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Inhibitory effects of cholinesterase inhibitor donepezil on the Kv1.5 potassium channel

View Article: PubMed Central - PubMed

ABSTRACT

Kv1.5 channels carry ultra-rapid delayed rectifier K+ currents in excitable cells, including neurons and cardiac myocytes. In the current study, the effects of cholinesterase inhibitor donepezil on cloned Kv1.5 channels expressed in HEK29 cells were explored using whole-cell recording technique. Exposure to donepezil resulted in a rapid and reversible block of Kv1.5 currents, with an IC50 value of 72.5 μM. The mutant R476V significantly reduced the binding affinity of donepezil to Kv1.5 channels, showing the target site in the outer mouth region. Donepezil produced a significant delay in the duration of activation and deactivation, and mutant R476V potentiated these effects without altering activation curves. In response to slowed deactivation time course, a typical crossover of Kv1.5 tail currents was clearly evident after bath application of donepezil. In addition, both this chemical and mutant R476V accelerated current decay during channel inactivation in a voltage-dependent way, but barely changed the inactivation and recovery curves. The presence of donepezil exhibited the use-dependent block of Kv1.5 currents in response to a series of depolarizing pulses. Our data indicate that donepezil can directly block Kv1.5 channels in its open and closed states.

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The blocking effect of donepezil on Kv1.5 currents.(a) Example traces of Kv1.5 currents were obtained in response to a 500 ms depolarizing pulse to potentials from −80 to +60 mV from a holding potential of −80 mV under control, 80 μM donepezil and washout conditions. Arrows denoted the zero-current level. (b) Current-voltage (I–V) relationships of Kv1.5 currents with and without donepezil (n = 11). (c) Bar graph of statistical analyses of block by donepezil in Kv 1.5 channels at +60 mV. *p < 0.05 compared with the control group. (d) The concentration-response relations of donepezil action on Kv1.5 currents. Data points with various concentration donepezil were fitted by a Hill equation (n = 6). (e) Mutant R476V but not H452G reduced the affinity of donepezil to Kv1.5 channels. Collected raw data were normalized to the control, and then were plotted with respect 80 μM donepezil (n = 5). Unless otherwise noted, WT indicates wild type in all figures. *p < 0.05 compared with the wild type group.
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f1: The blocking effect of donepezil on Kv1.5 currents.(a) Example traces of Kv1.5 currents were obtained in response to a 500 ms depolarizing pulse to potentials from −80 to +60 mV from a holding potential of −80 mV under control, 80 μM donepezil and washout conditions. Arrows denoted the zero-current level. (b) Current-voltage (I–V) relationships of Kv1.5 currents with and without donepezil (n = 11). (c) Bar graph of statistical analyses of block by donepezil in Kv 1.5 channels at +60 mV. *p < 0.05 compared with the control group. (d) The concentration-response relations of donepezil action on Kv1.5 currents. Data points with various concentration donepezil were fitted by a Hill equation (n = 6). (e) Mutant R476V but not H452G reduced the affinity of donepezil to Kv1.5 channels. Collected raw data were normalized to the control, and then were plotted with respect 80 μM donepezil (n = 5). Unless otherwise noted, WT indicates wild type in all figures. *p < 0.05 compared with the wild type group.

Mentions: After transfection of Kv1.5 channel plasmids in HEK293 cells, whole-cell recording was carried out to assess the effect of donepezil on corresponding currents. In the presence of 80 μM donepezil, a significant decrease was detected in Kv1.5 currents, which were elicited by depolarizing the cell from a holding potential of −80 mV to a test potentials ranging from −80 to +60 mV. This action was partially reversed following washout of the extracellular donepezil (Fig. 1a). The current-voltage (I–V) relationships of Kv1.5 currents in control, donepezil and washout are shown in Fig. 1b. The significant alterations of current amplitudes among the control, donepezil and washout were detected at different voltages between 0 mV to +60 mV (n = 11, ANOVA, p < 0.05). In Fig. 1c, the statistical analyses of block by donepezil at +60 mV are shown as the bar graph. Another cholinesterase inhibitor tacrine, however, barely affected the Kv1.5 currents, even in the higher concentration (200 μM, data not shown).


