Cellular pH recordings

Simultaneous recordings of whole‐cell current and associated intracellular pH changes were made using bath and pipette solutions with pH adjusted to 7.0, and the pipette contained a reduced concentration of HEPES (1 mM) and 100–150 μM 2′,7′‑bis‑(2‑carboxyethyl)‑5‑(and‑6)‑carboxyfluorescein (BCECF, free acid; Life Technologies) (Alekov & Fahlke, 2009). One to two minutes after establishing whole‐cell access, a brief control I–V was recorded (20–50 ms pulses, −80 to +160 mV potentials) to determine leak‐subtracted current density. Cellular BCECF was sequentially excited at 445 and 494 nm (50 ms, 4–5 Hz), and emitted fluorescence collected at 537 nm (Alekov & Fahlke, 2009), while applying 3.2 s depolarizing pulses (0, +40, +80, +120, +160 mV potentials). The final two pulses were preceded by ∼20 s rest intervals to allow intracellular pH re‐equilibration. BCECF fluorescence was calibrated in whole‐cell configuration by imaging the recorded cell in a series of pH standard buffers containing (in mM): 150 KCl, 10 HEPES, 10 μM nigericin (pH 6.5, 7.0, 7.5, 8.0). The ratio of integrated cellular fluorescence at the two excitation wavelengths (abbreviated here as F ₄₉₀/F ₄₄₀) was background‐corrected and measured offline. For each cell, F ₄₉₀/F ₄₄₀ values measured in the standard buffers was plotted vs. buffer pH, and the slope of this relationship was determined by a linear regression (Microsoft Excel 2010). Changes in cellular pH vs. time during depolarizing pulses were calculated as Slope × ΔF ₄₉₀/F ₄₄₀, relative to prepulse baseline level. The initial rate of pH change (ΔpH/s) was determined by linear fits to the first 1.0–2.5 s of each response. Statistical comparison was made between ΔpH/s values measured at +120 mV applied potential. Average ΔV _m values in pH experiments are indicated in Table ​Table2.2. Relative coupling ratios were determined from the slope of the relationship between alkalization rate and current density (ΔpH/s vs. I _SS), which is independent of voltage.

ClC‐3 proton coupling and turnover

Values are means ± SEM; numbers in parentheses indicate number of experiments or observations. ^aCellular alkalization rate at +115 mV applied potential. ^bAverage steady‐state R _S‐associated voltage error at +115 mV; ^cMean current magnitude at a corrected voltage of +115 mV, relative to ClC‐5/3 wild‐type. ^dΔpH/s per 100 pA/pF conductance. ^eEstimated anion:H⁺ coupling ratio assuming a ratio of 2.0 for ClC‐5/3 wild‐type at pH 7.35 in external Cl⁻. ^fRelative H⁺ transport efficiency vs. ClC‐5/3 wild‐type in control saline. ^g130 mM Cl⁻ control saline at pH 7.35. ^h122 mM SCN⁻/8 mM Cl⁻ saline at pH 7.35. ⁱValue of 2.0 determined by Zifarelli & Pusch (2009). ^jThe relationship for ClC‐5/3 displays a slope equal to that of ClC‐5 (Fig. ​(Fig.33 I). Symbols: ^* P ≤ 0.03 and ^† P ≤ 0.001, respectively, vs. wild‐type ClC‐5/3, control saline. All values in external SCN⁻ differ significantly (P ≤ 0.01) from corresponding values in external Cl⁻. ^‡Not significant (P ≥ 0.05) vs. wild‐type ClC‐5/3, control saline; ^¶not significant (P ≥ 0.05) vs. value in 130 mM Cl⁻; ^§voltage errors exceeding 5% of applied potential.