PMID: 9405384

Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PubMed]
Sentences
No. Mutations Sentence Comment
21 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:21:88
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:21:62
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:21:72
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:21:95
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:21:106
status: NEW
view ABCB1 p.Ala985Cys details
We show that the drug-stimulated ATPase activities of mutants L339C and A342C (TM6) and L975C, V982C, and A985C (TM12) were particularly sensitive to inhibition by dBBn and that the inhibition was prevented by various drug substrates. Login to comment
62 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:62:23
status: NEW
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The activity of mutant S344C was not determined (ND) due to our inability to express enough enzyme. Login to comment
65 ABCB1 p.Asn91Ala
X
ABCB1 p.Asn91Ala 9405384:65:96
status: NEW
view ABCB1 p.Asn91Ala details
ABCB1 p.Asn94Ala
X
ABCB1 p.Asn94Ala 9405384:65:101
status: NEW
view ABCB1 p.Asn94Ala details
ABCB1 p.Asn99Ala
X
ABCB1 p.Asn99Ala 9405384:65:106
status: NEW
view ABCB1 p.Asn99Ala details
B, whole cell extracts of HEK 293 cells expressing wild-type, Cys-less, glycosylation-deficient N91A/N94A/N99A and single Cys mutants that exhibited little or no verapamil-stimulated ATPase activity were subjected to immunoblot analysis as described under "Experimental Procedures." Login to comment
67 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:67:11
status: NEW
view ABCB1 p.Ser344Cys details
For mutant S344C, three times the normal amount of lysate was loaded onto the gel. Login to comment
81 ABCB1 p.Phe335Cys
X
ABCB1 p.Phe335Cys 9405384:81:12
status: NEW
view ABCB1 p.Phe335Cys details
ABCB1 p.Phe978Cys
X
ABCB1 p.Phe978Cys 9405384:81:97
status: NEW
view ABCB1 p.Phe978Cys details
One mutant, F335C (TM6) showed enhanced activity (280%), whereas the equivalent residue in TM12 (F978C) showed decreased activity (31%). Login to comment
83 ABCB1 p.Gly341Cys
X
ABCB1 p.Gly341Cys 9405384:83:53
status: NEW
view ABCB1 p.Gly341Cys details
ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 9405384:83:101
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 9405384:83:94
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly989Cys
X
ABCB1 p.Gly989Cys 9405384:83:115
status: NEW
view ABCB1 p.Gly989Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:83:87
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Gly984Cys
X
ABCB1 p.Gly984Cys 9405384:83:64
status: NEW
view ABCB1 p.Gly984Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:83:108
status: NEW
view ABCB1 p.Ala985Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:83:46
status: NEW
view ABCB1 p.Ser344Cys details
ABCB1 p.Gln990Cys
X
ABCB1 p.Gln990Cys 9405384:83:126
status: NEW
view ABCB1 p.Gln990Cys details
There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%). Login to comment
86 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 9405384:86:43
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly989Cys
X
ABCB1 p.Gly989Cys 9405384:86:57
status: NEW
view ABCB1 p.Gly989Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:86:50
status: NEW
view ABCB1 p.Ala985Cys details
ABCB1 p.Gln990Cys
X
ABCB1 p.Gln990Cys 9405384:86:68
status: NEW
view ABCB1 p.Gln990Cys details
A similar pattern was observed for mutants G346C, A985C, G989C, and Q990C, suggesting that the low ATPase activity in these mutants was not due to a processing defect. Login to comment
87 ABCB1 p.Gly341Cys
X
ABCB1 p.Gly341Cys 9405384:87:8
status: NEW
view ABCB1 p.Gly341Cys details
ABCB1 p.Gly984Cys
X
ABCB1 p.Gly984Cys 9405384:87:18
status: NEW
view ABCB1 p.Gly984Cys details
Mutants G341C and G984C, however, appeared to be degraded quite rapidly. Login to comment
89 ABCB1 p.Gln347Cys
X
ABCB1 p.Gln347Cys 9405384:89:18
status: NEW
view ABCB1 p.Gln347Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:89:8
status: NEW
view ABCB1 p.Ala342Cys details
Mutants A342C and Q347C also showed enhanced degradation in the absence of cyclosporin A, with the 120-kDa protein as the major product. Login to comment
90 ABCB1 p.Asn91Ala
X
ABCB1 p.Asn91Ala 9405384:90:162
status: NEW
view ABCB1 p.Asn91Ala details
ABCB1 p.Asn94Ala
X
ABCB1 p.Asn94Ala 9405384:90:167
status: NEW
view ABCB1 p.Asn94Ala details
ABCB1 p.Asn99Ala
X
ABCB1 p.Asn99Ala 9405384:90:172
status: NEW
view ABCB1 p.Asn99Ala details
This appeared to be a degradation product rather than a nonglycosylated product because it had a higher mobility than the glycosylation-deficient P-glycoprotein (N91A/N94A/N99A) (Fig. 2B, lanes 23 and 24). Login to comment
92 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:92:7
status: NEW
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Mutant S344C consistently yielded very low levels of immunoreactive P-glycoprotein in the presence or the absence of cyclosporin A (Fig. 2B, lanes 13 and 14). Login to comment
96 ABCB1 p.Gly341Cys
X
ABCB1 p.Gly341Cys 9405384:96:8
status: NEW
view ABCB1 p.Gly341Cys details
ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 9405384:96:22
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly989Cys
X
ABCB1 p.Gly989Cys 9405384:96:40
status: NEW
view ABCB1 p.Gly989Cys details
ABCB1 p.Gly984Cys
X
ABCB1 p.Gly984Cys 9405384:96:29
