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

Sabina Casula, Boris E. Shmukler, Sabine Wilhelm, Alan K. Stuart-Tilley, Wanfang Su, Marina N. Chernova, Carlo Brugnara, Seth L. Alper

Research output: Contribution to journalArticle

87 Scopus citations

Abstract

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl co-transport 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 (ΔN117) 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 Δ N117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. ΔN117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.

Original languageEnglish (US)
Pages (from-to)41870-41878
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number45
DOIs
StatePublished - Nov 9 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Casula, S., Shmukler, B. E., Wilhelm, S., Stuart-Tilley, A. K., Su, W., Chernova, M. N., Brugnara, C., & Alper, S. L. (2001). A dominant negative mutant of the KCC1 K-Cl cotransporter: Both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity. Journal of Biological Chemistry, 276(45), 41870-41878. https://doi.org/10.1074/jbc.M107155200