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Science 312 (5773): 589-592

Copyright © 2006 by the American Association for the Advancement of Science

A Voltage Sensor-Domain Protein Is a Voltage-Gated Proton Channel

Mari Sasaki1,2,3, Masahiro Takagi1,3, and Yasushi Okamura1,2,3,4*

1 Section of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.
2 The Graduate University for Advanced Studies, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.
3 National Institute for Physiological Sciences, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan.
4 Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.


Figure 1 Fig. 1.. Depolarization-activated whole-cell current induced by mVSOP. (A) A family of current traces recorded from an mVSOP-transfected tsA201 cell under voltage clamp in the whole-cell patch configuration. Pulses stepped by 3 s were applied in 10-mV increments ranging from –30 mV to 100 mV from a holding potential of –60 mV. NMDG solutions were used as patch and bath solutions. pHin/pHout = 7.0/7.0. (B) The current-voltage relationship of the current traces shown in (A). [View Larger Version of this Image (8K GIF file)]
 

Figure 2 Fig. 2.. Evidence that VSOP-induced currents are proton selective. (A) Plots of tail current amplitude against membrane potential under various pH conditions. Reversal potentials were determined from the intercept of the current-voltage relationship of tail currents. (B) Comparison of the reversal potentials obtained from tail currents with proton equilibrium potentials (EH) predicted from Nernst equation. The solid line indicates linear fitting of reversal potentials against {Delta}pH (56.0 mV/{Delta}pH). The dashed line shows EH calculated by the Nernst equation (59.3 mV/{Delta}pH). Junction potentials ranging up to 4 mV were corrected in the plot. In the normal solution, a mixture of sodium, potassium, and calcium was substituted for NMDG. (C) Ratiometric fluorescence measurements with pH-sensitive dye of pHin in mVSOP-transfected cells [beads (+), red] and nontransfected cells [beads (–), blue]. (D) Differences of pHin before and after depolarization were quantified. pHin immediately after intracellular acidification by NH4Cl [time 0 in (C)] and that at 10 min after the start of perfusion [arrow in (C)] of high-potassium solution were measured. Transfection (–) denotes results from cells without transfection (n = 14). Beads (+) denotes results from transfected cells (n = 25). Beads (–) denotes cells that did not express CD8 in the same dish for beads (+) cells (n = 18). [View Larger Version of this Image (29K GIF file)]
 

Figure 3 Fig. 3.. pH-dependent gating and inhibition by divalent metal cations of mVSOP-induced currents. (A) The current-voltage relationships evoked by a series of voltage steps in 10-mV increments (–80 to 100 mV) under pHin = 6.5 and pHout = 7.0, 6.5, or 6.1. The pulse duration was 500 ms. Currents were measured from the same sets of cells. Current amplitudes at the end of the depolarizing pulse obtained under each condition of pHout were normalized by those at 20 mV recorded under pHout = 7.0 for individual cells. (B) Voltage dependence and pH dependence of the time required for half-maximal activation. Maximal current was measured as the amplitude at the end of depolarizing pulse. The pulse duration was 500 ms. Representative current traces for (A) and (B) are shown in fig. S2. Averaged values ± SE are shown (n = 9) in (A) and (B). (C) Dose-response curves of inhibition by zinc and cadmium ions. Small circles are plots from individual cells, and squares denote average values. The holding potential was –80 mV. (D) Tissue distributions of VSOP mRNA examined by real-time RT-PCR. The expression level of VSOP mRNA was normalized by expression in the spleen. L8 ribosomal protein was used as internal control. [View Larger Version of this Image (28K GIF file)]
 

Figure 4 Fig. 4.. Mutation in the S4-like segment alters the voltage dependence of channel gating. (A) Current traces recorded from cells expressing wild-type mVSOP (wt) (left) or R201Q (right). Traces with depolarizing steps (–20 to 100 mV) are superimposed. pHin/pHout = 7.0/7.0. The pulse protocol is shown in fig. S2. (B) Voltage dependency of conductance is plotted for wt (n = 5; circles) and R201Q (n = 9; squares) and fitted by the Boltzmann equation Formula where k is the Boltzmann constant, e is the elementary electric charge, T is temperature, and z is the valence. V1/2 values are 63.7 ± 7.6 mV and 14.8 ± 8.0 mV, and z values are 1.4 ± 0.15 and 1.9 ± 0.25 for wt and R201Q, respectively. Error bars indicate SD. (C) Current traces under pHin/pHout = 7.0/6.1 for wt (left) and R201Q (right). Traces with depolarizing steps (20 to 90 mV) are superimposed. In R201Q, tail currents are scaled out in this magnification. (D) The current-voltage relationships for wt (n = 8) and R201Q mutant (n = 12) in pHin/pHout = 7.0/6.1. Averaged current densities at the end of depolarization pulses are plotted. Inward current is evident for R201Q. In [(A) to (D)], the holding potential was –60 mV. Error bars indicate SD. [View Larger Version of this Image (22K GIF file)]
 


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