Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter.
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.
Casula
Sabina
S
Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, USA.
Zolotarev
Alexander S
AS
Stuart-Tilley
Alan K
AK
Wilhelm
Sabine
S
Shmukler
Boris E
BE
Brugnara
Carlo
C
Alper
Seth L
SL
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051379:Mice; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009865:Oocytes; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects; D012414:Rubidium Radioisotopes / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000187:drug effects; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
D003432:Cross-Linking Reagents; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D012414:Rubidium Radioisotopes; D027981:Symporters; D000096922:K Cl- Cotransporters