Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium

Abstract

The voltage-activated H+ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D2O, for water, H2O, on both the conductance and the pH dependence of gating were explored. D+ was able to permeate proton channels, but with a conductance only about 50% that of H+. The conductance in D2O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D+ than H+), suggesting that D+ interacts specifically with the channel during permeation. Evidently the H+ or D+ current is not diffusion limited, and the H+ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H+ (or D+) and not OH- is the ionic species carrying current. The voltage dependence of H- channel gating characteristically is sensitive to pH0 and pHi and was regulated by pD0 and pDi in an analogous manner. shifting 40 mV/U change in the pD gradient. The time constant of H+ current activation was about three times slower (T(act) was larger) in D2O than in H2O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H+ channel activation requires deprotonation of the channel. In contrast, deactivation (T(tail)) was slowed only by a factor < or = 1.5 in D2O. The results are interpreted within the context of a model for the regulation of H+ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861-896). Most of the kinetic effects of D2O can be explained if the pKa of the external regulatory site is approximately 0.5 pH U higher in D2O.

Figure 1

The solvent in the bath replaces the solvent in the cell. Measurement of the tail current reversal potential, V _rev, is illustrated in a cell studied with a pipette solution containing D₂O at pD 7.0. The bath solution was D₂O at pD 7.0 (A), or H₂O at pH 7.0 (B) or pH 6.5 (C). (D) Schematic diagram of a didactic experiment in which the pipette solution is D₂O at pD 7.0 and the bath contains H₂O at pH 7.0 (or pH 6.5). The solvent in the bath permeates the membrane and fills the cell faster than the pipette solvent diffuses into the cell. Buffered pipette solution enters the cell, but H₂O replaces D₂O, and the effective pH_i is 0.5 U lower than was the original pD of the pipette solution. In the table the composition of the bath and pipette solutions, and the presumed effective composition of the solution in the cell is given, and a comparison of observed V _rev values with the Nernst potential, E _L. E _L was calculated assuming that the bath solvent fills the cell and that the pK _a of all buffers is 0.5 U higher in D₂O than in H₂O (see text for details). (E) Instantaneous current-voltage relationships for the measurements in parts A (▪), B (⋄), and C (▿). The amplitude of a single exponential fitted by eye to the tail current at each voltage is plotted. V _rev was determined by interpolation.

Figure 2

Reversal potentials, V _rev, measured in bilateral D₂O. V _rev was estimated from tail currents as illustrated in Fig. 1 A–C, and E. Symbols indicate mean ± SD of 2–12 measurements (48 in all) with pD_i 6.0 (♦), pD_i 7.0 (▪), or pD_i 8.0 (▴), and pD_o 6–10. The dark line shows the Nernst potential.

Figure 3

The effect of pD_o on D⁺ currents is similar to the effect of pH_o on H⁺ currents. Families of currents are shown for a cell studied with pD_i 6.0, and pD_o 8.0 (A), pD_o 7.0 (B), and pD_o 6.0 (C). Calibration bars apply to all three families. The holding potential, V _hold, was −60 mV (A) or −40 mV (B and C). Illustrated currents are for pulses applied in 20-mV increments to −40 through +40 mV (A), 0 to +80 mV (B), and +40 to +120 mV (C). Filter, 100 Hz.

Figure 4

Dependence of the current-voltage relationship on pD_o. The current amplitude measured at the end of the 8-s pulses illustrated in Fig. 3 A–C is plotted, without leak correction (solid symbols). Also plotted is the amplitude of a single exponential (Eq. 2, Fig. 5) fitted to the same currents (open symbols). This idealized amplitude is increased over the raw value when the currents did not reach steady-state during the pulses but reduced by the leak correction inherent in this procedure.

Figure 5

Time constant of activation, τ_act, measured in the same cell as in Figs. 3 and 4, at pD_o 8, 7, and 6, with pD_i 6.0. Inset shows the fit of a single exponential after a delay to the D⁺ current (shown as points) at +20 mV at pD_o 8//pD_i 6 in this cell. The amplitude was 135 pA, τ_act was 1.17 s, and the delay was 116 ms.

