ABCMdb

ABCB1 p.Asn508Cys

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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

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

109 The time course of [3H]-NEM association with the N508C Pgp was determined by incubating protein (250 ng) for 1-240 min (20 °C) with [3H]-NEM (1 µM). Login to comment
111 To determine the effect of nucleotide on accessibility of N508C Pgp to covalent modification the protein (250 ng) was incubated with the non-hydrolysable ATP analogue AMP-PNP (0-10 mM) in buffer comprising 100 mM MOPS pH 6.8 and 5 mM MgCl2. Login to comment
114 Labeling efficiency of the N508C Pgp trapped in a posthydrolytic conformation was tested following a vanadate trapping procedure based on previously published methods (12). Login to comment
118 The photoaffinity labeling of wild type and N508C Pgp was based on previously published methods (12-14). Login to comment
123 To assess the effect of NEM, the wild type or N508C isoforms were incubated with 100 µM NEM, in the dark for 1 h prior to the addition of [R-32 P]-azido-ATP. Login to comment
127 The time-course for [3H]-NEM labeling of the N508C mutant Pgp isoform was fitted by a single phase association equation: where B ) fraction bound; Bmax ) maximal binding; k ) association rate constant (min-1 ); t ) time (min). Login to comment
145 This parent protein is termed Cys-less, and single-cysteine isoforms derived from this are referred to as, for example, N508C, denoting that the asparagine at position 508 has been mutated to cysteine. Login to 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
173 The degree of inhibition varied from 78 ( 3% for S452C to 87 ( 1% for N508C and the IC50 values were in the range 0.55-0.8 µM. Similarly to the stimulatory drugs, the potency for XR9576 to affect ATP hydrolysis was not different in any mutant compared to Cys-less. Login to comment
175 Vanadate produced between 91 ( 3% (N508C) and 99 ( 1% (Cys-less) inhibition of overall ATPase activity. 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
194 Figure 2 shows data depicting the effects of covalent modification on drug stimulated ATPase activity in wild type, Cys-less and N508C Pgp. Login to comment
198 In contrast, NEM was able to inhibit the ATPase activity of N508C characterized by a potency of IC50 ) 15 ( 3 µM. Login to comment
199 These results suggest that either (i) only mutant N508C isoform is accessible to labeling with NEM or (ii) only residue 508C is involved in the catalytic process in Pgp. Login to comment
202 Figure 3a demonstrates representative autoradiograms of the relative labeling of wild-type Pgp and the mutant N508C isoform with varying concentrations of radiolabel (0.01-1 µM). Login to comment
212 Western immunoblotting analysis determined that these proteins were FIGURE 2: The effects of [3H]-NEM labeling on the ATPase activity of wild-type Pgp, cysteine-less Pgp (Cys-less) and the N508C mutant. Login to comment
216 (a) A representative autoradiogram showing the relative labeling of purified wild type and N508C Pgp. Login to comment
219 (b) Purified, reconstituted N508C (250 ng) was incubated with 1 µM [3H]-NEM for indicated times at 20 °C. (c) Quantitation of the Pgp associated band intensity was performed as described in the text and the mean graph obtained from four such experiments is plotted. 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
226 [3 H]-NEM was able to efficiently label mutants I500C and N508C to approximately 30% of that observed for the wild type Pgp (Table 4). Login to comment
229 Figure 3b shows a representative autoradiograph of the time dependent increase in [3 H]-NEM labeling of N508C Pgp. Login to comment
233 The Ability of NEM to Perturb Drug Interaction with N508C-Pgp. Login to comment
234 The data in Figure 2 demonstrated that treatment of mutant N508C with NEM produced a reduction in nicardipine stimulated ATPase activity. Login to comment
236 To address this question the N508C protein was derivitized with 100 µM NEM and the ability of nicardipine, vinblastine, and paclitaxel to stimulate ATP hydrolysis measured. Login to comment
239 This was corroborated by the unaltered extent of N508C labeling by [3H]-NEM (1 µM) in the presence of a fixed 30 µM concentration of nicardipine, vinblastine or paclitaxel (data not shown). Login to comment
240 These results may indicate that the inhibition of ATP hydrolysis by NEM in N508C Pgp occurs during the catalytic cycle itself rather than perturbation of allosteric influences produced by drug binding. Login to comment
248 The initial event in the catalytic cycle is binding of nucleotide and the ability of NEM treatment to affect this parameter in wild type and N508C Pgp is shown in Figure 5. Login to comment
