Scanning mutagenesis of the alpha repeats and of the transmembrane acidic residues of the human retinal cone Na/Ca-K exchanger.
The Na/Ca-K exchanger (NCKX) utilizes the inward sodium gradient and outward potassium gradient for Ca(2+) extrusion; two distinct NCKX isoforms are expressed in the outer segments of retinal rod (NCKX1) and cone (NCKX2) photoreceptors, respectively, where NCKX extrudes Ca(2+) that enters photoreceptors via the cGMP-gated channels. We carried out the first systematic NCKX mutagenesis study in which 96 residues were mutated in the human cone NCKX2 cDNA, and functional consequences of these mutations were measured; the residues selected for mutagenesis are conserved between rod and cone NCKX, the large majority are also conserved in NCKX paralogs found in lower organisms, and finally, they include the few residues conserved between members of the NCKX and members of the NCX (potassium-independent Na/Ca exchange) gene families. Twenty-five residues were identified for which mutagenesis reduced NCKX function to <20% of wild-type cone NCKX2 activity, while protein expression and plasma membrane targeting were not affected. Three classes of residues were found to be most sensitive toward mutagenesis: acidic (glutamate/aspartate) residues, polar (serines/threonine) residues, and glycine residues. These results are discussed with respect to residues that may contribute to the NCKX cation binding site(s).
Winkfein
Robert J
RJ
Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, N.W. Calgary, Alberta, T2N 4N1, Canada.
Szerencsei
Robert T
RT
Kinjo
Tashi G
TG
Kang
KyeongJin
K
Perizzolo
Marco
M
Eisner
Lynn
L
Schnetkamp
Paul P M
PP
D024342:Amino Acids, Acidic / Q000235:genetics* / Q000502:physiology; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000502:physiology; D004285:Dogs; D018698:Glutamic Acid / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry* / Q000502:physiology; D011188:Potassium / Q000737:chemistry / Q000502:physiology; D011392:Proline / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics* / Q000502:physiology; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry* / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D018411:Spodoptera; D014162:Transfection
D024342:Amino Acids, Acidic; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D001224:Aspartic Acid; D018698:Glutamic Acid; D011392:Proline; D011188:Potassium; D005998:Glycine