ABCMdb

ABCB1 p.Lys578Cys

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Publications

PMID: 10581253 [PubMed] Blott EJ et al: "Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein."

No. Sentence Comment

39 Four SC mutants (P549C, V569C, K578C and R580C) were expressed at significantly lower levels than wild-type P-gp, but use of an alternative transfection reagent (lipofectamine) allowed sufficient expression for further analysis. Login to comment
59 Cells were transiently transfected with the relevant cDNA, using lipofectin or lipofectamine (for SC mutants P549C, V569C, K578C and R580C), and 75 µg of whole cell lysate (150 µg for K578C and R580C) was added per lane. Login to comment
190 Oligonucleotides used for site-directed mutagenesis pSC-name Diagnostic restriction site Mutagenic oligonucleotide sequence 5Ј-3Ј N280C ϩAvaI GGTACAACAAATGTCTCGAGGAAGCTAAAAG G324C -AflIII CCTTGGTCTTATCATGTGAATATTCTATTGG S403C ϩPstI CTTCAGTTACCCCTGCAGAAAAGAAGTTAAG S419C ϩEco57I GAACCTGAAAGTGCAGTGTGGGCAGACG Q456C ϩBstEI GTTGATGGATGCGATATCCGGACCATAAATG F465C ϩBanI ATGTAAGGTGCCTACGGGAA I469C ϩNsiI CTACGGGAATGCATTGGTGT S474C ϩHpaII GGTGTCGTGTGTCAGGAACCGGTATTGTTT T482C ϩBsrGI GTATTGTTTGCCTGTACAATAGCTGAAAAC I488C ϩEclXI GCTGAAAACTGTCGCTACGGCCGTGAAAATG Y490C none ACATTCGCTGTGGCCGTGA V495C ϩBsrGI GGCCGTGAAAATTGTACAATGGATGAGATTG D498C -NcoI GTCACCATGTGTGAGATTGAG I500C ϩBsmI GTCACCATGGATGAATGCGAGAAAGCTGTC K502C ϩBsmI GGATGAGATTGAATGCGCTGTCAAGGAAG N508C ϩFspI GTCAAGGAAGCATGCGCATATGACTTTATC D511C ϩAflIII GGAAGCCAACGCGTATTGCTTTATCATG K515C ϩAflIII GAAGCCAACGCGTATGACTTTATCATGTGCCTGCCTCAT K519C ϩStyI GAAACTGCCTCATTGCTTTGACACCTTGGTTGGAGAGAGAG A529C ϩBanI GAGAGAGGGTGCCAGTTGAG K536C ϩBlpI GAGAGAGAGGCGCCCAGTTGAGTGGTGGGCAGTGCCAGAGGATCG R547C ϩBssSI GCACGTGCCCTCGTGTGCAACCCCAAG P549C ϩSspI TGGTTCGCAACTGCAAAATATTCCTGCTGGA L554C ϩSspI CGCAACCCCAAAATATTGCTGTGCGATGAGGCCACG S565C ϩBsmI GACACAGAATGCGAAGCAG V569C ϩBsmI GACACAGAATGCGAAGCAGTGTGTCAGGTGG K578C ϩBsiEI GATAAGGCCAGATGCGGCCGGACCACC R580C -MslI GCCACAAAAGGTTGCACGACCATTGTGATA T581C ϩApaLI GAAAAGGTCGGTGCACCATTGTG E638C ϩEclXI GAAGTTGAATTATGCAATGCGGCCGATGAATC 'ϩ` represents the introduction of a new restriction endonuclease site; '-` represents the removal of an existing site. Login to comment

PMID: 12820887 [PubMed] Gabriel MP et al: "Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508."