status: NEW
view ABCB1 p.Gly984Cys details
ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:96:15
status: NEW
view ABCB1 p.Ser344Cys details
Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B). Login to comment
98 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:98:35
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:98:21
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:98:28
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:98:42
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:98:53
status: NEW
view ABCB1 p.Ala985Cys details
In contrast, mutants L339C, A342C, L975C, V982C, and A985C were significantly inhibited by dBBn, because they retained only 10, 40, 13, 25, and 32% of their activities, respectively. Login to comment
99 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:99:96
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:99:89
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:99:121
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:99:103
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:99:110
status: NEW
view ABCB1 p.Ala985Cys details
The concentration of dBBn required to give 50% inhibition of ATPase activity for mutants L339C, L975C, V982C, A985C, and A342C were 90, 112, 320, 480, and 700 ␮M, respectively. Login to comment
106 ABCB1 p.Ser344Cys
X
ABCB1 p.Ser344Cys 9405384:106:37
status: NEW
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Inhibition of the activity of mutant S344C was not determined (ND) due to low expression, and mutants indicated by asterisks were not assayed due to proteolytic digestion. Login to comment
107 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 9405384:107:26
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly989Cys
X
ABCB1 p.Gly989Cys 9405384:107:36
status: NEW
view ABCB1 p.Gly989Cys details
The inhibition of mutants G346C and G989C were not determined (ND) due to their low activities. Login to comment
111 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:111:65
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:111:51
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:111:58
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:111:72
status: NEW
view ABCB1 p.Val982Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:111:83
status: NEW
view ABCB1 p.Ala985Cys details
The P-glycoproteins(His)10 of Cys-less and mutants L339C, A342C, L975C, V982C, and A985C were mixed with lipid and then preincubated for 15 min at 4 °C without drug or in the presence of 2 mM verapamil (Ver. Login to comment
114 ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:114:8
status: NEW
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ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:114:18
status: NEW
view ABCB1 p.Ala985Cys details
Mutants A342C and A985C were preincubated with verapamil only. Login to comment
121 ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:121:44
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:121:54
status: NEW
view ABCB1 p.Ala985Cys details
Due to the low ATPase activities of mutants A342C and A985C, their protection assays were done only in the presence of verapamil. Login to comment
123 ABCB1 p.Ala342Cys
X
ABCB1 p.Ala342Cys 9405384:123:28
status: NEW
view ABCB1 p.Ala342Cys details
ABCB1 p.Ala985Cys
X
ABCB1 p.Ala985Cys 9405384:123:38
status: NEW
view ABCB1 p.Ala985Cys details
As shown in Fig. 4, mutants A342C and A985C were protected from dBBn inactivation by verapamil. Login to comment
124 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:124:26
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:124:19
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:124:37
status: NEW
view ABCB1 p.Val982Cys details
Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates. Login to comment
126 ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:126:56
status: NEW
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Colchicine was also very effective in protecting mutant L339C from dBBn inactivation because it retained about 80% of its colchicine-stimulated ATPase activity. Login to comment
127 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:127:58
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:127:68
status: NEW
view ABCB1 p.Val982Cys details
More modest protection by colchicine was seen for mutants L975C and V982C. Login to comment
129 ABCB1 p.Leu975Cys
X
ABCB1 p.Leu975Cys 9405384:129:100
status: NEW
view ABCB1 p.Leu975Cys details
ABCB1 p.Leu339Cys
X
ABCB1 p.Leu339Cys 9405384:129:90
status: NEW
view ABCB1 p.Leu339Cys details
ABCB1 p.Val982Cys
X
ABCB1 p.Val982Cys 9405384:129:46
status: NEW
view ABCB1 p.Val982Cys details
It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C. Login to comment
141 ABCB1 p.Gly346Cys
X
ABCB1 p.Gly346Cys 9405384:141:44
status: NEW
view ABCB1 p.Gly346Cys details
ABCB1 p.Gly989Cys
X
ABCB1 p.Gly989Cys 9405384:141:50
status: NEW
view ABCB1 p.Gly989Cys details
ABCB1 p.Pro350Cys
X
ABCB1 p.Pro350Cys 9405384:141:61
status: NEW
view ABCB1 p.Pro350Cys details
ABCB1 p.Ser993Cys
X
ABCB1 p.Ser993Cys 9405384:141:67
status: NEW
view ABCB1 p.Ser993Cys details
ABCB1 p.Phe343Cys
X
ABCB1 p.Phe343Cys 9405384:141:31
status: NEW
view ABCB1 p.Phe343Cys details
ABCB1 p.Met986Cys
X
ABCB1 p.Met986Cys 9405384:141:37
status: NEW
view ABCB1 p.Met986Cys details
Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment