Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter

Sabina Casula, Alexander S. Zolotarev, Alan K. Stuart-Tilley, Sabine Wilhelm, Boris E. Shmukler, Carlo Brugnara, Seth L. Alper

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Oligomerization, function, and regulation of unmodified mouse Kcc1 K-Cl cotransporter were studied by chemical crosslinking. Treatment of Xenopus oocytes and 293T cells expressing K-Cl cotransporter Kcc1 with several types of chemical cross-linkers shifted Kcc1 polypeptide to higher molecular weight forms. More extensive studies were performed with the amine-reactive disuccinyl suberate (DSS) and with the sulfhydryl-reactive bis-maleimidohexane (BMH). Kcc1 cross-linking was time-dependent in intact oocytes, and was independent of protein concentration in detergent lysates from oocytes or 293T cells. Kcc1 cross-linking by the cleavable cross-linker DTME was reversible. The N-terminal and C-terminal cytoplasmic tails of Kcc1 were not essential for Kcc1 crosslinking. PFO-PAGE and gel filtration revealed oligomeric states of uncrosslinked KCC1 corresponding in mobility to that of cross-linked protein. DSS and BMH each inhibited KCC1-mediated 86Rb+ uptake stimulated by hypotonicity or by N-ethylmaleimide (NEM) without reduction in nominal surface abundance of KCC1. These data add to evidence supporting the oligomeric state of KCC polypeptides.

Original languageEnglish
Pages (from-to)233-240
Number of pages8
JournalBlood Cells, Molecules, and Diseases
Volume42
Issue number3
DOIs
StatePublished - May 1 2009
Externally publishedYes

    Fingerprint

Keywords

  • Bismaleimidohexane
  • Disuccinimidyl suberate
  • Pentadecafluorooctanoic acid PAGE
  • Potassium chloride cotransport
  • Xenopus oocyte

ASJC Scopus subject areas

  • Molecular Biology
  • Molecular Medicine
  • Hematology
  • Cell Biology

Cite this

Casula, S., Zolotarev, A. S., Stuart-Tilley, A. K., Wilhelm, S., Shmukler, B. E., Brugnara, C., & Alper, S. L. (2009). Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter. Blood Cells, Molecules, and Diseases, 42(3), 233-240. https://doi.org/10.1016/j.bcmd.2009.01.021