Direct Activation of the Large-Conductance Calcium-Activated Potassium Channel by Flavonoids Isolated from Sophora flavescens

Abstract

In our recent study, we reported that kurarinone, one of the most abundant flavonoids found in the dry root of Sophora flavescens (Kushen), is a potent activator of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel. Herein, we isolated and characterized other flavonoid components from Kushen. Among the 13 compounds tested, six flavonoids were found to activate the BK_Ca channel, three of which, 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone, kuraridin, and kuraridinol, are new activators of the BK_Ca channel.

The large-conductance Ca²⁺-activated K⁺ channel known as the BK_Ca channel or Slo1 requires membrane depolarization and/or increased intracellular Ca²⁺ for its activation.^1,2) Channel opening creates a rapid efflux of K⁺ ions across the cell membrane. The BK_Ca channel is related to relaxation of urinary bladder smooth muscle (UBSM) and represents a therapeutic target for overactive bladder (OAB).^3,4) In our previous study, kurarinone was established as a novel activator of BK_Ca channel with therapeutic potential for treating OAB.⁵⁾ This plant flavonoid contains a flavanone backbone and is abundant in the root of Sophora flavescens AITON (Kushen, Leguminosae).^6,7)

S. flavescens AIT. is a medicinal herb widely distributed in northeast Asia.⁸⁾ Kushen contains diverse flavonoids including kurarinone, kushenol C, and leachianone G, and these individual compounds display anti-inflammatory, anti-microbial, and cytotoxic activities.^9,10) In the present study, we tested other flavonoids in Kushen for their potentiating effects on BK_Ca channel function. Of thirteen different flavonoids and alkaloids purified from Kushen, six compounds including kurarinone significantly increased channel activity. The BK_Ca channel was activated differentially by the six flavonoids, which variously caused a shift in the conductance–voltage (G-V) relationship to more negative voltages.

MATERIALS AND METHODS

Materials

Purification and verification of 13 individual compounds were reported previously.^11,12) A voucher specimen has been deposited at the Department of Food Science and Nutrition, Pukyong National University. Individual compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, U.S.A.) to generate stock solutions.

Cell-Based Fluorescence Assay and Data Analysis

In the cell-based assay, hyperactive mutant BK_Ca channels (G803D/N806K) were stably overexpressed in AD-293 cells derived from human embryonic kidney 293 cells, as previously described.^5,13) The FluxOR potassium channel assay (Invitrogen, U.S.A.) was used to measure the activity of the BK_Ca channel following the manufacturer’s instructions. Fluorescence signals were measured using FlexStation 3 (Molecular Devices, U.S.A.) and SoftMax Pro software was used for data transformation. Fluorescence signals were recorded every 2 s for 2 min. Measured fluorescence values were normalized against the basal level of each trace to give normalized values in relative fluorescence units (RFU). Activation by individual compounds was compared in terms of the rate of increase in the initial fluorescence estimated by linear fitting of the first three timepoints after addition of the stimulus solution. Data analysis was performed using the OriginPro 9.1 program (OriginLab Corp., U.S.A.).

Electrophysiological Recording and Data Analysis

Electrophysiological recording was performed using the α-subunit of the rat BK_Ca channel (Slo1) expressed in Xenopus laevis oocytes. The Gigaohm-seal patch-clamp method was used for current recordings in an outside-out configuration as previously described.^5,14) Currents were digitized at a rate of 10 or 20 points/ms. The resting potential was −100 mV, and BK_Ca channels were activated by voltage pulses from −80 to 200 mV in 10 mV increments. For recording, KMHG [120 mM potassium gluconate, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 4 mM KCl, and 5 mM ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (pH 7.2)] was used as both an extra- and intra-cellular (pipette) solution to suppress endogenous Ca²⁺-activated chloride currents in Xenopus oocytes. To provide the precise free concentration of intracellular Ca²⁺ ([Ca²⁺]_i), the appropriate amount of total Ca²⁺ to be added to the intracellular solution was calculated using MaxChelator software (http://maxchelator.stanford.edu/).¹⁵⁾ Data acquisition and analysis were performed using Clampex 8.0 and Origin 9.1. Data are presented as means±standard error of the mean (S.E.M.) with n replicates, and statistical differences were determined using the two-sample t-test in Origin 9.1. A p-value of <0.05 was considered statistically significant.

RESULTS

Cell-Based Fluorescence Assays of the Effects of Kushen Flavonoids on BK_Ca Activity

In the present study, we tested flavonoids purified from Kushen for their effects on BK_Ca channel activity using the aforementioned cell-based fluorescence assay (Fig. 1A). Among the 13 flavonoids tested, four compounds were described in our previous study.⁷⁾ At a final concentration of 10 µM, five compounds (7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone, kuraridin, kuraridinol, kushenol C, and leachianone G) were found to significantly activate the channel. Kuraridinol, a prenylated chalcone, exhibited the highest potency for channel activation among the Kushen flavonoids (Figs. 1A, 1B). The chemical structures of these compounds are shown in Fig. 1C and supplementary Fig. 1.

Fig. 1. Effects of Kushen Flavonoids on BK_Ca Channel Activity Based on Cell-Based Fluorescence Assays

A. Initial increase in RFU following addition of each of 13 flavonoids (n=4). B. Time-dependent RFU increase by the each of the six compounds exhibiting the strongest effect on BK_Ca channel activity. Symbols are as follows: 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (▼), kuraridin (◆), kuraridinol (hexagon), kurarinone (■), kushenol C (▲) and leachianone G (●) C. Chemical structures of the flavonoids. D–I. Initial RFU increases were plotted for 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (D), kuraridin (E), kuraridinol (F), kurarinone (G), kushenol C (H), and leachianone G (I). (n=4; * p<0.05, ** p<0.01, *** p<0.001).

The potentiating effects of six compounds including kurarinone were further investigated at different concentrations (1−30 µM). All compounds except kuraridinol progressively increased the fluorescence signal in a dose-dependent manner (Figs. 1D–1I). The fluorescence signal evoked by each compound was completely blocked by co-treatment with 1 µM paxilline, a known BK_Ca channel inhibitor, confirming that the Tl⁺ fluorescence induced by the compounds was due to the activation of BK_Ca channels.¹⁶⁾

Effects of Kushen Flavonoids on Macroscopic Currents of the BK_Ca Channel

We further validated the effects of the six Kushen flavonoids electrophysiologically. Macroscopic BK_Ca channel currents were evoked by voltage pulses from −80 to 200 mV in the absence and presence of extracellular flavonoids. Both outward and inward tail currents were increased by all six flavonoids at a concentration of 10 µM (Fig. 2A). However, the degree of potentiation varied among the different flavonoids. The effects of each flavonoid were further quantified by plotting the G-V relationship (Figs. 2B, 2C). All compounds shifted the G-V curve toward more negative voltage to varying degrees. In the presence of 10 µM compound, the shift in V_1/2 was −27 mV for 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone, −38 mV for kuraridin, −102 mV for kuraridinol, −54 mV for kurarinone, −12 mV for kushenol C, and −66 mV for leachianone G. All six flavonoids also increased the maximum conductance (G_max) by 1.12-fold for 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone, 1.16-fold for kuraridin, 1.33-fold for kuraridinol, 1.28-fold for kurarinone, 1.28-fold for kushenol C, and 1.38-fold for leachianone G (Fig. 2E). Thus, the results indicate that all six Kushen flavonoids can potentiate BK_Ca channel activation with varying degrees of potency.

Fig. 2. Effects of Kushen Flavonoids on BK_Ca Channel Macroscopic Currents

A. Representative BK_Ca channel current traces in the presence of different Kushen flavonoids. [Ca²⁺]_i was 3 µM and the pulse duration was 100 ms for each step. B,C. Effects of the flavonoid on the G-V relationship. Conductance was recorded at 0.8 s after termination of the stimulus pulse. Symbols are as follows: vehicle (□, n=25), 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (▼, n=6), kuraridin (hexagon, n=5), kuraridinol (◆, n=4), kurarinone (■, n=5), kushenol C (▲, n=5), and leachianone G (●, n=5). The Boltzmann equation (G/G_Max={(G_Max−G_Min)/(1+exp[(V_1/2−V) / k])}+G_Min) was used for data fitting. D. Effects of flavonoids on V_1/2 values. E. Effects of flavonoids on G_Max values (* p<0.05, ** p<0.01, *** p<0.001).

