Molecular determinants of Kv1.5 channel block by diphenyl phosphine oxide-1.

Kv1.5 channels conduct the ultra-rapid delayed rectifier current (I(Kur)) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Diphenyl phosphine oxide-1 (DPO-1) is a novel and potent inhibitor of Kv1.5 potassium channels. The present study was undertaken to characterize the mechanisms and molecular determinants of channel block by DPO-1. Experiments were carried out on wild-type and mutant Kv1.5 channels expressed in Xenopus laevis oocytes using the standard two microelectrode voltage clamp technique. DPO-1 blocked Kv1.5 current in oocytes with an IC(50) of 0.78+/-0.12 microM at +40 mV. Block was enhanced by higher rates of stimulation, consistent with preferential binding of the drug to the open state of the channel. Ala-scanning mutagenesis of the pore domain of Kv1.5 identified the residues Thr480, Leu499, Leu506, Ile508, Leu510 and Val514 as components of the putative binding site for DPO-1, partially overlapping the site previously defined for the Kv1.5 channel blockers AVE0118 and S0100176. Block of Kv1.5 by DPO-1 was significantly reduced in the presence of Kvbeta1.3.

Quercetin activates human Kv1.5 channels by a residue I502 in the S6 segment.

1. The aims of the present study were to investigate the pharmacological effects of quercetin on wild-type (WT) and mutant (I502A) human (h) Kv1.5 channel currents (I(kur)) and to identify whether mutation in the S6 segment is critical to activation of I(kur) by quercetin. 2. Experiments were performed on WT and site-directed mutant hKv1.5 channels, which were stably expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. 3. Quercetin increased WT hKv1.5 channel current in a concentration-, voltage- and time-dependent manner, with an EC(50) of 37.8 micromol/L and a negative shift in the steady state activation and inactivation curves. Quercetin accelerated channel activation and inactivation, significantly decreasing activation and inactivation time constants. However, mutating the I502 residue to Ala abolished the activating effect of quercetin. Quercetin did not modify the activation and inactivation kinetics of I502A channels. As an anti-oxidant, tanshinone IIA (4 micromol/L) inhibited the H(2)O(2)-induced activation of WT hKv1.5 channels. In contrast, quercetin had no significant effect. 4. We conclude that: (i) quercetin preferentially binds to and increases the current amplitude of WT hKv1.5 channels; (ii) Ile502, an aliphatic and neutral amino acid residue residing in the S6 segment, is important in quercetin binding; and (iii) quercetin-induced changes in the properties of WT hKv1.5 channels may be foreign to its own anti-oxidant action.

Electropharmacological properties of telmisartan in blocking hKv1.5 and HERG potassium channels expressed on Xenopus laevis oocytes.

AIM: The objectives of this study were to investigate the inhibitory action of telmisartan, a selective angiotensin II type 1 receptor antagonist, on hKv1.5 and human ether-a-go-go-related gene (HERG) channels expressed on Xenopus laevis oocytes. METHODS: hKv1.5 and HERG channels were expressed on Xenopus laevis oocytes and studied using the 2-microelectrode voltage clamp technique. RESULTS: In hKv1.5 channels, telmisartan produced a voltage- and concentration-dependent inhibition; the efficacies of blockade were different at peak and 1.5 s end-pulse currents, which were 7.75%+/-2.39% (half-maximal inhibition concentration [IC50]=2.25+/-0.97 micromol/L) and 52.64%+/-3.77% (IC50=0.82+/-0.39 micromol/L) at 1 micromol/L telmisartan, respectively. Meanwhile, telmisartan accelerated the inactivation of the channels. However, telmisartan exhibited a low affinity for HERG channels (IC50=24.35+/-5.06 micromol/L); the blockade was voltage- and concentration-dependent. Telmisartan preferentially blocked open-state HERG channels. The slow time constants of deactivation were accelerated (n=6, P<0.05), which was inconsistent with the "foot-in-the-door"effect. CONCLUSION: Telmisartan blocks hKv1.5 potassium channels involving open and inactivated states at plasma concentration levels of therapeutic doses; whereas the blockade of HERG channels occurs only at supra plasma concentration levels of therapeutic doses and preferentially in open and closed-state channels.

Blockade of the human ether-a-go-go-related gene potassium channel by ketanserin.

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.

Pharmacological or genetic inhibition of LDHA reverses tumor progression of pediatric osteosarcoma.

Reprogrammed energy metabolism is an emerging hallmark of cancer. Lactate dehydrogenase A (LDHA), a key enzyme involved in anaerobic glycolysis, is frequently deregulated in human malignancies. However, limited knowledge is known about its roles in the progression of osteosarcoma (OS). In this study, we found that LDHA is commonly upregulated in four OS cell lines compared with the normal osteoblast cells (hFOB1.19). Treatment with FX11, a specific inhibitor of LDHA, significantly reduced LDHA activity, and inhibited cell proliferation and invasive potential in a dose dependent manner. Genetic silencing of LDHA resulted in a decreased lactate level in the culture medium, reduced cell viability and decreased cell invasion ability. Meanwhile, silencing of LDHA also compromised tumorigenesis in vivo. Furthermore, knockdown of LDHA remarkably reduced extracellular acidification rate (ECAR) as well as glucose consumption. In the presence of 2-DG, a glycolysis inhibitor, LDHA-mediated cell proliferation and invasion were completely blocked, indicating the oncogenic activities of LDHA may dependent on Warburg effect. Finally, pharmacological inhibition of c-Myc or HIF1α significantly attenuated LDHA expression. Taken together, upregulated LDHA facilitates tumor progression of OS and might be a potential target for OS treatment.

[The persistent expression of HERG channel in Xenopus oocyte and alteration of current].

AIM: 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. METHODS: 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. RESULTS: (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. CONCLUSION: 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.

High extracellular potassium ion concentration attenuates the blockade action of ketanserin on Kv1.3 channels expressed in xenopus oocytes.

BACKGROUND: 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. METHODS: Kv1.3 channels were expressed in xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. RESULTS: 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). CONCLUSIONS: 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.

Verapamil blocks HERG channel by the helix residue Y652 and F656 in the S6 transmembrane domain.

AIM: The objectives of this study were to investigate the inhibitory action of verapamil on wild-type(WT) and mutation HERG K+ channel current (I(HERG)), and to determine whether mutations in the S6 region are important for the inhibition of I(HERG) by verapamil. METHODS: HERG channels (WT, Y652A, and F656A) were expressed in oocytes of Xenopus laevis and studied using the 2-electrode voltage- clamp technique. RESULTS: WT HERG is blocked in a concentration-dependent manner by verapamil (half-maximal inhibition concentration [IC(50)]=5.1 micromol/L), and the steady state activation and inactivation parameters are shifted to more negative values. However, mutation to Ala of Y652 and F656 located on the S6 domain produced 16-fold and 20-fold increases in IC(50) for IHERG blockade, respectively. Simultaneously, the steady state activation and inactivation parameters for Y652A are also shifted to more negative values in the presence of the blockers. CONCLUSION: Verapamil preferentially binds to and blocks open HERG channels. Tyr-652 and Phe-656, 2 aromatic amino-acid residues in the inner (S6) helix, are critical in the verapamil-binding site.