Heterologous Expression of Pharaonis Halorhodopsin in Xenopus laevis Oocytes and Electrophysiological Characterization of Its Light-Driven Cl− Pump Activity

Abstract

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.

The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

“…The answer is that successive energy barriers encountered during the photocycle can be overcome faster if the starting energy level is higher (because it allows to raise the level of intermediates, thus lowering the relative barriers of escape from these intermediates). Indeed, experiments show that the photocycle turnover rate decreases linearly with an increase in opposing electrostatic membrane potential (6). In other words, hR can pump faster than it would if it had stored only as much as strictly necessary for working against the physiological membrane potential.…”

“…Understanding how this energy is stored thus constitutes an essential aspect of the mechanism and has been the focus of studies on visual rhodopsins (4) and bacteriorhodopsin (5). In the case of hR, the free energy available in the initial metastable state (called the K-state) corresponds to about 10 kcal/mol (6), essentially all from the enthalpic component (7). Interestingly, this is only 1 ⁄ 5 of the photon energy, raising the question of why the protein does not store more of the photon's energy (ϳ50 kcal/photon at 580 nm).…”

Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1

“…Electrophysiological studies were performed with the twoelectrode voltage clamp technique as described previously (17). SLC5A8-expresssing oocytes (kindly provided by Dr. Seiji Miyauchi, Toho University School of Pharmaceutical Sciences) were used as controls.…”

Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment

“…The Hr from Natronomonas pharaonis (NpHr) has been cloned and successfully expressed in heterologous systems such as E. coli, mammalian cells, and Xenopus laevis oocytes (Hohenfeld et al, 1999;Seki et al, 2007;Gradinaru et al, 2008). Expression of NpHr in X. laevis oocytes resulted in a light-dependent Cl 2 -inward current and consequently a negative shift in the membrane potential (Seki et al, 2007).…”