249 The two proteins were labeled with 3 µM [R-32P]- azidoATP, the maximal achievable concentration of this Table 5: The Effects of NEM on the Extent and Potency of Drugs to Alter the ATPase Activity of N508C Pgpa nicardipine vinblastine paclitaxel EC50 (µM) stimulation EC50 (µM) stimulation EC50 (µM) stimulation -NEM 5.3 ( 0.3 3.3 ( 0.1 8.3 ( 1.2 1.6 ( 0.1 5.1 ( 2.1 2.3 ( 0.1 +NEM 6.4 ( 0.8 3.0 ( 0.2 6.9 ( 1.7 1.5 ( 0.1 5.3 ( 2.5 2.2 ( 0.2 a ATP hydrolysis was measured for the N508C Pgp mutant protein (250 ng) in the presence or absence of 100 µM NEM at a range of drug concentrations (3 nM to 100 µM). Login to comment
253 FIGURE 4: The effect of sulfydryl modification on vinblastine stimulated and basal ATPase activity of mutant N508C. Login to comment
254 The basal and drug stimulated ATPase activities were determined for N508C Pgp (250 ng) as described in the experimental procedures at different ATP concentrations (0-2 mM). Login to comment
255 (a) ATPase activity of N508C Pgp in the presence (b) or absence (O) of 30 µM vinblastine. Login to comment
256 (b) ATPase activity of N508C Pgp that had been preincubated with 100 µM NEM for 30 min at 20 °C. Catalytic activities were determined in the presence (9) or absence (0) of 30 µM vinblastine. Login to comment
260 As demonstrated in Figure 5, both the wild type and N508C Pgp isoforms were labeled with the ATP analogue. Login to comment
262 In contrast, no effect was observed for the NEM derivitised N508C mutant isoform which displayed similar intensity of photoaffinity labeling with [R-32P]-azidoATP. Login to comment
263 Therefore, the inhibition of ATP hydrolysis caused by covalent attachment of NEM to the N508C isoform was not due to impaired binding of nucleotide. Login to comment
264 If the NEM derivitization of N508C has an inhibitory effect on ATP hydrolysis, one might expect that trapping of the N508C isoform with ADP and vanadate might exert a negative effect on the ability of NEM to label the protein. Login to comment
267 In contrast, the non-hydrolyzable nucleotide analogue AMP-PNP up to 10 mM did not alter labeling of the nucleotide-binding domain in N508C-Pgp by 1 µM [3H]- NEM (Figure 6a). Login to comment
287 Purified wild type and N508C Pgp isoforms were incubated with 3 µM Mg‚[32P]-azido-ATP for 20 min at 20 °C and then subjected to UV-light for 5 min at 4 °C. The extent of labeling for each isoform was compared with that following preincubation of the protein for 60 min with 100 µM NEM. Login to comment
289 FIGURE 6: The efficiency of N508C labeling by [3H]-NEM during different stages of the catalytic cycle. Login to comment
290 (a) N508C (250 ng) was labeled with [3H]-NEM (1 µM) in the presence of increasing concentrations of AMP-PNP and the inset shows a representative autoradiogram. Login to comment
291 (b) N508C-Pgp was trapped with 2 mM Mg‚ATP and 200 µM vanadate for 20 min at 37 °C. Control (b) and trapped (O) protein was then labeled with varying concentrations of [3H]- NEM and the autoradiograms were analyzed to determine the extent of labeling at each concentration of [3H]-NEM. Login to comment
295 However, the ATPase activity of the N508C mutant isoform was inhibited following covalent attachment of the maleimide-containing probe. Login to comment
297 Therefore, a more detailed examination of individual stages of the catalytic cycle was undertaken to provide a molecular mechanism for the nature of NEM induced inhibition of the N508C isoform. Login to comment
299 On first inspection, derivitization of the N508C protein with NEM caused a large decrease in the maximal velocity of drug stimulated ATP hydrolysis. Login to comment
307 In addition, albeit at low concentrations of nucleotide, the extent of [R32 P]-8-azido-ATP labeling of N508C Pgp was unaffected by prior interaction with NEM. Login to comment
309 The demonstration that the non-hydrolyzable ATP analogue AMP-PNP did not impair labeling of N508C suggests that this residue does not become buried upon ATP binding, despite the considerable conformational changes the NBD undergoes (49-51). Login to comment
310 The vanadate trapping technique (17) provides a useful tool to determine whether the inhibitory effect of NEM on the N508C Pgp isoform occurs immediately posthydrolysis of the γ-phosphate group. Login to comment
311 The extent to which N508C Pgp was covalently labeled by [3H]-NEM was significantly impaired following vanadate trapping of the protein. Login to comment
313 By inference, the prior covalent attachment of NEM to N508C would be expected to prevent or retard the previously reported conformational changes immediately following ATP hydrolysis (12, 13, 52). Login to comment