Figure 2. Extracellular D 2 O slows the... | Rockefeller University Press

Figure 2.

Extracellular D2O slows the inactivation kinetics of T449A, T449A/I470A, and T449K/I470C Shaker-IR mutants.(A and C) Macroscopic currents were measured in voltage-clamped inside-out (A) and outside-out patches (C) excised from tsA201 cells expressing the T449A Shaker-IR mutant. Displayed current traces were normalized to the respective peak currents. (A) Normalized inside-out patch currents obtained in different patches are displayed. The holding potential was −120 mV, and patches were repeatedly depolarized to +50 mV for 2.0 s every 45 s to measure currents. Control current was recorded in symmetric H2O//H2O (intra/extracellular, black trace); traces obtained in extracellular D2O, intracellular H2O (H2O//D2O, red trace), and intracellular D2O and extracellular H2O (D2O//H2O, green trace) are also displayed. (B) Inactivation time constants (τinact) measured at +50 mV in inside-out patches for the indicated mutants in the absence (H2O//H2O, filled symbols) or presence of extracellular D2O (H2O//D2O, empty symbols). The intracellular solution was H2O based. (C) Outside-out patches were repeatedly depolarized from a holding potential of −120 mV to obtain the currents. The first depolarization was in a H2O-based extracellular solution, followed by 60-s-long period at −120 mV, which was sufficient to ensure full recovery of the inactivated channels. Thereafter, another 2.0-s-long depolarizing pulse was applied in the presence of D2O; the solution exchange was initiated 1 s before the start of the depolarization. The voltage pulse protocol is shown above the corresponding normalized current traces; the color coding is the same as in A. For the composition of solutions, see Materials and methods. (D) The same data as in B, except that currents were recorded in outside-out configuration. Bars and error bars represent mean ± SEM for n ≥ 4 independent experiments (B and D). Symbols indicate individual data points (circles, T449A; down triangles, T449A/I470A; up triangles, T449K/I470C). Differences were considered significant (*, P < 0.05; **, P < 0.01; ****, P < 0.0001) compared with control (H2O//H2O).

Extracellular D₂O slows the inactivation kinetics of T449A, T449A/I470A, and T449K/I470C Shaker-IR mutants. (A and C) Macroscopic currents were measured in voltage-clamped inside-out (A) and outside-out patches (C) excised from tsA201 cells expressing the T449A Shaker-IR mutant. Displayed current traces were normalized to the respective peak currents. (A) Normalized inside-out patch currents obtained in different patches are displayed. The holding potential was −120 mV, and patches were repeatedly depolarized to +50 mV for 2.0 s every 45 s to measure currents. Control current was recorded in symmetric H₂O//H₂O (intra/extracellular, black trace); traces obtained in extracellular D₂O, intracellular H₂O (H₂O//D₂O, red trace), and intracellular D₂O and extracellular H₂O (D₂O//H₂O, green trace) are also displayed. (B) Inactivation time constants (τ_inact) measured at +50 mV in inside-out patches for the indicated mutants in the absence (H₂O//H₂O, filled symbols) or presence of extracellular D₂O (H₂O//D₂O, empty symbols). The intracellular solution was H₂O based. (C) Outside-out patches were repeatedly depolarized from a holding potential of −120 mV to obtain the currents. The first depolarization was in a H₂O-based extracellular solution, followed by 60-s-long period at −120 mV, which was sufficient to ensure full recovery of the inactivated channels. Thereafter, another 2.0-s-long depolarizing pulse was applied in the presence of D₂O; the solution exchange was initiated 1 s before the start of the depolarization. The voltage pulse protocol is shown above the corresponding normalized current traces; the color coding is the same as in A. For the composition of solutions, see Materials and methods. (D) The same data as in B, except that currents were recorded in outside-out configuration. Bars and error bars represent mean ± SEM for n ≥ 4 independent experiments (B and D). Symbols indicate individual data points (circles, T449A; down triangles, T449A/I470A; up triangles, T449K/I470C). Differences were considered significant (*, P < 0.05; **, P < 0.01; ****, P < 0.0001) compared with control (H₂O//H₂O).