Claydon TW , Vaid M , Rezazadeh S , Kwan DC , Kehl SJ , Fedida D .

	Figure 1. Low pH reduces the conductance of Shaker channels. (A) G-V relations of Shaker Δ6–46 channels recorded with the indicated external pH (n = 3). Conductance was calculated from currents recorded during 100-ms voltage pulses applied from −80 to +100 mV at 10-s intervals in 10-mV increments (holding potential −80 mV). (B–E) Typical ionic currents (B and C) and fluorescence signals (D and E) recorded from Shaker A359C channels in response to 100-ms voltage pulses applied from −80 to +100 mV at 10-s intervals (holding potential −80 mV) at pH 7.5 and pH 4.0. Although pulses were applied in 10-mV increments, only selected pulses are shown for clarity. Current and fluorescence traces at pH 4.0 (C and E) are shown at more depolarized potentials than in B and D to account for the charge screening effect of protons. Contributions of the fast and slow phases to the overall fluorescence signal were measured from biexponential fits of the fluorescence traces at +60 mV for pH 7.5 and at +100 mV for pH 4.0. Arrows and percentages in D and E show the contribution of the slow phase at pH 7.5 and pH 4.0. The mean slow phase contribution was 14 ± 2% at pH 7.5 and 71 ± 3% at pH 4.0 (n = 8).
	Figure 2. Acidic pH does not alter total gating charge movement in Shaker channels. (A) Typical wild-type Shaker Δ6–46 (Shaker A359) gating current records obtained from tsa201 cells in the whole-cell patch clamp configuration during 20-ms voltage-clamp pulses from −80 to +60 mV (in 10-mV increments) from a holding potential of −80 mV. Currents during every other pulse are shown for clarity and only the first 6 ms of the depolarizing pulses are shown to highlight the on-gating current. (B) Typical integrals of on-gating currents, such as those in A, recorded in the same cell under control conditions (pH 7.5) and at pH 4.0. Acidic pH did not alter the total gating charge movement in these channels. (C) Mean Q-V relationships recorded at pH 7.5 and pH 4.0 (n = 2–4). At pH 4.0, the Q-V relation was shifted by ∼+35 mV from −27.9 ± 0.4 mV at pH 7.5 to +5.9 ± 1.2 mV.
	Figure 3. Acidic pH enhances the inactivation report from Shaker A359C. Typical ionic currents (A) and fluorescence signals (B) recorded from Shaker A359C channels during 7-s voltage-clamp pulses to +60 mV at the indicated external pH (holding potential of −80 mV). (C) Typical ionic currents and fluorescence signals recorded at pH 7.5 and pH 4.0 from TMRM-labeled channels in which a cysteine residue was not engineered at position A359 (Shaker A359). These channels lack an externally labelable cysteine residue (the only external cysteine, in the S1–S2 linker [C245], has been removed) and act as a control demonstrating the lack of a voltage-dependent fluorescence deflection in the absence of an introduced cysteine and also the pH independence of emission of the TMRM dye.
	Figure 4. Voltage sensor detection of pH-induced enhancement of inactivation is site specific. Typical fluorescence signals from TMRM attached at different sites in the S4 voltage sensor and S3–S4 linker from M356C to R362C recorded during a 7-s pulse to +60 mV at pH 7.5 and pH 4.0 (holding potential −80 mV). Robust fluorescence deflections were recorded from each site in the scan with the exception of I360C and L361C, from which we were unable to record voltage-dependent fluorescence signals. Only A359C and R362C report the pH-induced enhancement of inactivation.
	Figure 5. Two effects of acidic pH are detected from TMRM attached within the pore. Typical ionic currents (A) and fluorescence signals (B) recorded from Shaker S424C channels during 42-s voltage-clamp pulses to +60 mV at the indicated external pH (holding potential −80 mV). Acidic pH decreased peak and accelerated inactivation of the ionic current, and this was detected in the fluorescence signals as a decrease in the peak fluorescence amplitude and an enhancement of the slow phase of the remaining fluorescence deflection. (C) Shaker S424C channel fluorescence deflections from B scaled to the maximum signal amplitude to highlight the pH-induced enhancement of the slow phase of fluorescence deflection.
