title: Regions in the cytosolic C-terminus of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 are required for its homodimerization.
abstract: The "secretory" Na+-K+-2Cl- cotransporter, NKCC1, is a member of a small gene family of electroneutral cation-chloride cotransporters (CCCs) with 9 homologues in vertebrates. A number of these transporters, including NKCC1 itself, have been shown to exist as homodimers in the membrane, suggesting that this may be a common feature of the CCCs. Here we employ chemical cross-linking studies, a novel co-immunoprecipition assay, and NKCC1/CCC chimeras to further explore the basis and significance of NKCC1 dimerization. An N-terminally truncated NKCC1 (nttNKCC1), in which the first 20 kDa of the 28 kDa cytosolic N-terminus are deleted, forms homodimers as well as heterodimers with full-length NKCC1, indicating that this region of N-terminus is not required for dimerization. On the other hand, replacing the 50 kDa NKCC1 C-terminus with that of several other non-NKCC1 homologues results in chimeric proteins that form homodimers but show little or no heterodimerization with NKCC1, demonstrating that the C-terminus of NKCC1 plays an essential role in dimerization and that NKCC1 dimerization exhibits definite homologue-specificity. Using additional chimeras we find that the residues required for dimer formation lie between amino acids 751 and 998 of (rat) NKCC1. We also show that dramatically overexpressing the nonfunctional truncated protein nttNKCC1 relative to the endogenous NKCC1 in the HEK293 cells results in a modest inhibition of fluxes via the endogenous transporter and a change in its sensitivity to the specific inhibitor bumetanide. These latter results indicate that there is a functional interaction between dimer subunits but that nonfunctional subunits do not necessarily have a dominant negative effect as has been previously proposed.
authors.affiliation: Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, Maryland 20892-1190, USA.
authors.forename: Most Nahid
authors.initials: MN
authors.lastname: Parvin
authors.forename: Tudevdagva
authors.initials: T
authors.lastname: Gerelsaikhan
authors.forename: R James
authors.initials: RJ
authors.lastname: Turner
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D047468:Immunoprecipitation; D051381:Rats; D011993:Recombinant Fusion 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; D013388:Succinimides / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D011993:Recombinant Fusion 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; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)