Further characterization of the sixth transmembrane domain of Pgp1 by site-directed mutagenesis

Citation
Jm. Song et Pw. Melera, Further characterization of the sixth transmembrane domain of Pgp1 by site-directed mutagenesis, CANC CHEMOT, 48(5), 2001, pp. 339-346
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Oncology,"Onconogenesis & Cancer Research
Journal title
CANCER CHEMOTHERAPY AND PHARMACOLOGY
ISSN journal
0344-5704 → ACNP
Volume
48
Issue
5
Year of publication
2001
Pages
339 - 346
Database
ISI
SICI code
0344-5704(200111)48:5<339:FCOTST>2.0.ZU;2-F
Abstract
Purpose: Several studies have identified amino acid residues located on the hydrophobic side of the helix that forms transmembrane domain 6 (TM6) of t he ABC transporter P-glycoprotein (Pgp) as being important for function. Th e purpose of this study was to deter-mine if alterations to residues on the hydrophilic side could also affect function and to determine the extent to which altering the hydrophobic nature of residues on the hydrophobic side would impair the protein. Methods: A full-length cDNA encoding wild-type Pg p1 from CHL cells was used as a template for site-directed mutagenesis. Eig ht different mutations, three on the hydrophilic side and five on the hydro phobic side, were prepared and transfected into drug-sensitive host cells. Wild-type transfectants served as controls. Drug resistance levels, RD50 va lues for cyclosporin A (CsA) and verapamil, iodoarylazidoprazosin (IAAP) ph otolabeling and verapamil-stimulated ATPase activity were evaluated. Result s: Substitution of any one of three amino acid residues on the hydrophilic side of TM6 disrupted function and led to alterations in drug resistance, C sA sensitivity, IAAP photoaffinity labeling, and in one case verapamil-stim ulated ATPase activity. Replacement of a hydrophobic residue on the hydroph obic face of the helix with increasingly hydrophilic side-chains led to fun ctional changes, the extent of which did not correlate with the degree of s ide-chain hydrophilicity. Finally, while the placement of a proline residue along either face of the helix had varying effects on function, in all cas es its presence interfered with verapamil-stimulated ATPase activity. Concl usions: Taken together these results indicate that both faces of TM6 mediat e Pgp1 function and that the expected conformational changes resulting from proline substitutions at a variety of locations within the helix can alter the protein's enzymatic activity.