5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel

Figures

• 

  Figure 1. Inhibition of hHV1 currents by ClGBI. Peak currents were determined using protocols and experimental conditions as described in Materials and Methods. (A) Whole-cell currents were recorded on CHO cells expressing HV1 in response to slow voltage ramps (0.12 mV/ms) from −60 to +60 mV in the absence (pHec = 7.4, black) and presence of 200 μM ClGBI (ClGBI, red). ECS at a pH of 6.4 was used as an indicator of both the ion channel and the proper operation of the perfusion system (pHec = 6.4, blue). (B) The effect of ClGBI at 200 μM was determined as remaining current fraction (RCF, see Materials and Methods) and plotted as a function of time as 200 μM ClGBI was applied to the bath solution. Pulses were delivered every 10 s. (C) Concentration-dependent inhibition of the HV1 channel by ClGBI. Points on the dose–response curves represent the mean of 4–5 independent measurements. Data points were fitted with a two-parameter Hill equation (see Materials and Methods). The best fit yielded Kd of 15.9 ± 2.0 μM and nH of 1.0 ± 0.1. Error bars represent SEM.

• 

  Figure 2. The effect of ClGBI on human lymphocyte proliferation. (A) Representative flow cytometry histograms of CFSE-labeled lymphocytes in the absence and presence of ClGBI (200 μM, 20 μM, and 2 μM). (B) Histogram overlays of samples presented in (A) showing the percentage of proliferating cells by CFSE dilution.

• 

  Figure 3. Block of KV1.3 channels by ClGBI. (A) Whole-cell potassium currents through hKV1.3 channels evoked from a human T cell by depolarizing pulses to +50 mV every 15 s. Traces indicate K+ current in the absence (control, black) and presence of 200 µM ClGBI at equilibrium block (ClGBI, red), with 10 mM TEA+ (blue) used as a positive control. (B) Kinetics of the block. Normalized peak currents as remaining current fraction (RCF) are plotted as a function of time. The boxes indicate the application of the control ECS and ClGBI-containing ECS, respectively. Solid line indicates the fitting of a single-exponential decaying function to the data points. Perfusion with ClGBI-free ECS resulted in complete recovery from block. Data points were recorded every 15 s. (C) Dose–response relationship for ClGBI on KV1.3. The dose response was obtained by plotting the remaining current fraction as a function of ClGBI concentration and fitting it with a two-parameter Hill equation (see Materials and Methods). Error bars indicate SEM throughout the figure.

• 

  Figure 4. Selectivity profile of ClGBI. Effect of 200 μM ClGBI was assayed in CHO cells transfected with different ion channel genes or using HEK293 cell line stably expressing hKV11.1 and hNaV1.4 channels. The representative current traces shown here were measured before the application of ClGBI (control, black) and after reaching equilibrium block upon application of 200 μM ClGBI (red). (A–D) Currents were evoked from holding potentials of –100 mV by depolarizations to +50 mV for the time interval indicated on the panels. The time between depolarizing pulses was 15 s or 30 s (for KV10.1). (E) hKV11.1 currents were measured with a voltage step from a holding potential of –80 mV to +20 mV, followed by a step to –40 mV, during which the peak current was measured. Pulses were delivered every 30 s. (F) hKCa3.1 currents were elicited every 15 s with voltage ramps to +50 mV from a holding potential of −120 mV at a rate of 0.85 mV/ms. (G,H) hNaV1.4 and hNaV1.5 currents were measured by applying depolarizing pulses to 0 mV from a holding potential of –120 mV every 15 s. (I) The effect of 200 μM ClGBI on the peak currents was reported as the remaining current fraction (RCF = I/I0, where I0 and I…