Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H⁺ pumping

Abstract

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

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

“…Similarly, 0.1-to 0.3-unit ⌬pH changes can result in a similar accumulation ratio. Such modest changes in the membrane potential or ⌬pH are consistent with the proton-pumping capacity of PR (27,29), suggesting that illumination leads to increased lactate uptake, increased reduction of electron carriers such as NAD ϩ /NADH and menaquinone/menaquinol, and a need for faster regeneration of oxidized electron carriers (Fig. 7).…”

Theoretical principles underlying optical stimulation of a channelrhodopsin-2 positive pyramidal neuron

“…10A. Nonetheless, we elected to present the majority of our results using the only available quantitative measurement of photosensitive channel density (Nagel et al 1995), albeit from bacteriorhodopsin in Xenopus oocytes.…”

“…The ChR2 model was typically inserted in all compartments of the neuron model, with a uniform ChR2 channel density of 130 m Ϫ2 (Nagel et al 1995). To simulate the effect of nonuniform distributions, ChR2 was distributed either in specific compartments (Table 2), or by distributing its density based on distance from the soma (see Fig.…”

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

“…The voltage-clamp setup was essentially that of Nagel et al (1,2). If not mentioned otherwise, the membrane potential was clamped at Ϫ40 mV (50 Hz signal filtering) in the case of pSRII, pSRII-F86D, and pSRII(F86D)͞pHtrII and at Ϫ20 mV (20 Hz signal filtering) for SRI and SRI͞HtrI.…”

“…In previous work, it has been demonstrated that bacteriorhodopsin (BR) from Halobacterium salinarum could also be functionally expressed into the plasma membrane of oocytes, which allowed the elucidation of the electrogenic characteristics of this proton pump under well-defined voltageclamp conditions (1,2). This work indicated that the other members of the bacterial rhodopsin family, the chloride pump halorhodopsin, and the sensory rhodopsins can also be analyzed by this approach.…”