Purified Human MDR 1 Modulates Membrane Potential in Reconstituted Proteoliposomes

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Abstract
Human multidrug resistance (hu MDR 1) cDNA was fused to a P. shermanii transcarboxylase biotin acceptor domain (TCBD), and the fusion protein was heterologously overexpressed at high yield in K+-uptake deficient Saccharomyces cerevisiae yeast strain 9.3, purified by avidin−biotin chromatography, and reconstituted into proteoliposomes (PLs) formed with Escherichia coli lipid. As measured by pH- dependent ATPase activity, purified, reconstituted, biotinylated MDR−TCBD protein is fully functional. Dodecyl maltoside proved to be the most effective detergent for the membrane solubilization of MDR−TCBD, and various salts were found to significantly affect reconstitution into PLs. After extensive analysis, we find that purified reconstituted MDR−TCBD protein does not catalyze measurable H+ pumping in the presence of ATP. In the presence of physiologic [ATP], K+/Na+ diffusion potentials monitored by either anionic oxonol or cationic carbocyanine are easily established upon addition of valinomycin to either control or MDR−TCBD PLs. However, in the absence of ATP, although control PLs still maintain easily measurable K+/Na+ diffusion potentials upon addition of valinomycin, MDR−TCBD PLs do not. Dissipation of potential by MDR−TCBD is clearly [ATP] dependent and also appears to be Cl- dependent, since replacing Cl- with equimolar glutamate restores the ability of MDR−TCBD PLs to form a membrane potential in the absence of physiologic [ATP]. The data are difficult to reconcile with models that might propose ATP-catalyzed “pumping” of the fluorescent probes we use and are more consistent with electrically passive anion transport via MDR−TCBD protein, but only at low [ATP]. These observations may help to resolve the confusing array of data related to putative ion transport by hu MDR 1 protein.

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