Detailed comparison of expressed and native voltage-gated proton channel currents

Abstract

Two years ago, genes coding for voltage-gated proton channels in humans, mice and Ciona intestinalis were discovered. Transfection of cDNA encoding the human HVCN1 (H(V)1) or mouse (mVSOP) ortholog of HVCN1 into mammalian cells results in currents that are extremely similar to native proton currents, with a subtle, but functionally important, difference. Expressed proton channels exhibit high H(+) selectivity, voltage-dependent gating, strong temperature sensitivity, inhibition by Zn(2+), and gating kinetics similar to native proton currents. Like native channels, expressed proton channels are regulated by pH, with the proton conductance-voltage (g(H)-V) relationship shifting toward more negative voltages when pH(o) is increased or pH(i) is decreased. However, in every (unstimulated) cell studied to date, endogenous proton channels open only positive to the Nernst potential for protons, E(H). Consequently, only outward H(+) currents exist in the steady state. In contrast, when the human or mouse proton channel genes are expressed in HEK-293 or COS-7 cells, sustained inward H(+) currents can be elicited, especially with an inward proton gradient (pH(o) < pH(i)). Inward current is the result of a negative shift in the absolute voltage dependence of gating. The voltage dependence at any given pH(o) and pH(i) is shifted by about -30 mV compared with native H(+) channels. Expressed H(V)1 voltage dependence was insensitive to interventions that promote phosphorylation or dephosphorylation of native phagocyte proton channels, suggesting distinct regulation of expressed channels. Finally, we present additional evidence that speaks against a number of possible mechanisms for the anomalous voltage dependence of expressed H(+) channels.

Figure 1. Measured g_H is altered profoundly by flux and cannot be uniquely determined

A, the g_H–V relationship estimated by three methods at each of three pH_o values in a COS-7 cell transfected with the mouse proton channel gene. The pipette contained pH_i 6.5 with 100 mm BisTris buffer. At pH_o 8.0, 2 s pulses were applied in 5 mV increments every 20 s from V_hold = −100 mV. At pH_o 7.0 or 6.0, V_hold was −60 and −40 mV, respectively, and 6 s pulses were applied every 20 s. As indicated in the inset, g_H was estimated from proton current measured at the end of each pulse (I_end, circles) or at the start of the tail current (I_tail, diamonds) assuming a single constant measured value for V_rev. Alternatively, g_H was calculated as the slope conductance (squares) between I_end and I_tail, which does not require an estimate of V_rev. B, actual V_rev estimated by the X axis intercept of a line connecting I_end and I_tail for the indicated pulses in the same cell at pH_o 8.0.

Figure 3. Relationship between V_threshold and V_rev for H_V1 expressed in HEK-293 or COS-7 cells compared with native proton channels in HEK-293 and other cells

Endogenous proton currents in 5 non-transfected HEK-293 cells () studied in whole-cell configuration with pH_i 6.5, fitted by the red dashed line: V_threshold = 0.71 V_rev + 27 mV. Data for H_V1 at various pH_o values with pH_i 7.5 or 6.5 in whole-cell configuration, or at various pH_i values with pH_o 7.5 in inside-out patches. Dashed lines are drawn by linear regression on the points, according to V_threshold = 0.66 V_rev – 11 mV for pH_i 7.5 (), V_threshold = 0.73 V_rev – 9 mV for pH_i 6.5 (), and V_threshold = 0.67 V_rev – 10 mV for pH_o 7.5 () in inside-out patches. Data are from 21 cells and 8 patches for H_V1. The continuous green line indicates the relationship for native proton currents that includes published data from 15 different types of cells (from DeCoursey, 2003). The continuous black line indicates equality between V_threshold and V_rev; data below this line exhibit inward H⁺ current at V_threshold. HEK-293 or COS-7 cells co-transfected with H_V1 and GFP (), fitted by: V_threshold = 0.66 V_rev – 16 mV. HEK-293 cells expressing low levels of H_V1, ‘tet’ (), fitted by: V_threshold = 0.82 V_rev – 9 mV. HEK-293 cells expressing H_V1 after pretreatment with 10 nm staurosporine (), fitted by: V_threshold = 0.77 V_rev – 14 mV. See text for more details.

Figure 2. Inward proton currents are large at inward ΔpH

Whole-cell currents near V_rev in a HEK-293 cell transfected with H_V1. Pipette pH was 6.5, pH_o was 7.0 (A), 6.5 (B) or 5.5 (C). No leak correction has been applied. Illustrated currents are for pulses in 10 mV increments from −40 to −10 mV (A), −10 to +10 mV (B) or +30 to +60 mV (C), from V_hold −100 mV (A), −50 mV (B) or −20 mV (C). D, chord conductance–voltage (g_H–V) relationships from this cell, calculated by extrapolation of a single exponential fitted to the tail currents to the start of the pulse, using V_rev measured in each solution. E, activation time constants, τ_act, in the same cell, from single exponential fits. For pH_o 6.5 and 7.0, values for τ_act from two different families of pulses were averaged; other data are individual measurements.

Figure 4. The g_H–V relationship is shifted negatively by about 30 mV in expressed proton channels compared with native proton channels

The human proton channel was expressed in HEK-293 cells (downward green triangles), the mouse channel in COS-7 cells (diamonds). Native proton currents are from other studies in THP-1 cells (upward triangles, DeCoursey & Cherny, 1996a), PLB-985 cells (squares, DeCoursey et al. 2001b), human basophils (open circles, unpublished studies by B. Musset, V. V. Cherny, D. Morgan and T. E. DeCoursey), and mean values from 6 to 9 rat alveolar epithelial cells (hexagons, taken from Fig. 6 in Cherny et al. 1995). Proton currents were fitted with single exponential curves extrapolated to infinite time to obtain the current amplitude, and chord conductance was calculated using V_rev measured in each solution, all studied at pH_o 7.0 and pH_i 6.5 in whole-cell configuration. Native g_H was normalized to its value at +80 mV, expressed g_H at +40 mV. Except for alveolar epithelium, data are from individual cells.

Figure 5. Inward proton currents also occur in excised patches

Proton currents in inside-out patches from a COS-7 cell (A) and a HEK-293 cell (B), both transfected with H_V1, and a COS-7 cell transfected with the mouse proton channel mVSOP (C). Pipette and bath solutions were at pH 7.5 for all. Pulses are in 10 mV increments as indicated. In C the arrows indicate the inward current during the pulse to −10 mV as well as the corresponding tail current.