2+)-K(+) Exchanger NCKX2.
Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.
Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Haider F
HF
Altimimi
Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Eric H
EH
Fung
Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Robert J
RJ
Winkfein
Department of Physiology & Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada. Electronic address: pschnetk@ucalgary.ca.
Paul P M
PPM
Schnetkamp
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012964:Sodium