	Figure 6. Acidic pH decreases open probability without altering single channel current amplitude. Typical recordings of single channel events obtained from outside-out patches of ltk− cells expressing Shaker Δ6–46 channels during consecutive 500-ms voltage-clamp pulses to +100 mV (pulse interval 15 s) from a holding potential of −80 mV (the voltage protocol is shown). In this example the patch contains a single channel. Channel activity from the same patch was recorded during control conditions (pH 7.4) and at pH 4.0. Dashed lines mark the zero current level. Acidic pH markedly reduced channel availability (calculated by determining the proportion of sweeps showing channel activity during the first 50 ms of the pulse) from 0.87 at pH 7.4 to 0.14 at pH 4.0 without effect on the single channel current amplitude. At +100 mV, the single channel current amplitude was 2.7 ± 0.3 pA at pH 7.4 and 2.7 ± 0.3 pA at pH 4.0 (data were collected from 11 patches at pH 7.4 and 5 patches at pH 4.0). The 84% reduction in channel availability (from 0.87 to 0.14) was greater than the reduction of macroscopic conductance measured from Shaker channels expressed in Xenopus oocytes (Fig. 1), but similar to the decrease in peak macroscopic current measured from Shaker channels expressed in mammalian cells at pH 4.0 (91 ± 8%; n = 5). This difference between expression systems is most likely due to incomplete exchange of solution over the entirety of the large invaginated oocyte membrane.
	Figure 7. The loss of fluorescence at low pH is similar to the reduction of peak current amplitude. (A) G-V relations of Shaker S424C channels recorded with the indicated external pH (n = 3). Using Eq. 1 (the intracellular K+ concentration was assumed to be 99 mM), conductance was calculated from currents recorded during 100-ms voltage pulses applied from −80 to +100 mV at 10-s intervals in 10-mV increments (holding potential −80 mV). (B) F-V relations of the Shaker S424C channels recorded at the indicated pH during 42-s pulses from the same group of oocytes as A (n = 3). All fluorescence amplitudes were normalized to the fluorescence amplitude at +100 mV with pH 7.5. Lines represent no mathematical significance and are simply to guide the eye. The loss of fluorescence at low pH was similar to the decrease in conductance at all potentials. (C) Plot of the dependence on the external pH of Shaker S424C channel fluorescence amplitude at +60 mV or conductance at +60 mV (n = 2–7). Fluorescence and conductance values were normalized to those at pH 7.5. Conductance values were calculated from peak currents recorded at the same time as fluorescence deflections during 42-s pulses so as to directly compare the decrease in conductance with the loss of fluorescence from simultaneous measurements from the same oocyte. Data were fitted with a standard Hill equation (assuming a Hill coefficient, n, of 1). pKa values were 5.1 and 5.2 for the loss of fluorescence and reduction of conductance, respectively.
	Figure 8. Low pH enhances P-type inactivation. Typical ionic currents and fluorescence signals recorded at pH 7.5 (A) and pH 5.0 (B) from Shaker S424C channels during a 2-s test pulse to +60 mV applied either 200 ms, 12 s, or 42 s after a 5-s conditioning pulse to +60 mV (holding potential −80 mV). Similar records were obtained from five other oocytes.
	Figure 9. Low pH enhances C-type inactivation. (A) Typical fluorescence signals recorded from the same cell expressing Shaker S424C W434F channels during 7-s depolarizing pulses to +60 mV (applied after at least 3 min at the holding potential of −80 mV) at pH 7.5, pH 5.0, and on return to pH 7.5. Oocytes were held at −80 mV for 3 min between measurements. The W434F mutation permanently P-type inactivates channels enabling observation of C-type inactivation rearrangements during depolarizing pulses. The amplitude of the decaying component of fluorescence was approximately twofold larger at pH 5.0 than at pH 7.5. The dotted lines highlight the extent of the decay. The decay of fluorescence was biexponential with τ values of 21 ± 1 ms and 1.8 ± 0.1 s at pH 7.5, and 21 ± 1 ms and 0.36 ± 0.03 s at pH 5.0 (n = 3; P < 0.001, paired t test, when compared with pH 7.5). (B) Plot of the voltage dependence of the amplitude of the decaying component of fluorescence that reflects C-type inactivation (n = 3). The amplitude of the decaying component was measured at each potential and normalized to that at +100 mV (ΔF decay). Also plotted is the G-V relationship of Shaker A359C channels and F-V relationship of Shaker A359C W434F channels for comparison of the voltage dependence of pore opening and voltage sensor movement, respectively (n = 5–21).
