Scanning Mutagenesis of the Alpha Repeats and of the Transmembrane Acidic Residues of the Human Retinal Cone Na/Ca-K Exchanger
Abstract
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).
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The region containing the polymorphic residue of NCKX5 contains a number of residues that are critical for exchange function.
“This region contains a number of residues that are critical for exchange function (14 -16), and the experimentally determined topology for NCKX2 suggests that the polymorphic residue of NCKX5 sits in a transmembrane domain (17).”
From SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis (2008) · cites this paper as [14]
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- Authors
- Winkfein, R. J., Szerencsei, R. T., Kinjo, T. G., Kang, Perizzolo, M., Eisner, L., & Schnetkamp, P. P. M.
- Affiliations
- University of Calgary (CA)
- Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, N.W. Calgary, Alberta, T2N 4N1, Canada
- Corresponding author
- Schnetkamp, Paul P. M.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2002-12-20
- Volume
- 42
- Issue
- 2
- Pages
- 543-552
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2073227081
- Cited by
- 61
- References
- 10
- Citations by year
- 2026: 2 · 2025: 3 · 2023: 1 · 2022: 1 · 2019: 2 · 2018: 6 · 2017: 4 · 2016: 4 · 2015: 2 · 2014: 2 · 2013: 8 · 2012: 2
- FWCI
- 1.04
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion Transport and Channel Regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Keywords
- NCKX2, cone photoreceptors, calcium efflux, site-directed mutagenesis, cation binding sites, Na+/Ca2+ exchanger, photoreceptor outer segments, cGMP-gated channels
- MeSH terms
- Animals, Aspartic Acid, Cell Line, Cell Membrane, Dogs, Glycine, Humans, Potassium, Proline, Transfection, Mutagenesis, Site-Directed, Retinal Cone Photoreceptor Cells, Spodoptera, Glutamic Acid, Sodium-Calcium Exchanger, Photoreceptor Cells, Vertebrate, Repetitive Sequences, Amino Acid, Amino Acids, Acidic
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