Identification of IQM-266, a Novel DREAM Ligand That Modulates KV4 Currents

Figures

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  Figure 1. IQM-266 as DREAM ligand. (A) Chemical structure of IQM-266. (B) Sensograms for binding of IQM-266 using increasing concentrations of the ligand in the range 1–7 μM. RU, resonance units. (C) Surface plasmon resonance (SPR) binding plot adjusted with one site specific model with Hill slope.

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  Figure 2. Concentration-dependence of inhibition and/or increase of the current or the charge through KV4.3/DREAM channels produced by IQM-266. (A) Concentration-response curve of IQM-266 (continuous line). The dashed line represents the fit of the data to a Hill equation with nH = 1 (n = 34). (B) Inhibition or increase of current induced by IQM-266 in KV4.3/DREAM channels measured at the maximum peak current and in the charge (measured as the area of the current during the application of a 250 ms pulse to +60 mV; n = 34). (C) Original recordings obtained after applying a depolarizing pulse to +60 mV in the absence and in the presence of IQM-266 (3 μM). (D) Original recordings obtained after applying a depolarizing pulse to +60 mV in the absence and in the presence of IQM-266 (10 μM). *p < 0.05 when comparing the effect of IQM-266 on the peak current and on the charge through KV4.3/DREAM channels.

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  Figure 3. Activation and inactivation kinetics of KV4.3/DREAM currents in the absence and in the presence of IQM-266. (A) Normalized first 20 ms of original current recordings in control and in the presence of IQM-266 (3 μM). The current records were fitted to a monoexponential equation in order to obtain the activation time constant (τAc). (B) Normalized first 20 ms of original current recordings in control and in the presence of IQM-266 (10 μM). The current records were fitted to a monoexponential equation in order to obtain the τAc values. (C) Concentration-dependence of the τAc,IQM-266/τAc,Control. (D) Histogram representing the τAc in the different experimental conditions. (E) Voltage-dependent effects of IQM-266 (3 μM) on the time constant of activation (τAct).(F) Normalized first 250 ms of original current recordings in control and in the presence of IQM-266 (3 μM). The current records were fitted to a monoexponential equation in order to obtain the inactivation time constant (τInac). (G) Normalized first 250 ms of original current recordings in control and in the presence of IQM-266 (10 μM). The current records were fitted to a monoexponential equation in order to obtain the τInac…

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  Figure 4. Inactivation recovery kinetics of KV4.3/DREAM channels in the absence and in the presence of IQM-266. Original recordings after applying the pulse protocols shown in the upper part of the figure in the absence and in the presence of IQM-266 (A) 3 μM or (B) 10 μM. (C) Data obtained after plotting the It/I0 vs. the interpulse period separating both, the conditioning and the test pulse. (D) Histogram bar showing the time constant of recovery obtained under control and in the presence of IQM-266 (n = 4–5). **p < 0.01 when comparing data in presence of IQM-266 with control.

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  Figure 5. Voltage dependence interaction between IQM-266 and KV4.3/DREAM channels. (A) Original recordings obtained generated by KV4.3/DREAM channels in the absence and in the presence of IQM-266 after applying the pulse protocols shown in the upper part of the figure. (B) I-V relationship of the currents generated by KV4.3/DREAM channels in the absence and in the presence of IQM-266 at 3 μM (left) and 10 μM (right) when measured at the maximum peak current. It is also shown the relative current (IIQM-266/IControl) vs. membrane potential. Dotted and dashed lines show the activation and the inactivation curves, respectively (n = 4–6). (C) Q-V relationship of the charge through KV4.3/DREAM channels in the absence and in the presence of IQM-266 at 3 μM (left) and 10 μM (right) when measured at the area under the current during the application of depolarizing pulse protocol shown in the upper part of the figure. It is also shown the relative charge (QIQM-266/QControl) vs. membrane potential. Dotted and dashed lines show the activation and the inactivation curves, respectively (n = 4–6).

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  Figure 6. Effects of IQM-266 on the steady-state inactivation curve and on the closed-state inactivation. (A) Original recordings obtained generated by KV4.3/DREAM channels in the absence and in the presence of IQM-266 after applying the pulse protocols shown in the upper part of the figure. (B) Steady-state inactivation in cells expressing KV4.3/DREAM channels in the absence and in the presence of IQM-266 (3 μM) are shown. Peak currents in response to test step to +50 mV after various conditioning potentials (Vh) are shown. (C) Original recordings recorded from KV4.3/DREAM channels after applying the pulse protocol shown in the upper part of the figure generated by in the absence and in the presence of IQM-266 (3 μM). (D) Development of closed-state inactivation of KV4.3/DREAM channels at a membrane potential (−40 mV) where this current inactivates, but does not conduct. Note how IQM-266 increases the closed-state inactivation.

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  Figure 7. Effects of IQM-266 on KV4.3 channels. (A) Concentration-block of KV4.3 channels induced by IQM-266. The IC50 obtained after fitting the data points resulted to be 7.1 μM. (B) Percentage of block of KV4.3 channels measuring the peak current and the charge. (C) Current recordings from −80 to +60 mV in the absence and in the presence of IQM-266 (5 μM). (D) First 20 ms of original current recordings from −80 to +60 mV in control and in the presence of IQM-266 (5 μM). (E) First 250 ms of original current recordings from −80 to +60 mV in control and in the presence of IQM-266 (5 μM). (F) Recovery kinetics from inactivation in the absence and in the presence of IQM-266 (5 μM; n = 4). Note that this compound does not modifies this process. (G) I-V relationship of the currents generated by KV4.3 channels in the absence and in the presence of IQM-266 (5 μM) when measured at the maximum peak current. It is also shown the relative current (IIQM-266/IControl) vs. membrane potential. Dashed line shows the activation curve (n = 6). (H) Steady-state inactivation curve obtained in the same manner than shown in Figure 6 (n = 4). (I) Development of the closed-state inactivation of KV4.3 channels at…

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  Figure 8. Effects of IQM-266 on peak amplitude and inactivation kinetics of IA from dorsal root ganglia (DRG) neurons. (A) Representative recordings of IA isolated by using the voltage protocol depicted on top of the panels (see “Materials and Methods” section). Upper panels show original IA recordings in the absence (Control) and the presence of IQM-266 (3 μM); lower panels show IA recordings in the absence (Control) and the presence of IQM-266 (10 μM). (B) Bar graph of percent block of peak IA by IQM-266 (3 μM or 10 μM) at different potentials. Data were obtained from eight cells for IQM-266 (3 μM), and five cells for IQM-266 (10 μM). (C) Effect of IQM-266 (3 μM; upper panels) and (10 μM; lower panels) on inactivation kinetics of IA. Original recordings (left panels) have been normalized to peak IA in the absence of IQM-2666 (right panels) to better appreciate the change in inactivation kinetics. (D) Bar graph showing the effect of IQM-266 at 3 μM (upper graph) or 10 μM on time constants of inactivation at different potentials. The current records were fitted to a biexponential equation to obtain the τf and τs values. Data are from eight cells for IQM-266 (3 μM), and five cells for…