title: A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity.
abstract: K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.
authors.affiliation: Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
authors.forename: S
authors.initials: S
authors.lastname: Casula
authors.forename: B E
authors.initials: BE
authors.lastname: Shmukler
authors.forename: S
authors.initials: S
authors.lastname: Wilhelm
authors.forename: A K
authors.initials: AK
authors.lastname: Stuart-Tilley
authors.forename: W
authors.initials: W
authors.lastname: Su
authors.forename: M N
authors.initials: MN
authors.lastname: Chernova
authors.forename: C
authors.initials: C
authors.lastname: Brugnara
authors.forename: S L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D051379:Mice; D009154:Mutation; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000096922:K Cl- Cotransporters
chemical_list: D002712:Chlorides; D011188:Potassium; D027981:Symporters; D000096922:K Cl- Cotransporters