Figure 6

D⁺ currents in an inside-out patch, illustrating the effects of changes in pD_i. The pipette contained pD 8.0 solution, and the bath either pD 6.0 (A) or pD 7.0 (B). Superimposed in A are currents during two runs: from V _hold = −60 to −40 mV through +20 mV in 20-mV increments, and from V _hold = −70 to −50 mV through +30 mV in 20-mV increments, as indicated. (B) Currents in the same patch at pD_o 8//pD_i 7. V _hold was −40 mV, and pulses were to −20 mV through +60 mV in 20-mV increments, as indicated. Filter, 20 Hz.

Figure 7

Appearance of whole-cell D⁺ currents at different pD_i with constant pD_o 7.5. (A) The NH₄ ⁺ gradient was 1//50 and V _rev was −66 mV. From V _hold = −60 mV pulses were applied in 20-mV increments at 30-s intervals to −40 through +40 mV. (B) In the same cell at a 15//50 NH₄ ⁺ gradient, V _rev was −27 mV. From V _hold = −40 mV, pulses were applied to 0 through +80 mV. Calibration bars apply to both families.

Figure 8

Families of currents in the same cell at pH_o 6.5 (A) and pD_o 7.0 (B). The pipette contained pD 7.0 solution, and we assume that with H₂O in the bath the membrane in A effectively “sees” pH_o 6.5//pH_i,eff 6.5. In both parts, pulses were applied from V _hold = −40 mV in 20-mV increments up to +100 mV. The observed V _rev in this cell was −1 mV (A) and +3 mV (B). Filter, 100 Hz; families recorded 66 and 80 min after achieving whole-cell configuration.

Figure 9

Ratio of the extrapolated or “steady-state” current amplitude in H₂O to that in D₂O in the same cell at the corresponding pL. Symbols show the mean ± SEM of the ratio of pD_o 8 to pH_o 7.5 in 3–5 cells (▴), pD_o 7 to pH_o 6.5 in 8–10 cells (▪), and pD_o 6 to pH_o 5.5 in 4 cells (♦). Corresponding τ_act ratios from the same data set are plotted in Fig. 13. The steady-state current amplitude was obtained by extrapolation of a single exponential (after a delay) fitted to the outward currents (Eq. 2). The effective pL_i in each case is 0.5 U higher in D₂O than in H₂O (see Practical Considerations). The only significant differences between mean values were at +80 mV and +100 mV at pD_o 6 vs. pD_o 8 (P < 0.05).

Figure 10

Steady-state voltage-gated conductance in D₂O (filled symbols) or H₂O (open symbols) in various NH₄ ⁺ gradients in the same cell at pL_o 7.5. The NH₄ ⁺ gradient was 15//50 mM (squares), 3//50 mM (diamonds), and 1//50 mM (triangles). The lines show the average limiting slope of 4.65 mV/e-fold change in conductance (see text). The chord conductance was calculated using V _rev measured in each solution. Currents recorded during voltage pulses 4–16 s long were fitted with a single rising exponential after a delay (Eq. 2). The amplitude of the exponential component is plotted. Longer pulses were used in D₂O and for small depolarizations above threshold, where τ_act was larger. Measurements were made in some solutions two or three different times during the experiment. The average V _rev (of 1–3 determinations) in H₂O and D₂O, respectively, at each NH₄ ⁺ gradient were: 15//50 (−32 mV, −27 mV), 3//50 (−65 mV, −58 mV), and 1//50 (−78 mV, −67 mV). The protons in 50 mM NH₄ ⁺ contaminate the D₂O by only ∼0.2%.