	Figure 10. Inhibition of inactivation by raising external K+ rescues the loss of fluorescence at low pH. (A–F) Typical fluorescence signals recorded from the same oocyte expressing Shaker S424C channels during 42-s depolarizing pulses to +60 mV during the indicated manipulations of the external K+ and proton concentration. After control fluorescence recordings were obtained with 3 mM K+ and pH 7.5 (A), the pH was reduced to pH 5.0 (with 3 mM K+) to demonstrate the decrease of fluorescence (B). Fluorescence signals were then recorded on return to pH 7.5 with 3 mM K+ (C) before application of solution with 99 mM K+ at pH 7.5 (D). The fluorescence was then measured with 99 mM K+ and pH 5.0 (E), before the oocyte was washed with control solution (3 mM K+ and pH 7.5) again (F). Similar records were obtained from three other oocytes.
	Figure 11. Inhibition of inactivation by the mutation T449V rescues the loss of fluorescence at low pH. Typical fluorescence signals recorded from Shaker S424C T449V channels during 7-s depolarizing pulses to +60 mV at pH 7.5 and pH 5.0 (holding potential −80 mV). Inhibition of inactivation prevented the loss of fluorescence at low pH.
	Figure 12. Acidic pH induces conformational changes in closed channels that are associated with inactivation. Typical fluorescence signals recorded from Shaker ILT S424C channels during 100-ms voltage pulses from −80 to 0 mV at pH 7.5 (A) and from −80 to +80 mV at pH 5.0 (B). Although pulses were applied in 10-mV increments, only every other pulse is shown for clarity. A voltage pulse to 0 mV at pH 7.5 or to +80 mV at pH 5.0 (shift in voltage dependence is due to surface charge screening by protons) evokes maximum voltage sensor movement without any channel opening. Reduction of the extracellular pH induced conformational changes in closed channels that are consistent with inactivation. (C) Mean F-V relationships of the fluorescence recorded from closed channels at pH 7.5 and pH 5.0 from Shaker ILT S424C channels (n = 4), and from Shaker ILT A359C W434F channels (n = 4). The V1/2 and k values were −37.1 ± 0.8 mV and 16.1 ± 0.7 mV for Shaker ILT S424C channels at pH 5.0, and −83.0 ± 0.4 mV and 15.7 ± 0.4 mV for Shaker ILT A359C W434F channels at pH 7.5, respectively. (D) Mean τ-V relationships for the fast phase (fitted with a single exponential) of the fluorescence deflection from Shaker ILT S424C channels at pH 5.0, and from Shaker ILT A359C W434F channels (n = 4). The voltage dependence and kinetics of the fluorescence report from Shaker ILT S424C channels was similar to that of voltage sensor movement reported by Shaker A359C W434F.
	Figure 13. Demonstration of pH-induced closed-state rearrangements. Typical diary plots of the effect of changing the pH on the fluorescence emission from Shaker ILT S424C (A) or Shaker A359 (C, as a control) channels held continuously at −80 mV. To minimize bleaching of the fluorophore, fluorescence was sampled every second (although only every fifth recording is shown for clarity) by opening the shutter for 100 ms. Low pH induced rearrangements at −80 mV that were detected by TMRM at S424C. (B) Plot of the dependence of the closed-state fluorescence changes on the external pH (n = 3). The relative fluorescence change from that at pH 7.5 is plotted. Data were fitted with a standard Hill equation (assuming a Hill coefficient, n, of 1). The pKa for the closed-state fluorescence change was pH 5.9. The value at pH 7.5 represents the mean of all pre- and post-treatment conditions since the order of pH changes was not necessarily consistent.

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