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Figure 1. Voltage- and concentration-dependent blockade of fKv1.4ΔN currents by diltiazem. (A) Channels were expressed in Xenopus oocytes and recorded with the two electrode voltage clamp technique. Currents were obtained by applying 5 s pulses to potentials (P1) ranging from -100 mV to +50 mV and were followed by the tail currents obtained upon repolarization to +50 mV (P2) under control conditions (a), then in the presence of 250 μmol/L diltiazem (b), and finally after 10 min of washout (c). (B) Current-voltage relationships of fKv1.4ΔN channels under control conditions, in the presence of 250 μmol/L diltiazem, and after the drug washout for 10 min. Currents were normalized to the peak current at +50 mV under control conditions. The IDIL/ICON ratio was plotted as a function of the membrane potential. Data are shown as means±SEM. (n=5). (C) Dose-response relationships for diltiazem inhibition of fKv1.4ΔN channels at 2 mmol/L [K+]o. Data were obtained upon repolarization to -90 mV after 1 s pulses to +50 mV, holding potential -90 mV. All values shown were normalized to the peak current in the absence of drug in 2 mmol/L [K+]o. Continuous line was derived by fitting the data to the Hill equation: f=KD/(KD+D), where f is fractional current, KD is the apparent dissociation constant, and D is the diltiazem concentration. Symbols and error bar are means±SEM. fKv1.4ΔN current was reduced to 50% by 241.04±23.06 μmol/L.
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Figure 2. Frequency-dependent block of fKv1.4ΔN channels by diltiazem. Currents were elicited by applying a series of depolarising pulses from -90 mV to +50 mV with a frequency of 1 Hz in the absence (A) and in the presence of 250 μmol/L diltiazem (B). The peak currents shown in Panel A and B were normalized to the maximum control value without drug and plotted in Panel C. As pulse number increased, currents in both control and diltiazem-treated groups decreased. In control cells, there was a use-dependent reduction in the magnitude of the peak current. When cells were exposed to 250 μmol/L diltiazem for 10 min before stimulation, there was a reduction in the magnitude of the first peak current compared to the control value and then a use-dependent component. The use-dependent reduction in current with diltiazem was much greater than that seen in control.
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Figure 3. Effect of diltiazem on the steady inactivation of peak Kv1.4ΔN currents. (A) The time-dependent progression of channel currents. Currents were elicited by applying 1 s pulses from -90 mV to +50 mV in the absence and presence of increasing concentrations of diltiazem. For comparison, all current traces were normalized to the peak values under control conditions. The smooth continuous line superimposed on each trace is the best fit of an exponential function, used to determine the inactivation time constant (s). The control trace was best fitted by a mono-exponential function (Chebyshev method) (a), whereas in the presence of 10 μmol/L–1000 μmol/L diltiazem, inactivation was best fitted by a bi-exponential function (Levenberg-Marquardt) (b–f). (B) Steady-state inactivation relationships (a). The steady-state inactivation for each P1 voltage was calculated as the magnitude of the peak current in P2 compared with that from the maximum of the P2 obtained when P1 was -100 mV. Average data are shown as mean±SEM. Steady-state inactivation relationships are shown: fKv1.4ΔN (▪) and fKv1.4ΔN+250 μmol/L diltiazem (•). Continuous lines represent the fit of the data to a Boltzmann equation: f=1/{1+exp*[(V–V1/2)/k)]}. Steady-state inactivation relationships were re-normalized (b).
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Figure 4. Comparison of the voltage for half-inactivation (V1/2) and slope factor (k) from fKv1.4ΔN without diltiazem and fKv1.4ΔN with 250 μmol/L diltiazem (A(a) and A(b)). (A (a)) V1/2, control=-38.38±0.81 mV (n=6), V1/2, diltiazem=-39.23±0.85 mV (n=6), (A(b)) Kcontrol=4.58±0.75 (n=6), Kdiltiazem=5.06±0.78 (n=6). Average data are shown as means±SEM (aP>0.05 vs control), (A(c)) The effect of diltiazem on the rate of inactivation of fKv1.4ΔN channels. The time constant of inactivation was acquired by fitting the current trace elicited at +50 mV (P1) ranging from the beginning of the peak of P1 to the end of 5 s. τinactivation, control=2.32±0.41 s (n=6). In the presence of diltiazem, τfast=0.41±0.04 s and τslow=1.78±0.29 s (n=6). Average data are shown as means±SEM (bP<0.05 vs control). (B) Diltiazem alters the rate of inactivation for fKv1.4ΔN channels. Inactivation of fKv1.4ΔN channels is well fitted by a single exponential function (Chebyshev method), and is voltage independent (▪) over the range 0 mV to +50 mV. In the presence of diltiazem, the inactivation of fKv1.4ΔN is best fitted with a bi-exponential function (Levenberg-Marquardt). Over the range 0 mV to +50 mV, τfast is voltage independent (•), whereas τslow is voltage dependent (▴). (C) The reciprocal of the diltiazem-induced fast time constant (1/τblock) at +50 mV as a function of the diltiazem concentration for data obtained at concentrations in the range between 10 μmol/L and 1000 μmol/L. The straight line is the least-squares fit to equation: 1/τblock=k+1[d]+k-1, where τblock is the time constant of development of block, k+1 and k-1 are the apparent association rate constant and the apparent dissociation rate constant, respectively. The dotted lines is the 95% confidence interval of the fit, each point represents the means±SEM of 6 experiments.