Inhibitory effects of cholinesterase inhibitor donepezil on the Kv1.5 potassium channel
The blocking effect of donepezil on Kv1.5 currents.(a) Example traces of Kv1.5 currents were obtained in response to a 500 ms depolarizing pulse to potentials from −80 to +60 mV from a holding potential of −80 mV under control, 80 μM donepezil and washout conditions. Arrows denoted the zero-current level. (b) Current-voltage (I–V) relationships of Kv1.5 currents with and without donepezil (n = 11). (c) Bar graph of statistical analyses of block by donepezil in Kv 1.5 channels at +60 mV. *p < 0.05 compared with the control group. (d) The concentration-response relations of donepezil action on Kv1.5 currents. Data points with various concentration donepezil were fitted by a Hill equation (n = 6). (e) Mutant R476V but not H452G reduced the affinity of donepezil to Kv1.5 channels. Collected raw data were normalized to the control, and then were plotted with respect 80 μM donepezil (n = 5). Unless otherwise noted, WT indicates wild type in all figures. *p < 0.05 compared with the wild type group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304190&req=5

f1: The blocking effect of donepezil on Kv1.5 currents.(a) Example traces of Kv1.5 currents were obtained in response to a 500 ms depolarizing pulse to potentials from −80 to +60 mV from a holding potential of −80 mV under control, 80 μM donepezil and washout conditions. Arrows denoted the zero-current level. (b) Current-voltage (I–V) relationships of Kv1.5 currents with and without donepezil (n = 11). (c) Bar graph of statistical analyses of block by donepezil in Kv 1.5 channels at +60 mV. *p < 0.05 compared with the control group. (d) The concentration-response relations of donepezil action on Kv1.5 currents. Data points with various concentration donepezil were fitted by a Hill equation (n = 6). (e) Mutant R476V but not H452G reduced the affinity of donepezil to Kv1.5 channels. Collected raw data were normalized to the control, and then were plotted with respect 80 μM donepezil (n = 5). Unless otherwise noted, WT indicates wild type in all figures. *p < 0.05 compared with the wild type group.
Mentions: After transfection of Kv1.5 channel plasmids in HEK293 cells, whole-cell recording was carried out to assess the effect of donepezil on corresponding currents. In the presence of 80 μM donepezil, a significant decrease was detected in Kv1.5 currents, which were elicited by depolarizing the cell from a holding potential of −80 mV to a test potentials ranging from −80 to +60 mV. This action was partially reversed following washout of the extracellular donepezil (Fig. 1a). The current-voltage (I–V) relationships of Kv1.5 currents in control, donepezil and washout are shown in Fig. 1b. The significant alterations of current amplitudes among the control, donepezil and washout were detected at different voltages between 0 mV to +60 mV (n = 11, ANOVA, p < 0.05). In Fig. 1c, the statistical analyses of block by donepezil at +60 mV are shown as the bar graph. Another cholinesterase inhibitor tacrine, however, barely affected the Kv1.5 currents, even in the higher concentration (200 μM, data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Kv1.5 channels carry ultra-rapid delayed rectifier K+ currents in excitable cells, including neurons and cardiac myocytes. In the current study, the effects of cholinesterase inhibitor donepezil on cloned Kv1.5 channels expressed in HEK29 cells were explored using whole-cell recording technique. Exposure to donepezil resulted in a rapid and reversible block of Kv1.5 currents, with an IC50 value of 72.5&thinsp;&mu;M. The mutant R476V significantly reduced the binding affinity of donepezil to Kv1.5 channels, showing the target site in the outer mouth region. Donepezil produced a significant delay in the duration of activation and deactivation, and mutant R476V potentiated these effects without altering activation curves. In response to slowed deactivation time course, a typical crossover of Kv1.5 tail currents was clearly evident after bath application of donepezil. In addition, both this chemical and mutant R476V accelerated current decay during channel inactivation in a voltage-dependent way, but barely changed the inactivation and recovery curves. The presence of donepezil exhibited the use-dependent block of Kv1.5 currents in response to a series of depolarizing pulses. Our data indicate that donepezil can directly block Kv1.5 channels in its open and closed states.

No MeSH data available.


Related in: MedlinePlus