title: Identification of key residues involved in the dimerization of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1.
abstract: The "secretory" Na(+)-K(+)-2Cl(-) cotransporter, NKCC1, belongs to the SLC12 gene family of electroneutral cation-chloride cotransporters. A number of these proteins, including NKCC1 itself, exist as homodimers in the membrane, suggesting that this may be a common feature of the SLC12 family. We have previously demonstrated that replacing the C-terminus of NKCC1 with that of its close homologue NKCC2 produced a fully functional chimeric protein that formed homodimers but did not dimerize with NKCC1. Here we employ a novel co-immunoprecipitation assay to study the dimerization interaction of NKCC1 using additional NKCC1/NKCC2 C-terminal chimeras and point mutants. Our results indicate that the substitution of a number of regions of the C-terminus of NKCC1 with the corresponding sequence from NKCC2 results in weakened dimerization with wild-type NKCC1, demonstrating that various residues play a role in this interaction. Most interestingly, however, we find that the replacement of a single NKCC1 residue, G812, with cysteine, the corresponding amino acid in NKCC2, results in a point mutant that displays no significant dimerization with the wild-type protein. In addition to this effect on heterodimer formation, we also find that G812 mutants can nevertheless form homodimers but that this interaction can be weaker than that observed for wild-type NKCC1. We demonstrate that our results are consistent with at least one established mechanism of protein dimer formation, that of "domain swapping", as well as with a recently reported crystal structure of the C-terminus of a bacterial SLC12 homologue.
authors.affiliation: Membrane Biology Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, Maryland 20892, United States.
authors.forename: Most Nahid
authors.initials: MN
authors.lastname: Parvin
authors.forename: R James
authors.initials: RJ
authors.lastname: Turner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry* / Q000235:genetics; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C578714:SLC12A1 protein, human; C578877:SLC12A2 protein, human; C578716:Slc12a1 protein, rat; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D064506:Solute Carrier Family 12, Member 2