TY - JOUR
T1 - Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter
AU - Casula, Sabina
AU - Zolotarev, Alexander S.
AU - Stuart-Tilley, Alan K.
AU - Wilhelm, Sabine
AU - Shmukler, Boris E.
AU - Brugnara, Carlo
AU - Alper, Seth L.
N1 - Funding Information:
This work was supported by NIH grants HL15157 (CB and SLA) and DK61051 (SLA). Aspects of this article were presented in the 2008 Red Cell Conference held at the University of Rochester, New York, in memory of Philip Knauf.
PY - 2009/5
Y1 - 2009/5
N2 - 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.
AB - 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.
KW - Bismaleimidohexane
KW - Disuccinimidyl suberate
KW - Pentadecafluorooctanoic acid PAGE
KW - Potassium chloride cotransport
KW - Xenopus oocyte
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U2 - 10.1016/j.bcmd.2009.01.021
DO - 10.1016/j.bcmd.2009.01.021
M3 - Article
C2 - 19380103
AN - SCOPUS:64649102228
VL - 42
SP - 233
EP - 240
JO - Blood Cells, Molecules, and Diseases
JF - Blood Cells, Molecules, and Diseases
SN - 1079-9796
IS - 3
ER -