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

Several studies have identified amino acid residues located on the hydrophobic side of the helix that forms transmembrane domain 6 (TM6) of the ABC transporter P-glycoprotein (Pgp) as being important for function. The purpose of this study was to determine 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. A full-length cDNA encoding wild-type Pgp1 from CHL cells was used as a template for site-directed mutagenesis. Eight different mutations, three on the hydrophilic side and five on the hydrophobic side, were prepared and transfected into drug-sensitive host cells. Wild-type transfectants served as controls. Drug resistance levels, RD50 values for cyclosporin A (CsA) and verapamil, iodoarylazidoprazosin (IAAP) photolabeling and verapamil-stimulated ATPase activity were evaluated. Substitution of any one of three amino acid residues on the hydrophilic side of TM6 disrupted function and led to alterations in drug resistance, CsA sensitivity, IAAP photoaffinity labeling, and in one case verapamil-stimulated ATPase activity. Replacement of a hydrophobic residue on the hydrophobic face of the helix with increasingly hydrophilic side-chains led to functional changes, the extent of which did not correlate with the degree of side-chain hydrophilicity. Finally, while the placement of a proline residue along either face of the helix had varying effects on function, in all cases its presence interfered with verapamil-stimulated ATPase activity. Taken together these results indicate that both faces of TM6 mediate 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.