Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter

Blood Cells Mol Dis. 2009 May-Jun;42(3):233-40. doi: 10.1016/j.bcmd.2009.01.021.

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 (86)Rb(+) 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Chromatography, Gel
  • Cross-Linking Reagents / pharmacology*
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Humans
  • Ion Transport / drug effects
  • K Cl- Cotransporters
  • Kidney / cytology
  • Kidney / embryology
  • Mice
  • Microscopy, Fluorescence
  • Molecular Weight
  • Oocytes
  • Protein Structure, Tertiary
  • RNA, Complementary / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / drug effects
  • Rubidium Radioisotopes / metabolism
  • Symporters / chemistry*
  • Symporters / drug effects
  • Xenopus laevis

Substances

  • Cross-Linking Reagents
  • RNA, Complementary
  • Recombinant Fusion Proteins
  • Rubidium Radioisotopes
  • Symporters