ABCMdb

ABCB1 p.Gly324Cys

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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

36 Two additional single-cysteine (SC) mutants (N280C and G324C), known to be located in an intracellular and extracellular loop of TMD1, respectively (Loo and Clarke, 1995), were also generated as controls. Login to comment
38 P-gp-cys- and 21 of the 30 SC mutants (including the control mutants SC-N280C and SC-G324C) expressed full-length protein in both the lower molecular weight non-glycosylated and the higher molecular weight glycosylated forms (Figure 2). Login to comment
66 To establish the validity of the assay, SC-P-gp mutants G324C (known to be exposed in an extracellular loop of TMD1) and N280C (known to be located in an intracellular loop of TMD1) were used as positive and negative controls, respectively. Login to comment
67 In intact cells, the higher molecular weight, glycosylated P-gp-G324C was biotinylated while neither form of P-gp- N280C was biotinylated (Figure 4A), as expected. Login to comment
69 In this case, both the mature and immature (nonglycosylated) forms of G324C and N280C P-gp were labelled equivalently (Figure 4B): immature P-gp is likely to be confined to intracellular membranes and so accessible to BM only after permeabilization of the cell. 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: 11598005 [PubMed] Rosenberg MF et al: "Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle."

No. Sentence Comment

138 As expected, the drug-stimulated ATPase activities of cysteine-less P-gp and the control (G324C, which has a single cysteine in an extracellular loop; Blott et al., 1999) were not signi®cantly affected by either reagent. Login to comment

PMID: 15192095 [PubMed] Rothnie A et al: "The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein."

No. Sentence Comment

149 Mutant G324C, which contains a cysteine in an accessible extracellular loop, was used as a positive control for labeling. Login to comment
153 Nonspecific labeling of Cys-less P-gp by FM was 4 Ϯ 1% (Fig. 2a), whereas G324C labeled to 100% with a half-life of 12 Ϯ 1 min (data not shown). Login to comment
155 In contrast, F343C in the native protein conformation was accessible to labeling with FM as adjudged by the 81 Ϯ 2% labeling extent (Lext), which was characterized by a half-life (t1/2 ϭ 47 Ϯ 8 min) almost 4 times slower than observed for G324C. Login to comment
158 The level of nonspecific interaction with the Cys-less P-gp was higher (Lext ϭ 25 Ϯ 4%), and G324C was also labeled by CM (Lext ϭ 102 Ϯ 4%), although the half-life (t1/2 ϭ 31 Ϯ 4 min) was almost 3 times slower than observed for FM. Login to comment
162 Nonspecific interaction with Cys-less protein was 19 Ϯ 1% and G324C labeled fully (Lext ϭ 97 Ϯ 3%) with a half-life approximately double (t1/2 ϭ 23 Ϯ 2 min) that obtained for FM. Login to comment

PMID: 18303860 [PubMed] Storm J et al: "Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling."

No. Sentence Comment

115 The positive control lane refers to labeling of the G324C isoform for 300 min and was assigned a value of 100%. Login to comment
137 The "+ve" lane refers to labeling of the G324C isoform for 300 min (b) The SDS-PAGE data was quantified using densitometric analysis, and the values were expressed as a percentage of that obtained for the G324C isoform. Login to comment

PMID: 20731718 [PubMed] Crowley E et al: "Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1."

No. Sentence Comment

47 Labelling was time dependent during the 300 min incubation, and the extents of labelling were quantified in comparison with that found with cysteine-less ABCB1 and the G324C isoform. Login to comment
48 The G324C mutant was assigned as the positive control and given a value of 100%, as this residue is located on an external loop and is freely accessible to each of the probes used [25,28]. Login to comment
49 Furthermore, the complete labelling of the G324C mutant with the zwitterionic and hydrophilic probes BODIPY maleimide (BM) and fluorescein maleimide (FM), respectively, demonstrated that the protein was not preferentially oriented in one direction within the proteoliposomes. Login to comment
50 Consequently, labelling of TM12 isoforms was determined as a percentage of G324C labelling, as outlined in Experimental procedures. Login to comment
74 Lane (vii) contains the G324C isoform, which has been assigned a 100% value for labelling with BM. Login to comment
103 These were then expressed as a percentage of the maximal extent of G324C labelling. Login to comment
104 The degree of labelling (% of G324C level) was plotted as a function of time (min) and fitted with an exponential reaction curve, using nonlinear least squares regression. Login to comment
136 The Lext for labelling is expressed as the fraction of specific labelling of single-cysteine isoforms over the specific labelling of the G324C positive control. Login to comment
257 The G324C mutation, located on a freely accessible extracellular loop, has previously been demonstrated to be freely accessible to each maleimide probe [28]; labelling of the isoform containing this mutation was therefore assigned the value of 100% after 300 min. Login to comment
258 Both the cysteine-less and G324C isoforms were incubated with 10 lm probe for 300 min and treated identically to the other isoforms. Login to comment
259 The extent of labelling for each single-cysteine mutant was therefore determined by comparison with G324C. Login to comment
261 The propensity for labelling was calculated with the following equation: L ¼ Liso À Lcys À Á L324C À Lcys À Á 100 where L is extent of labelling (%), Liso is the extent of isoform labelling, Lcys is labelling of the cysteine-less isoform, and L324C is labelling of the G324C isoform. Login to comment