Blockade of the human ether-a-go-go-related gene potassium channel by ketanserin / 生理学报

In the present study, we investigated the inhibitory action of ketanserin on wild-type (WT) and Y652 mutant human ether-a-go-go-related gene (HERG) potassium channels expressed in Xenopus oocytes and the effects of changing the channel molecular determinants characteristics on the blockade with and without ketanserin intervention using standard two-microelectrode voltage-clamp techniques. Point mutations were introduced into HERG gene (Y652A and Y652R) and subcloned into the pSP64 plasmid expression vector. Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe after linearization of the expression construct with EcoR I. Clampfit 9.2 software was employed for data collection and analysis. Origin 6.0 software was used to fit the data, calculate time constants and plot histograms. The results showed that ketanserin blocked WT HERG currents in voltage- and concentration-dependent manner and showed minimal tonic blockade of HERG current evaluated by the envelope of tails test. The IC50 value was (0.38+/-0.04) micromol/L for WT HERG potassium channel. The peaks of the I-V relationship for HERG channel suggested a negative shift in the voltage-dependence of activation after using ketanserin, whose midpoint of activation values (V1/2) were (-16.59+/-1.01) mV (control) vs (-20.59+/-0.87) mV (ketanserin) at 0.1 micromol/L, (-22.39+/-0.94) mV at 1 micromol/L, (-23.51+/-0.91) mV at 10 micromol/L, respectively (P<0.05, n=6). Characteristics of blockade were consistent with an open-state channel blockade, because the extent and rate of onset of blockade was voltage-dependent, increasing at more potentials even in the condition of leftward shift of activation curve. Meanwhile, in the different depolarization duration, the fractional blockade of end-pulse step current and peak tail current at 100 ms duration was significantly lower than that at 400 ms and 700 ms, which indicated that following the channel activation fractional blockade was enhanced by the activated channels. Ketanserin could also modulate the inactivation of HERG channel, which shifted the voltage-dependence of WT HERG channel inactivation curve from (-51.71+/-2.15) mV to (-80.76+/-14.98) mV (P<0.05, n=4). The S6 mutation, Y652A and Y652R, significantly attenuated the blockade by ketanserin. The IC50 value were (27.13+/-9.40) micromol/L and (20.20+/-2.80) micromol/L, respectively, increased by approximately 72-fold for Y652A and 53-fold for Y652R compared to that of WT HERG channel blockade [(0.38+/-0.04) micromol/L]. However, between the inhibitory effects of Y652A and Y652R, there was no significant difference. In conclusion, ketanserin blocks WT HERG currents in voltage- and concentration-dependent manner and preferentially blocks open-state HERG channels. Tyr-652 is one of the critical residues in the ketanserin-binding sites.

High extracellular potassium ion concentration attenuates the blockade action of ketanserin on Kv1.3 channels expressed in xenopus oocytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ketanserin (KT), a selective serotonin (5-HT) 2-receptor antagonist, reduces peripheral blood pressure by blocking the activation of peripheral 5-HT receptors. In this study electrophysiological method was used to investigate the effect of KT and potassium ion on Kv1.3 potassium channels and explore the role of blocker KT in the alteration of channel kinetics contributing to the potassium ion imbalances.</p><p><b>METHODS</b>Kv1.3 channels were expressed in xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique.</p><p><b>RESULTS</b>KCl made a left shift of activation and an inactivation curve of Kv1.3 current and accelerated the activation and inactivation time constant. High extracellular [K(+)] attenuated the blockade effect of KT on Kv1.3 channels. In the presence of KT and KCl the activation and inactivation time constants were not influenced significantly no matter what was administered first. KT did not significantly inhibit Kv1.3 current induced by tetraethylammonium (TEA).</p><p><b>CONCLUSIONS</b>KT is a weak blocker of Kv1.3 channels at different concentrations of extracellular potassium and binds to the intracellular side of the channel pore. The inhibitor KT of ion channels is not fully effective in clinical use because of high [K(+)](o) and other electrolyte disorders.</p>

The persistent expression of HERG channel in Xenopus oocyte and alteration of current / 中国应用生理学杂志

<p><b>AIM</b>To explore a method of the stable and persistent expression of HERG(human ether-a-go-go-related gene) channels in Xenopus oocytes, and investigate the alteration of rest membrane potential of oocytes and electrophysiological properties of expressed channel in different culture duration.</p><p><b>METHODS</b>HERG mRNA for injection was prepared with in intro transcription using vector plasmid pSP64 containing HERG cDNA fragment. Expressed HERG current was recorded using standard two-microelectrode voltage-clamp technique.</p><p><b>RESULTS</b>(1) Functional channels, with electrophysiological properties consistent with those of HERG channels were persistently expressed in oocytes membrane with this method. Furthermore, channel current could be recorded stably in 10-15 days. (2) The negative value of rest membrane potential increased gradually in the 3, 6, and 9 days of culture, and then decreased in the 12 days. The potential of peak value of inward rectification shifted gradually to the positive direction in 3, 6 and 9 days, and recovered in 12 days. Half-maximal activation potential (V1/2) of heterological expressed current shifted gradually to the negative direction in 3, 6 and 9 days of culture and then recovered in 12 days, the tendency of change was coincident with that of membrane rest potential.</p><p><b>CONCLUSION</b>The investigation provides a method of persistent expression of HERG channel in Xenopus oocytes and offers evidences for the difference of electrophysiological experimental data of studies of molecular site and drugs effect of HERG channel in different experimental conditions.</p>