Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1).
P-glycoprotein (P-gp) is an ABC transporter that exports many amphipathic or hydrophobic compounds, including chemically and functionally dissimilar anticancer drugs, from cells. To understand the role of transmembrane helices (TMH) 1 and 7 in drug-binding and transport, we selected six residues fro...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC6161881?pdf=render |
| _version_ | 1811192997314822144 |
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| author | Andaleeb Sajid Sabrina Lusvarghi Eduardo E Chufan Suresh V Ambudkar |
| author_facet | Andaleeb Sajid Sabrina Lusvarghi Eduardo E Chufan Suresh V Ambudkar |
| author_sort | Andaleeb Sajid |
| collection | DOAJ |
| description | P-glycoprotein (P-gp) is an ABC transporter that exports many amphipathic or hydrophobic compounds, including chemically and functionally dissimilar anticancer drugs, from cells. To understand the role of transmembrane helices (TMH) 1 and 7 in drug-binding and transport, we selected six residues from both TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732); and substituted them with alanine by gene synthesis to generate a variant termed "TMH1,7 mutant P-gp". The expression and function of TMH1,7 mutant P-gp with twelve mutations was characterized using the BacMam baculovirus-HeLa cell expression system. The expression and conformation of TMH1,7 mutant P-gp was not altered by the introduction of the twelve mutations, as confirmed by using the human P-gp-specific antibodies UIC2, MRK16 and 4E3. We tested 25 fluorescently-labeled substrates and found that only three substrates, NBD-cyclosporine A, Rhod-2-AM and X-Rhod-1-AM were transported by the TMH1,7 mutant. The basal ATPase activity of TMH1,7 mutant P-gp was lower (40-50%) compared to wild-type (WT) P-gp, despite similar level of expression. Although most of the substrates modulate ATPase activity of P-gp, the activity of TMH1,7 mutant transporter was not significantly modulated by any of the tested substrates. Docking of selected substrates in homology models showed comparable docking scores for the TMH1,7 mutant and WT P-gp, although the binding conformations were different. Both the ATPase assay and in silico docking analyses suggest that the interactions with residues in the drug-binding pocket are altered as a consequence of the mutations. We demonstrate that it is possible to generate a variant of P-gp with a loss of broad substrate specificity and propose that TMH1 and TMH7 play a critical role in the drug efflux function of this multidrug transporter. |
| first_indexed | 2024-04-12T00:00:00Z |
| format | Article |
| id | doaj.art-d9cc898ba5ea49af912fd029622038b0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T00:00:00Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d9cc898ba5ea49af912fd029622038b02022-12-22T03:56:15ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-01139e020469310.1371/journal.pone.0204693Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1).Andaleeb SajidSabrina LusvarghiEduardo E ChufanSuresh V AmbudkarP-glycoprotein (P-gp) is an ABC transporter that exports many amphipathic or hydrophobic compounds, including chemically and functionally dissimilar anticancer drugs, from cells. To understand the role of transmembrane helices (TMH) 1 and 7 in drug-binding and transport, we selected six residues from both TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732); and substituted them with alanine by gene synthesis to generate a variant termed "TMH1,7 mutant P-gp". The expression and function of TMH1,7 mutant P-gp with twelve mutations was characterized using the BacMam baculovirus-HeLa cell expression system. The expression and conformation of TMH1,7 mutant P-gp was not altered by the introduction of the twelve mutations, as confirmed by using the human P-gp-specific antibodies UIC2, MRK16 and 4E3. We tested 25 fluorescently-labeled substrates and found that only three substrates, NBD-cyclosporine A, Rhod-2-AM and X-Rhod-1-AM were transported by the TMH1,7 mutant. The basal ATPase activity of TMH1,7 mutant P-gp was lower (40-50%) compared to wild-type (WT) P-gp, despite similar level of expression. Although most of the substrates modulate ATPase activity of P-gp, the activity of TMH1,7 mutant transporter was not significantly modulated by any of the tested substrates. Docking of selected substrates in homology models showed comparable docking scores for the TMH1,7 mutant and WT P-gp, although the binding conformations were different. Both the ATPase assay and in silico docking analyses suggest that the interactions with residues in the drug-binding pocket are altered as a consequence of the mutations. We demonstrate that it is possible to generate a variant of P-gp with a loss of broad substrate specificity and propose that TMH1 and TMH7 play a critical role in the drug efflux function of this multidrug transporter.http://europepmc.org/articles/PMC6161881?pdf=render |
| spellingShingle | Andaleeb Sajid Sabrina Lusvarghi Eduardo E Chufan Suresh V Ambudkar Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). PLoS ONE |
| title | Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). |
| title_full | Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). |
| title_fullStr | Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). |
| title_full_unstemmed | Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). |
| title_short | Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1). |
| title_sort | evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human p glycoprotein abcb1 |
| url | http://europepmc.org/articles/PMC6161881?pdf=render |
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