title: Molecular mechanisms of Cl- transport by the renal Na(+)-K(+)-Cl- cotransporter. Identification of an intracellular locus that may form part of a high affinity Cl(-)-binding site.
abstract: The 2nd transmembrane domain (tm) of the secretory Na(+)-K(+)-Cl(-) cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were approximately 8 mm for saF/A and saA, and approximately 70 mm for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all approximately 20 mM, and Km(Rb+) all approximately 2 mM. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.
authors.affiliation: Nephrology Research Group, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6, Canada.
authors.forename: Edith
authors.initials: E
authors.lastname: Gagnon
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Bergeron
authors.forename: Geneviève M
authors.initials: GM
authors.lastname: Brunet
authors.forename: Nikolas D
authors.initials: ND
authors.lastname: Daigle
authors.forename: Charles F
authors.initials: CF
authors.lastname: Simard
authors.forename: Paul
authors.initials: P
authors.lastname: Isenring
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002034:Bumetanide / Q000494:pharmacology; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002713:Chlorine / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005091:Exons; D005822:Genetic Vectors; D007477:Ions; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012413:Rubidium / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012754:Sharks; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry* / Q000378:metabolism; D064466:Solute Carrier Family 12, Member 1; D014981:Xenopus; D014982:Xenopus laevis
chemical_list: D002412:Cations; D018076:DNA, Complementary; D007477:Ions; D020033:Protein Isoforms; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D002034:Bumetanide; D002713:Chlorine; D012413:Rubidium