Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven Cl- pump activity.
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.
Seki
Akiteru
A
Graduate School of Pharmaceutical Sciences, Laboratory of Biomolecular Systems, Creative Research Initiative Sosei, and Department of Biomolecular Science, Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan.
Miyauchi
Seiji
S
Hayashi
Saori
S
Kikukawa
Takashi
T
Kubo
Megumi
M
Demura
Makoto
M
Ganapathy
Vadivel
V
Kamo
Naoki
N
D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D002713:Chlorine / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D025602:Halorhodopsins / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology* / Q000528:radiation effects; D008027:Light; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D019633:Natronobacterium / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology*; D011829:Radiation Dosage; D011994:Recombinant Proteins / Q000378:metabolism; D014982:Xenopus laevis
D018118:Chloride Channels; D025602:Halorhodopsins; D011994:Recombinant Proteins; D002713:Chlorine