Figure 1.
Figure 1. State-independent block of Slo2.1 channels by verapamil and D890. (A) Activation of ISlo2.1 during repetitive pulsing to 0 mV (0.3-s duration, 10-s interpulse interval) by 6 mM NFA under control conditions (i), and after a 10-min equilibration with 100 µM verapamil (ii). (B) Time course of ISlo2.1 in a single oocyte during activation with 6 mM NFA, washout of NFA, incubation with 100 µM verapamil for 10 min, and reapplication of 6 mM NFA in the continued presence of verapamil. The oocyte was clamped at −90 mV and pulsed to 0 mV every 10 s except during the 10-min incubation with verapamil. The notations “i” and “ii” correspond to the currents shown in A. (C and D) External application of 100 µM D890 does not block ISlo2.1. Protocol and notations are the same as noted for A and B. (E) Plot of normalized and mean peak ISlo2.1 induced by 6 mM NFA alone and by 6 mM NFA in the presence of 100 µM verapamil (n = 6; *, P < 0.0001) or D890 (n = 8) after a 10-min pulse-free equilibration period. Error bars indicate mean ± SEM. (F and G) Mean currents (18–25 consecutive sweeps) recorded from inside-out excised macropatches during voltage ramps from +80 to −140 mV. Currents were recorded in the absence (control) and presence of 100 µM verapamil (F) or 100 µM D890 (G). Arrows indicate 0 current level. (H) Time course of E275D ISlo2.1 block by 100 µM verapamil, added to the extracellular solution, measured in response to a train of test pulses applied to +60 mV. Two pulse train protocols are compared. One protocol used 3-s pulses that were applied once every 5 s (open circle, n = 6). The other protocol used 0.3 s pulses that were applied once every 30 s (red circle, n = 6). Currents were normalized to ISlo2.1 measured before drug treatment, and each oocyte was subjected to only one pulse train. Error bars indicate mean ± SEM. (I) Diagram illustrating proposed mechanism of Slo2.1 channel block by verapamil and its quaternary amine derivative D890. Verapamil, but not D890, can cross the cell membrane into the cytoplasm. When applied to the bathing solution of inside/out patches, both compounds can block channels in a state-independent manner, which suggests that the S6 segments do not form a physical barrier to drug entry into the central cavity.

State-independent block of Slo2.1 channels by verapamil and D890. (A) Activation of ISlo2.1 during repetitive pulsing to 0 mV (0.3-s duration, 10-s interpulse interval) by 6 mM NFA under control conditions (i), and after a 10-min equilibration with 100 µM verapamil (ii). (B) Time course of ISlo2.1 in a single oocyte during activation with 6 mM NFA, washout of NFA, incubation with 100 µM verapamil for 10 min, and reapplication of 6 mM NFA in the continued presence of verapamil. The oocyte was clamped at −90 mV and pulsed to 0 mV every 10 s except during the 10-min incubation with verapamil. The notations “i” and “ii” correspond to the currents shown in A. (C and D) External application of 100 µM D890 does not block ISlo2.1. Protocol and notations are the same as noted for A and B. (E) Plot of normalized and mean peak ISlo2.1 induced by 6 mM NFA alone and by 6 mM NFA in the presence of 100 µM verapamil (n = 6; *, P < 0.0001) or D890 (n = 8) after a 10-min pulse-free equilibration period. Error bars indicate mean ± SEM. (F and G) Mean currents (18–25 consecutive sweeps) recorded from inside-out excised macropatches during voltage ramps from +80 to −140 mV. Currents were recorded in the absence (control) and presence of 100 µM verapamil (F) or 100 µM D890 (G). Arrows indicate 0 current level. (H) Time course of E275D ISlo2.1 block by 100 µM verapamil, added to the extracellular solution, measured in response to a train of test pulses applied to +60 mV. Two pulse train protocols are compared. One protocol used 3-s pulses that were applied once every 5 s (open circle, n = 6). The other protocol used 0.3 s pulses that were applied once every 30 s (red circle, n = 6). Currents were normalized to ISlo2.1 measured before drug treatment, and each oocyte was subjected to only one pulse train. Error bars indicate mean ± SEM. (I) Diagram illustrating proposed mechanism of Slo2.1 channel block by verapamil and its quaternary amine derivative D890. Verapamil, but not D890, can cross the cell membrane into the cytoplasm. When applied to the bathing solution of inside/out patches, both compounds can block channels in a state-independent manner, which suggests that the S6 segments do not form a physical barrier to drug entry into the central cavity.

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