A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity

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 cotransp...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2001-11, Vol.276 (45), p.41870-41878
Main Authors: Casula, S, Shmukler, B E, Wilhelm, S, Stuart-Tilley, A K, Su, W, Chernova, M N, Brugnara, C, Alper, S L
Format: Article
Language:English
Subjects:
Animals
Chlorides - metabolism
Cytoplasm - chemistry
Humans
K Cl- Cotransporters
KCC gene
Mice
Mutation
Potassium - metabolism
potassium/chloride cotransporter
Rats
Structure-Activity Relationship
Symporters - chemistry
Symporters - genetics
Symporters - physiology
Xenopus
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0021-9258
DOI:10.1074/jbc.M107155200