Chemical crosslinking studies with the mouse Kcc1 K–Cl cotransporter

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• 作者：Sabina Casula ; Alex ; er S. Zolotarev ; Alan K. Stuart-Tilley ; Sabine Wilhelm ; Boris E. Shmukler ; Carlo Brugnara ; Seth L. Alper
• 关键词：Potassium chloride cotransport ; Disuccinimidyl suberate ; Bismaleimidohexane ; Xenopus oocyte ; Pentadecafluorooctanoic acid PAGE
• 刊名：Blood Cells, Molecules, and Diseases
• 出版年：2009
• 出版时间：May-June 2009
• 年：2009
• 卷：42
• 期：3
• 页码：233-240
• 全文大小：660 K

文摘

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.