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Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl- pump. To characterize the electrophysiological features of the Cl- pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl- pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl-, Br-, I-, NO3-, and SCN-, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl- current via pHR and the light intensity was linear, demonstrating that transport of Cl- is driven by a single-photon reaction and that the steady-state current is proportional to the excited pHR molecule. The current-voltage relationship for pHR-mediated photoinduced currents was also linear between -150 mV and +50 mV. The slope of the line describing the current-voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl- as the substrate for the anion pump activity of pHR was about -400 mV. The value for VR was independent of light intensity, meaning that the VR reflects the intrinsic value of the excited pHR molecule. The value of VR changed significantly for the R123K mutant of pHR. We also show that the Cl- pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl- pump. We have also analyzed the kinetics of voltage-dependent Cl- pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl- transport via pHR is presented.

Halorhodopsin (HR) acts as a light-driven chloride pump which transports a chloride ion from the extracellular (EC) to the cytoplasmic space during a photocycle reaction that includes some photointermediates initiated by illumination. To understand the chloride uptake mechanisms, we focused on a basic residue Arg123 of HR from Natronomonas pharaonis (NpHR), which is the only basic residue located in the EC half ion channel. By the measurements of the visible absorption spectra in the dark and the light-induced inward current through the membrane, it was shown that the chloride binding and transport ability of NpHR completely disappeared by the change of arginine to glutamine. From flashphotolysis analysis, the photocycle of R123Q differed from that of wildtype NpHR completely. The response of the R123H mutant depended on pH. These facts imply that the positive charge at position 123 is essential for chloride binding in the ground state and for the chloride uptake under illumination. On the basis of the molecular structures of HR and the anion-transportable mutants of bacteriorhodopsin, the effects of the positive charge and the conformational change of the Arg123 side chain as well as the chloride-pumping mechanism are discussed.

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