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Figure 5. Effect of diltiazem on the rate of recovery from inactivation in fKv1.4ΔN. Recovery from inactivation was measured using a standard variable interval gapped pulse protocol. An initial 5 s pulse (P1) from -90 mV to +50 mV was followed by a second pulse (P2) to +50 mV after an interval of between 0.1 s and 20 s. (A) The ratio of the peak current elicited by the P1 and P2 pulses (P2/P1) is plotted against pulse interval to show the recovery from inactivation. The recovery of inactivation was best fitted using the function: f=1–A*exp(-τ/t), where t is duration (in s), τ is the time constant, A is the amplitude of the current. Recovery curves for fKv1.4ΔN and fKv1.4ΔN+diltiazem, holding potential=-90 mV. (B) Comparison of recovery rate data from fKv1.4ΔN without and with 250 μmol/L diltiazem. The mean time constants for recovery were 1.73±0.10 s (n=5) in control and 2.66±0.14 s (n=5) in the diltiazem treated group (bP<0.05 vs control).
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Figure 6. (A) Average recovery time course for fKv1.4ΔN without diltiazem and with 250 μmol/L diltiazem. Data were normalized between 0 and 1 presented with intervals on a log scale. (B) t1/2 for fKv1.4ΔN was 1.01±0.03 s (n=5) and t1/2 was 1.67±0.05 s (n=5) in the presence of 250 μmol/L diltiazem (bP<0.05 vs control).
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Figure 7. Voltage- and concentration-dependent blockade by propafenone on fKv1.4ΔN currents. (A) Current recordings from two-electrode voltage clamp of oocytes expressing fKv1.4ΔN. Currents were obtained by applying 5 s pulses to potentials (P1) ranging from -100 mV to +50 mV followed by the tail currents obtained upon repolarization to +50 mV (P2) under control conditions (a), then in the presence of 100 μmol/L propafenone (b), and finally after 10 min of washout (c). (B) Current-voltage relationships of fKv1.4ΔN channels under control conditions, in the presence of 100 μmol/L propafenone, and after the drug washout for 10 min. Currents were normalized to the peak current at +50 mV under control conditions. The IPRO/ICON ratio was plotted as a function of the membrane potential. Data are shown as means±SEM (n=5). (C) Dose-response relationships for propafenone inhibition of fKv1.4ΔN channels at 2 mmol/L [K+]o. Data were obtained upon repolarization to -90 mV after 1 s pulses to +50 mV, holding potential -90 mV. All values shown were normalized to the peak current in the absence of drug in 2 mmol/L [K+]o. Continuous line was derived by fitting the data to the Hill equation: f=KD/(KD+D), where f is fractional current, KD is the apparent dissociation constant, and D is the propafenone concentration. Symbols and error bar are means±SEM. fKv1.4ΔN current was reduced to 50% by 103.68±11.25 μmol/L.
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Figure 8. Frequency-dependent block of fKv1.4ΔN channel by propafenone. Currents were elicited by using a series of depolarising pulses from -90 mV to +50 mV with a frequency of 1 Hz in the absence (A) and in the presence of 100 μmol/L propafenone (B). The peak currents shown in Panel A and B were normalized to the maximum control value without drug and plotted in Panel C. As pulse number increased, currents in both control and propafenone-treated groups decreased. In control cells, there was a use-dependent reduction in the magnitude of the peak current. When cells were exposed to 100 μmol/L propafenone for 10 min before stimulation, there was a reduction in the magnitude of the first peak current compared to the control value and then a use-dependent component. The use-dependent reduction in current with propafenone was obviously greater than that seen in control.
