Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB − ) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB − was shown to be taken up by proteoliposomes. The PCB − uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca 2+ . It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing ( cis -side) compartment negatively and the trans -side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans -side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans -side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans -side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans -side solution. Through these pores, H + (pumped by bacteriorhodopsin from the cis -side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans -side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.