| A yeast optimized synthetic Pfmdr1 gene has been designed and constructed, and has been successfully over expressed PfMDR1 in yeast. The protein in both full length and half transporter form is well localized to the yeast plasma membrane, and is fully functional as evidenced by ATPase activity. We have expressed various drug resistance related isoforms and have assessed antimalarial drug effects on them using ATPase activity measurements. In relation to other member of the B subfamily of ATP binding cassette transporters, PfMDR1 has similar pH, [ATP] and Mg ++ dependencies but surprisingly much higher Km and V max for ATP hydrolysis. Drug resistance related isoforms show either elevated (N86Y; Dd2, D1246Y) or reduced (S1034C, S1034C/N1042D/D1246Y; TM, 184F/1034C/1042D/1246Y; 7G8) basal ATPase activity and different patterns of drug stimulation or inhibition relative to wild-type. Verapamil (1--300 muM) which stimulates P-gp ATPase activity does not stimulate PfMDR1. To decipher the low ATPase activity of the 7G8 mutant isoform various C-terminal mutants were constructed. Analysis of the ATPase activity of the C-terminal mutants provide evidence that the S1034C mutation contributes to the low basal ATPase activity of the 7G8 PfMDR1 isoforms this effect is reduced in the presence of other mutations. Wild-type PfMDR1 has also been purified and reconstituted into proteoliposomes which have drug stimulatable ATPase activity. It is now believed that PfMDR1 does not cause chloroquine resistance (CQR) in and of itself, but modulates CQR caused by PfCRT mutations. To this end, a yeast model system harboring both PfMDR1 and PfCRT has been devised which can be used to provide valuable information on the combined roles of these two major anti-malarial drug transporters. |
