ABCMdb

PMID: 14749322

Loo TW, Bartlett MC, Clarke DM
Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein.
J Biol Chem. 2004 Apr 30;279(18):18232-8. Epub 2004 Jan 28., 2004-04-30 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB1 p.Ala935Cys Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 °C, when thermal motion is reduced. Login to comment 84 ABCB1 p.Gly774Cys We recently showed that two halves of P-gp are close together at N296C(TM5)/ G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C (TM8), and G300C(TM5)/F770C(TM8), suggesting that the packing of the two halves shows rotational symmetry (i.e. head-to-tail arrangement) (31). Login to comment 100 ABCB1 p.Ala935Cys When cross-linking was carried out at 22 °C, only mutants V133C(TM2)/G939C(TM11), C137C(TM2)/A935C (TM11), and L138C(TM2)/A935C(TM11) formed detectable cross-linked products. Login to comment 106 ABCB1 p.Ala935Cys At 4 °C, time-dependent cross-linking was observed in mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), but not in mutant L138C(TM2)/A935C(TM11) even after 32 min (Fig. 3). Login to comment 115 ABCB1 p.Gly939Cys Disulfide cross-linking of P-gp mutants. Membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2), G939C(TM11), C137C(TM2), A935C(TM11), V133C(TM2)/G939C (TM11), or C137C(TM2)/A935C(TM11). Login to comment 119 ABCB1 p.Leu138Cys FIG. 3. Effect of temperature on cross-linking of mutants. Membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), or L138C- (TM2)/A935C(TM11). Login to comment 124 ABCB1 p.Gly939Cys ABCB1 p.Phe942Cys ABCB1 p.Ile131Cys ABCB1 p.Ser134Cys ABCB1 p.Ala935Cys ABCB1 p.Phe938Cys ABCB1 p.Ser943Cys ABCB1 p.Leu138Cys ABCB1 p.His936Cys ABCB1 p.Gln132Cys ABCB1 p.Ile940Cys ABCB1 p.Tyr130Cys ABCB1 p.Ile937Cys ABCB1 p.Trp136Cys ABCB1 p.Thr941Cys ABCB1 p.Phe944Cys ABCB1 p.Ala128Cys ABCB1 p.Ala129Cys TABLE I Cross-linking between residues in TM2 and TM11 -, no cross-linked product detected on SDS-polyacrylamide gels at 37 °C; ϩ, relatively weak cross-linking (Ͻ50% of P-gp cross-linked) at 37 °C; ϩϩ, relatively strong cross-linking (Ͼ50% of P-gp cross-linked) at 37 °C; *, cross-linked product also detected at 22 °C; **, cross-linked product also detected at 22 and 4 °C. TM2 TM11 A935C H936C I937C F938C G939C I940C T941C F942C S943C F944C A128C - - - - - - - - - - A129C - - - - - - - - - - Y130C - - - - ϩ ϩ - ϩ ϩ - I131C - - - - - - - - - - Q132C - - - - - - - - - - V133C - - - ϩ ϩϩ, ** - - ϩ ϩ - S134C ϩ ϩ - - ϩ ϩ - - - - F135C - - - - - - - - - - W136C - - - - - - - - - - C137C ϩϩ, ** - - - ϩ - - - - - L138C ϩϩ, * - - - - - - - - - TM11 are close to each other at their cytoplasmic ends. Login to comment 176 ABCB1 p.Asn296Cys A, membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2)/G939C(TM11), C137C(TM2)/A935C(TM11), or N296C- (TM5)/G774C(TM8). Login to comment