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The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of approximately 5 ms (tau1) and approximately 20 ms (tau2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau3 approximately 300 ms at neutral pH is observed. The fast processes (tau1, tau2) can be assigned to the decay of M2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau3 is due to the decay of M1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M1 and the M2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.

We report on the first successful expression of the light driven H ÷ pump, bacteriorhodopsin, into the plasma membrane of oocytes from Xenopus laevis. The light induced photocurrents which reflect the pumping of H ÷ by BR were analysed under voltage clamp conditions. At least 100 active BR molecules per/~m 2 were expressed in the plasma membrane so that both the voltage clamp and giant patch clump method could be applied. We show that H + pumping by BR is modulated by the membrane potential, i.e. the pump current shows strong voltage dependence in the range measured between -165 mV to +60 mV.Key words: Bacteriorhodopsin; H+-pump; Voltage dependence; Xenopus oocyte cedure to achieve this requirement reproducibly has been reported. To circumvent diffÉculties of mixed orientation and to measure the photocurrent elicited by the pump with electrophysiological methods, we expressed BR into the plasma membrane of oocytes of Xenopus laevis. We report here two methods to study the electrical properties of the pump: (a) the usual voltage clamp and (b) the so-called giant patch clamp method [12,13]. Under voltage clamp conditions the pump current induced by BR in the whole cell was studied. The giant patch clamp method allows to analyse the pump under cell-free conditions which give optimal control of the electrolyte on both sides of the oocyte plasma membrane. Materials and methods

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