Figure 11

The potential at which clearly time-dependent outward current was detected, V _threshold, is plotted as a function of V _rev measured in the same cell and the same solution. Only data in which V _threshold was examined using voltage increments of 5 mV or less are included. Open symbols indicate measurements with H₂O in the bath; filled symbols indicate measurements with D₂O. Pipette solutions are indicated by the shape of the symbol: pD 7.0 (▵), pD 8.0 (▿), pD 9.0 (open hexagons), pH 7.5 (⋄), pH 5.5 (○), 50 mM NH₄ ⁺ (□). The data with H₂O in the bath are considered to be essentially symmetrical H₂O (see Strategic Considerations), and those with D₂O in the bath symmetrical D₂O. The lines show the results of linear regression of the H₂O data (solid line), r = 0.963, slope = 0.760, y-intercept = 18.1 mV. The D₂O data (dashed line) were described by r = 0.926, slope = 0.750, y-intercept = 22.0 mV. Dotted line shows V _threshold = V _rev illustrating that V _threshold is positive to V _rev over the entire physiological range.

Figure 13

Slowing of activation by D₂O is illustrated as the ratio of τ_act measured in D₂O to that measured in the same cell in H₂O at effectively symmetrical pL. The effective pL in each case is 0.5 U higher in D₂O than in H₂O (see Practical Considerations). Symbols show the mean ± SEM of the ratio of pD_o 8 to pH_o 7.5 (▴), pD_o 7 to pH_o 6.5 (▪), and pD_o 6 to pH 5.5 (♦). Fitting procedure and numbers of cells are given in Fig. 9 legend. Solid symbols are from experiments with D₂O pipette solutions; 4 cells studied at pD_o 7 and pH_o 6.5 with pH_i 6.5 (H₂O) in the pipette are also plotted (□). Data points are connected by lines and a reference line at a ratio of 1.0 is also plotted. There was no significant difference between mean ratios at different pD at any potential.

Figure 12

Average time constant of activation, τ_act, in H₂O (open symbols) and D₂O (solid symbols). Symbols show the mean ± SEM at pD 8//pD 8 in 5–7 cells (▴), pH_o 7.5//pH_i,eff 7.5 in 5 cells (▵), pD_o 7//pD 7 in 9–12 cells (▪), pH_o 6.5//pH_i,eff 6.5 in 7–9 cells (□), pD_o 6//pD 6 in 3–6 cells (♦), and pH_o 5.5//pH_i,eff 5.5 in 4 cells (⋄).

Figure 14

Tail current time constant, τ_tail at effectively symmetrical pH (open symbols) or pD (solid symbols). Symbols indicate pD_o 8.0//pD_i 8.0 (▴), pH_o 7.5//pH_i,eff 7.5 (▵), pD_o 7.0//pD_i 7.0 (▪), pH_o 6.5//pH_i,eff 6.5 (□), pD_o 6.0//pD_i 6.0 (♦), or pH_o 5.5// pH_i,eff 5.5 (⋄). Plotted is the mean ± SEM of τ_tail obtained by fitting the decay of the tail current with a single exponential (Eq. 3). Means are for 4–10 cells for each condition, with fewer measurements at some potentials.

Figure 15

H⁺ and D⁺ currents in a cell-attached patch. During 16-s depolarizing pulses, there are slowly increasing outward currents, which we interpret as H⁺ currents. In both parts V _hold was −60 mV relative to the membrane potential, and pulses were applied to −40 mV through +100 mV in 20-mV increments. The bath contained KMeSO₃ solution, intended to clamp the membrane potential to near 0 mV, and the pipette contained pD 8.0 solution. (B). When the bath was changed to D₂O instead of H₂O, the outward currents were much smaller and, if anything, even slower to activate. The H⁺ currents are small, consistent with a small patch area and the membrane being near the pipette tip (cf. DeCoursey and Cherny, 1995). The completeness of exchange of solvent near the membrane cannot be determined, but the altered behavior when D₂O in the bath replaced H₂O suggests that the solvent near the membrane was changed substantially.