Extracellular D₂O slows the inactivation kinetics of T449A/I470A and T449K/I470C Shaker-IR constructs. (A and C) Macroscopic currents were measured in voltage-clamped inside-out patches excised from tsA201-expressing T449A/I470A (A) and T449K/I470C (C) Shaker-IR constructs, and displayed records were normalized to the respective peak currents. Control currents were recorded in symmetric H₂O//H₂O (intra/extracellular, black traces) and extracellular D₂O and intracellular H₂O (H₂O//D₂O, red traces). For the composition of solutions, see Materials and methods. The holding potential was −120 mV, and currents were evoked using 2.0-s-long (T449A/I470A) or 400-ms-long (T449K/I470C) test pulses to +50 mV every 60 s. The inactivation time constant of the currents at +50 mV was determined by fitting a single-exponential function to the decaying part of the currents. The analysis of the inactivation time constants is presented in Fig. 2 B. (B and D) Outside-out patches were repeatedly depolarized from a holding potential of −120 mV using the pulse protocols shown above the corresponding normalized current traces. 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, and the solution exchange was initiated 1 s before the start of the depolarization. Inactivation time constant (τ_inact) of the current at +50 mV was determined by fitting a single-exponential function to the decaying part of the currents; analysis of the inactivation time constants is presented in Fig. 2 D.