FIGURE 1.

Ba²⁺ blocks Kv1.5 in a concentration- and frequency-dependent manner. (A) With 0 mM pH_o 7.4 solution, 10-ms test pulses to +50 mV were applied at 1 Hz without (uppermost trace) and with 5 mM Ba²⁺. Shown are 65 consecutive traces from the time of Ba²⁺ application, which began immediately after the control trace. (Inset) The trace at the steady-state level of block was scaled 1.87× to match the peak amplitude of the control current. There was a small effect of Ba²⁺ on the onset of the K⁺ current. (B (i)) Trace 1 was recorded using a 10-ms pulse to +50 mV after a 1-min exposure to 1 mM Ba²⁺ in 0 mM at −80 mV. Traces 2–5 show the current from subsequent pulses applied at 1 Hz in the continued presence of Ba²⁺. Trace 25 shows the steady-state current in 1 mM Ba²⁺ with 1 Hz stimulation and Trace 100 is the steady-state current after recovery in 0 mM Ba²⁺. (B (ii)) Traces 1–5 and 25 are normalized with respect to Trace 100. There was a marked slowing of the rising phase of the current elicited by the first pulse after Ba²⁺ exposure, but this effect is diminished with subsequent pulses in the continued presence of Ba²⁺. (C) The time course of the onset and offset of the Ba²⁺ block. The average current amplitude in the last 1 ms of each pulse was normalized to the Ba²⁺-free control current. Down and up arrows indicate the time of fast Ba²⁺ application and withdrawal, respectively. Black circles (•) represent the normalized current amplitudes for the experiment shown in A. Single-exponential fits to the onset of and recovery from Ba²⁺ block give τ_block = 6.84 s and τ_unblock = 19.21 s, respectively. When the experiment was performed with the same stimulus protocol but with 20 mM Ba²⁺ (solid triangles), block onset was slightly faster (τ_block = 4.97 s) and the normalized steady-state current was smaller (0.37 vs. 0.50 for 5 mM Ba²⁺), but the time course of current recovery was the same. If the [Ba²⁺] was kept constant at 5 mM but the pulse frequency decreased to 0.2 Hz (open diamonds), the normalized steady-state level was also reduced (steady-state normalized current = 0.27). Although the onset of the block was not affected by the decrease in stimulation frequency, recovery with a 0.2-Hz pulse frequency was much slower (τ_unblock = 63.20 s) than that seen at 1 Hz. (D) The blocking rate (1/τ_block) is a nonlinear function of the [Ba²⁺]. τ_block values were assessed in 0 mM with 10-ms pulses to +50 mV applied at 1 Hz. The solid line represents the best fit of the data to a sequential two-site binding model (Eq. 1; see text for fit parameters). Data points are from a total of 46 cells, 3–8 cells/point. (E) The unblocking rate is linearly related to the pulse frequency. Values for τ_unblock were assessed with 0 mM and 20 mM using 10-ms pulses to +50 mV applied at 1, 0.7, 0.2, 0.066, or 0.033 Hz. The extrapolated off rate without pulsing (0 Hz) is 0.001 ± 0.002 s⁻¹. Data points are from a total of 53 cells, 5–24 cells/point.