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Figure 9. Effect of propafenone on the steady inactivation of peak Kv1.4ΔN currents. (A) The time-dependent progression of channel currents. Currents were recorded by using 1 s pulses from -90 mV to +50 mV in the absence and presence of increasing concentrations of propafenone. For comparison, all current traces were normalized to the peak values under control conditions. The smooth continuous line superimposed on each trace is the best fit of an exponential function, used to determine the inactivation time constant (s). The control trace was best fitted by a mono-exponential function (Chebyshev method) (a), whereas in the presence of 10–500 μmol/L propafenone, inactivation was best fitted by a bi-exponential function (Levenberg-Marquardt) (b–f). (B) Steady-state inactivation relationships (a). The steady-state inactivation for each P1 voltage was calculated as the magnitude of the peak current in P2 compared with that from the maximum of the P2 obtained when P1 was -100 mV. Average data are shown as mean±SEM. Steady-state inactivation relationships are shown: fKv1.4ΔN (▪) and fKv1.4ΔN+100 μmol/L propafenone (•). Continuous lines represent the fit of the data to a Boltzmann equation: f=1/{1+exp*[(V-V1/2)/k)]}. Steady-state inactivation relationships were re-normalized (b).
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Figure 10. Comparison of the voltage for V1/2 and K from fKv1.4ΔN without propafenone and fKv1.4ΔN with 100 μmol/L propafenone [A(a) and A(b)]. [A(a)] V1/2, control=-41.29±5.21 mV (n=6), V1/2, propafenone=-50.62±6.77 mV (n=6); [A(b)] Kcontrol=1.13±0.09 (n=6), Kpropafenone=1.62±0.27 (n=6); [A(c)] The effect of propafenone on the rate of inactivation of fKv1.4ΔN channels. The time constant of inactivation was acquired by fitting the current trace elicited at +50 mV (P1) ranging from the beginning of the peak of P1 to the end of 5 s. τinactivation, control=2.32±0.41 s (n=6). In the presence of propafenone, τfast=0.44±0.03 s and τslow=2.32±0.23 s (n=6). Average data are shown as means±SEM (aP>0.05, bP<0.05 vs control). (B) Effect of propafenone on the rate of inactivation for fKv1.4ΔN channels. Inactivation of fKv1.4ΔN channels is well fitted by a single exponential function (Chebyshev method), and is voltage independent (▪) over the range of 0 mV to +50 mV. In the presence of propafenone, the inactivation of fKv1.4ΔN is best fitted with a bi-exponential function (Levenberg-Marquardt). Over the range of 0 mV to +50 mV, both τfast (•) and τslow (▴) are voltage independent. (C) The reciprocal of the propafenone-induced fast time constant (1/τblock) at +50 mV as a function of the propafenone concentration for data obtained at concentrations in the range between 10 and 500 μmol/L. The straight line is the least-squares fit to equation: 1/τblock=k+1[d]+k-1, where τblock is the time constant of development of block, k+1 and k-1 are the apparent association rate constant and the apparent dissociation rate constant, respectively. The dotted lines is the 95% confidence interval of the fit, each point represents the means±SEM of 6 experiments.
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Figure 11. Effect of propafenone on the rate of recovery from inactivation in fKv1.4ΔN. Recovery from inactivation was measured using a standard variable interval gapped pulse protocol. An initial 5 s pulse (P1) from -90 mV to +50 mV was followed by a second pulse (P2) to +50 mV after an interval between 0.1 and 20 s. (A) The ratio of the peak current elicited by the P1 and P2 pulses (P2/P1) is plotted against pulse interval to show the recovery from inactivation. The recovery of inactivation was best fitted using the function: f=1–A*exp(-τ/t), where t is duration (in s), τ is the time constant, A is the amplitude of the current. Recovery curves for fKv1.4ΔN and fKv1.4ΔN+propafenone, holding potential=-90 mV. (B) Comparison of recovery rate data from fKv1.4ΔN without and with 100 μmol/L propafenone. The mean time constants for recovery were 1.78±0.09 s (n=5) in control and 1.86±0.14 s (n=5) in the propafenone treated group (aP>0.05 vs control).
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Figure 12. (A) Average recovery time course for fKv1.4ΔN without propafenone and with 100 μmol/L propafenone. Data were normalized between 0 and 1 presented with intervals on a log scale. (B) t1/2 for fKv1.4ΔN was 1.14±0.04 s (n=5) and t1/2 was 1.49±0.05 s (n=5) in the presence of 100 μmol/L propafenone (aP>0.05 vs control).
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