No. Sentence Comment

149 Therefore, our investigations concentrated on three mutations located on the R-helical subdomain (I500C, N508C, K578C) and two on the surface of the -sheet ABC-specific subdomain (E393C, S452C). Login to comment
151 Similar yields were obtained for Cys-less (105 ( 23 µg), N508C (121 ( 28 µg), K578C V ) (Vmax[S])/(Km + [S]) (1) V ) Vmin + (Vmax - Vmin)/(1 + 10(logEC50-L) ) (2) B ) Bmax(1 - exp-kt ) (3) FIGURE 1: Model of the N-terminal nucleotide binding subdomain of Pgp. Login to comment
182 To Table 2: The Effects of Cysteine Replacement on Characteristics of ATP Hydrolysisa basal ATPase activity stimulated ATPase activity Pgp isoform n Km (mM) Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) fold stimulation Cys-less 10 0.52 ( 0.05 0.29 ( 0.06 0.37 ( 0.04 0.83 ( 0.12 2.9 ( 0.2 E393C 3 0.39 ( 0.03 0.47 ( 0.25 0.18 ( 0.01 1.41 ( 0.48 3.4 ( 0.8 S452C 4 0.39 ( 0.03 0.49 ( 0.13 0.31 ( 0.04 1.19 ( 0.37 2.4 ( 0.7 I500C 4 0.20 ( 0.03 0.24 ( 0.07 0.24 ( 0.04 0.51 ( 0.21 2.6 ( 0.2 N508C 4 0.54 ( 0.12 0.21 ( 0.09 0.38 ( 0.03 0.52 ( 0.10 3.2 ( 0.5 K578C 4 0.64 ( 0.17 0.31 ( 0.13 0.35 ( 0.05 0.54 ( 0.21 2.0 ( 0.2 a ATPase activity was measured for each mutant isoform of Pgp as described in Materials and Methods. Login to comment
188 Table 3: The Effects of Cysteine Replacement on Drug Stimulation or Inhibition of ATP Hydrolysisa nicardipine vinblastine XR9576 vanadate Pgp isoform EC50 (µM) EC50 (µM) IC50 (µM) IC50 (µM) Cys-less 6.3 ( 0.1 4.6 ( 1.3 0.79 ( 0.27 3.4 ( 1.3 E393C 4.4 ( 1.4 10.8 ( 4.2 0.79 ( 0.16 4.6 ( 1.1 S452C 2.9 ( 0.8 8.2 ( 3.4 0.61 ( 0.24 4.9 ( 0.4 I500C 3.0 ( 0.6 5.4 ( 0.7 0.80 ( 0.19 5.1 ( 0.9 N508C 5.8 ( 0.9 6.2 ( 2.9 0.55 ( 0.15 7.4 ( 1.8 K578C 7.7 ( 0.5 12.4 ( 2.1 0.57 ( 0.11 4.3 ( 0.8 a ATPase activity was measured for each Pgp isoform in the presence of 2 mM Mg‚ATP with varying concentrations (3 nM to 100 µM) of either stimulatory drugs (nicardipine, vinblastine) or inhibitors (XR9576, vanadate). Login to comment
197 None of the mutants E393C (2.3 ( 2.1%), S452C (2.2 ( 1.9%), I500C (1.9 ( 1.2%), nor K578C (3.1 ( 1.1%) displayed significant inhibition of the drug stimulated ATPase activity in the presence of up to 100 µM NEM (data not shown). Login to comment
206 Negligible labeling was observed for the Cys-less and the K578C mutant Pgp isoforms. Login to comment
207 The inability to label mutant K578C correlates with the lack of effect of NEM on the ATPase activity of this mutant Pgp isoform. Login to comment
220 Table 4: The Ability of [3H]-NEM to Label Pgp Isoformsa Pgp isoform fraction labeledc wild type 1.00 ( 0.06 Cys-less 0.08 ( 0.05 E393C n/db S452C n/d I500C 0.29 ( 0.08 N508C 0.33 ( 0.02 K578C 0.03 ( 0.05 a The various purified Pgp (250 ng) isoforms were incubated with 1 µM [3 H]NEM for 1 h at 20 °C and then subjected to SDS-PAGE (8%) and autoradiography to determine the relative accessibilities of the introduced cysteine residues to covalent modification. Login to comment
292 The K578C mutant isoform did not display inhibition of ATPase activity by NEM, and this was attributed to the lack of detectable labeling by this maleimide-containing compound. Login to comment