ABCMdb

PMID: 10681495

Loo TW, Clarke DM
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis.
J Biol Chem. 2000 Feb 25;275(8):5253-6., 2000-02-25 [PubMed]

Sentences

No. Mutations Sentence Comment

35 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys If the TM segments of P-gp possess enough flexibility to accommodate substrates of varying sizes, then it was reasonable to assume that residues in other TMs could be cross-linked to P350C or S993C. Login to comment 51 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Printed in U.S.A. This paper is available on line at http://www.jbc.org atUniversityofNorthCarolinaatChapelHill,onSeptember29,2011www.jbc.orgDownloadedfrom ation was that P350C (TM6) and S993C (TM12) are predicted to be close to the cytoplasmic side of the membrane. Login to comment 53 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Accordingly, mutants were constructed that had either P350C (TM6) and a cysteine residue introduced into predicted TMs 7 to 11, or between S993C (TM12) and a cysteine introduced into predicted TMs 1 to 5. Login to comment 58 ABCB1 p.Ser993Cys ABCB1 p.Gly54Cys All of the mutants, except mutant G54C/S993C, had greater than 70% of the activity of Cys-less P-gp. Login to comment 59 ABCB1 p.Ser993Cys ABCB1 p.Gly54Cys The level of expression of mutant G54C/S993C was adequate for cross-linking analysis, but was too low for measurement of ATPase activity. Login to comment 64 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys No cross-linked product was detected between S993C (TM12) and cysteines introduced into TMs 1, 2, or 3 or between P350C (TM6) and cysteines introduced into TMs 7, 8, or 9 (Table I). Login to comment 65 ABCB1 p.Glu875Cys ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ala233Cys ABCB1 p.Ile299Cys ABCB1 p.Leu236Cys ABCB1 p.Met876Cys ABCB1 p.Trp232Cys ABCB1 p.Leu227Cys ABCB1 p.Val874Cys ABCB1 p.Ile235Cys ABCB1 p.Ala295Cys Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant. Login to comment 66 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys A representative result (mutant G939C/P350C) is shown in Fig. 1A. Login to comment 74 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Leu227Cys ABCB1 p.Val874Cys Table II shows the minimum temperature required for cross-linking was 4 °C for mutants A233C(TM4)/ S993C(TM12), I235C(TM4)/S993C(TM12), L236C(TM4)/ S993C(TM12), and I299C(TM5)/S993(TM12); 21 °C for mutants V231C(TM4)/S993C(TM12), W232C(TM4)/S993C(TM12), A295C(TM5)/S993C(TM12), V874C (TM10)/P350C (TM6), M876C(TM10)/P350C(TM6), and G939C(TM11)/P350C(TM6); and 37 °C for mutants L227C (TM4)/S993C/(TM12) and E875C(TM10)/P350C(TM6). Login to comment 77 ABCB1 p.Glu875Cys ABCB1 p.Gly939Cys ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Gly54Cys ABCB1 p.Ala233Cys ABCB1 p.Ile299Cys ABCB1 p.Leu236Cys ABCB1 p.Met876Cys ABCB1 p.Trp232Cys ABCB1 p.Leu227Cys ABCB1 p.Val874Cys ABCB1 p.Ile235Cys ABCB1 p.Ala295Cys ABCB1 p.Ile131Cys ABCB1 p.Thr837Cys ABCB1 p.Asp188Cys ABCB1 p.Ala230Cys ABCB1 p.Ser134Cys ABCB1 p.Arg832Cys ABCB1 p.Phe194Cys ABCB1 p.Ile60Cys ABCB1 p.Phe711Cys ABCB1 p.Ala935Cys ABCB1 p.Ala718Cys ABCB1 p.Val715Cys ABCB1 p.Ile840Cys ABCB1 p.Ala302Cys ABCB1 p.Val52Cys ABCB1 p.Gly774Cys ABCB1 p.Ala834Cys ABCB1 p.Phe938Cys ABCB1 p.Phe775Cys ABCB1 p.Leu879Cys ABCB1 p.Met878Cys ABCB1 p.Ala58Cys ABCB1 p.Ser943Cys ABCB1 p.Val835Cys ABCB1 p.Ser831Cys ABCB1 p.Lys234Cys ABCB1 p.Thr294Cys ABCB1 