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 Citations (Scopus)

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

Oocytes
HEK293 Cells
Peptides
Ethylmaleimide
Xenopus
Detergents
Amines
Gel Chromatography
Tail
Proteins
Molecular Weight
potassium-chloride symporters

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

Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter. / Casula, Sabina; Zolotarev, Alexander S.; Stuart-Tilley, Alan K.; Wilhelm, Sabine; Shmukler, Boris E.; Brugnara, Carlo; Alper, Seth L.

In: Blood Cells, Molecules, and Diseases, Vol. 42, No. 3, 01.05.2009, p. 233-240.

Research output: Contribution to journalArticle

Casula, S, Zolotarev, AS, Stuart-Tilley, AK, Wilhelm, S, Shmukler, BE, Brugnara, C & Alper, SL 2009, 'Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter', Blood Cells, Molecules, and Diseases, vol. 42, no. 3, pp. 233-240. https://doi.org/10.1016/j.bcmd.2009.01.021
Casula S, Zolotarev AS, Stuart-Tilley AK, Wilhelm S, Shmukler BE, Brugnara C et al. Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter. Blood Cells, Molecules, and Diseases. 2009 May 1;42(3):233-240. https://doi.org/10.1016/j.bcmd.2009.01.021
Casula, Sabina ; Zolotarev, Alexander S. ; Stuart-Tilley, Alan K. ; Wilhelm, Sabine ; Shmukler, Boris E. ; Brugnara, Carlo ; Alper, Seth L. / Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter. In: Blood Cells, Molecules, and Diseases. 2009 ; Vol. 42, No. 3. pp. 233-240.
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