p.Glu782Cys ABCB1 p.Gln946Cys ABCB1 p.Ile829Cys ABCB1 p.Gln773Cys ABCB1 p.Phe770Cys ABCB1 p.Leu138Cys ABCB1 p.Met192Cys ABCB1 p.Ala828Cys ABCB1 p.Gln195Cys ABCB1 p.Ile836Cys ABCB1 p.Leu772Cys ABCB1 p.Thr776Cys ABCB1 p.His936Cys ABCB1 p.Asn839Cys ABCB1 p.Ile297Cys ABCB1 p.Gln132Cys ABCB1 p.Gly141Cys ABCB1 p.Leu56Cys ABCB1 p.Lys189Cys ABCB1 p.Ala869Cys ABCB1 p.Lys877Cys ABCB1 p.Ala139Cys ABCB1 p.Phe303Cys ABCB1 p.Gly714Cys ABCB1 p.Asn721Cys ABCB1 p.Gly722Cys ABCB1 p.Gly226Cys ABCB1 p.Ile719Cys ABCB1 p.Ile940Cys ABCB1 p.Leu304Cys ABCB1 p.Ile867Cys ABCB1 p.Phe771Cys ABCB1 p.Met51Cys ABCB1 p.Ala140Cys ABCB1 p.Ile868Cys ABCB1 p.Gly778Cys ABCB1 p.Ile720Cys ABCB1 p.Ala780Cys ABCB1 p.Tyr130Cys ABCB1 p.Ile937Cys ABCB1 p.Phe193Cys ABCB1 p.Ala301Cys ABCB1 p.Ser298Cys ABCB1 p.Val53Cys ABCB1 p.Asn296Cys ABCB1 p.Ser237Cys ABCB1 p.Trp136Cys ABCB1 p.Gly191Cys ABCB1 p.Gly185Cys ABCB1 p.Ile783Cys ABCB1 p.Ala57Cys ABCB1 p.Val713Cys ABCB1 p.Gly872Cys ABCB1 p.Ser228Cys ABCB1 p.Gly781Cys ABCB1 p.Gln838Cys ABCB1 p.Thr941Cys ABCB1 p.Leu833Cys ABCB1 p.Gly723Cys ABCB1 p.Phe777Cys ABCB1 p.Lys779Cys ABCB1 p.Phe716Cys ABCB1 p.Ile293Cys ABCB1 p.Phe944Cys ABCB1 p.Ala229Cys ABCB1 p.Gly830Cys ABCB1 p.Ala871Cys ABCB1 p.Ile186Cys ABCB1 p.Ile870Cys ABCB1 p.Val873Cys ABCB1 p.Ala947Cys ABCB1 p.Ile190Cys ABCB1 p.Val712Cys In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment 79 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys A representative result (mutant G939C/P350C) is shown in Fig. 1C. Login to comment 82 ABCB1 p.Glu875Cys Cyclosporin A inhibited the cross-linking of mutants L227C(TM4)/S993C(TM12), W232C(TM4)/ S993C(TM12), I299C(TM5)/S993(TM12), E875C (TM10)/ P350C(TM6), and M876C(TM10)/P350C(TM6). Login to comment 86 ABCB1 p.Ser993Cys Vinblastine enhanced the cross-linking of mutant L227C(TM4)/S993C(TM12) and inhibited that of mutants I299C(TM5)/S993C (TM12), E875C(TM10)/P350C(TM6), and G939C(TM11)/P350C(TM6). Login to comment 99 ABCB1 p.Glu875Cys ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ala233Cys ABCB1 p.Ile299Cys ABCB1 p.Leu236Cys ABCB1 p.Met876Cys ABCB1 p.Trp232Cys ABCB1 p.Leu227Cys ABCB1 p.Val874Cys ABCB1 p.Ile235Cys ABCB1 p.Ala295Cys Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively. Login to comment 110 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys Oxidative cross-linking of P-gp mutant G939C/P350C. Login to comment 111 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys A, membranes from HEK 293 cells transfected with vector alone (Control), mutant G939C/P350C, or Cys-less (C-less) P-gp cDNA were treated for 10 min at 37 °C in the presence (ϩ) or absence (-) of 2 mM Cu2ϩ (phenanthroline)3. Login to comment 115 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys C, membranes from HEK 293 cells expressing mutant G939C/P350C were treated with 0.2 mM Cu2ϩ (phenanthroline)3 in the presence of no drug (None), 1 mM colchicine (Colch), 0.15 mM verapamil (Ver), 10 ␮M cyclosporin A (Cyclo) or 35 ␮M vinblastine (Vin) or in the presence of no addition (None), 5 mM ATP (ATP), 5 mM ATP and 0.1 mM vanadate (ATP/VO4) or 0.1 mM vanadate (VO4). Login to comment 118 ABCB1 p.Glu875Cys ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ser993Cys ABCB1 p.Ala233Cys ABCB1 p.Ile299Cys ABCB1 p.Leu236Cys ABCB1 p.Met876Cys ABCB1 p.Trp232Cys ABCB1 p.Leu227Cys ABCB1 p.Val874Cys ABCB1 p.Ile235Cys ABCB1 p.Ala295Cys TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE. Login to comment