DISCUSSION

Kushen contains various natural compounds including flavonoids and alkaloids.¹⁷⁾ Since flavonoids are the most abundant class of chemicals in Kushen, and since kurarinone, a major flavonoid in Kushen, can potently active the BK_Ca channel, we investigated the effects of other Kushen flavonoids on channel activity.^5,18)

In addition to flavonoids with a flavanone backbone reported previously, two other flavonoids with a chalcone backbone, kuraridinol and kuraridin, were found to potently activate the BK_Ca channel.⁵⁾ Among these flavanones and chalcone pairs, we found that the chalcone displayed higher activity. In fact, no potentiation was observed for kurarinol in the present study or in our previous report.⁵⁾ Thus, opening of the C ring between O1 and C2 may allow freedom of rotation and the adoption of a conformation better suited to channel binding.

It is worth noting that the effect of kuraridinol on BK_Ca channel is biphasic. While kuraridinol potently activates the channel below 5 µM, higher concentrations of the compound inhibit the channel (Fig. 1F). This biphasic effect could be explained by the presence of more than two or more binding sites for the compound with opposite effects. Assuming two distinct binding sites, an ‘activation site’ of high affinity and an ‘inhibition site’ of lower affinity, we can envisage kuraridinol activating the channel by occupying the activation binding site at low concentrations and inhibiting the channel by binding to the inhibition site at higher concentrations. It remains to be further examined if such binding sites exist and how occupancy of these sites can evoke opposite functional consequences in terms of channel activity.

In summary, we showed that the chalcones kuraridinol and kuraridin activate the channel from the extracellular side. Since activation of the BK_Ca channel by kurarinone can induce the relaxation of UBSM and relieve the symptoms of OAB,⁵⁾ the flavonoid components described in this study could represent new BK_Ca channel-targeting agents for the treatment of diseases such as OAB syndrome.

Acknowledgments

This work was supported by Grants from the National Leading Research Laboratories supported by the National Research Foundation of Korea [2011-0028665] and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) [2017-352] to CSP.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Barrett JN, Magleby KL, Pallotta BS. Properties of single calcium-activated potassium channels in cultured rat muscle. J. Physiol., 331, 211–230 (1982).
• 2) Yang H, Zhang G, Cui J. BK channels: multiple sensors, one activation gate. Front Physiol., 6, 29 (2015).
• 3) Meredith AL, Thorneloe KS, Werner ME, Nelson MT, Aldrich RW. Overactive bladder and incontinence in the absence of the BK large conductance Ca²⁺-activated K⁺ channel. J. Biol. Chem., 279, 36746–36752 (2004).
• 4) dela Peña IC, Yoon SY, Kim SM, Lee GS, Ryu JH, Park CS, Kim YC, Cheong JH. Bladder-relaxant properties of the novel benzofuroindole analogue LDD175. Pharmacology, 83, 367–378 (2009).
• 5) Lee S, Chae MR, Lee BC, Kim YC, Choi JS, Lee SW, Cheong JH, Park CS. Urinary bladder-relaxant effect of kurarinone depending on potentiation of large-conductance Ca²⁺-activated K⁺ channels. Mol. Pharmacol., 90, 140–150 (2016).
• 6) Sun M, Han J, Duan J, Cui Y, Wang T, Zhang W, Liu W, Hong J, Yao M, Xiong S, Yan X. Novel antitumor activities of Kushen flavonoids in vitro and in vivo. Phytother. Res., 21, 269–277 (2007).
• 7) Shi YQ, Xin XL, Yuan QP, Wang CY, Zhang BJ, Hou J, Tian Y, Deng S, Huang SS, Ma XC. Microbial biotransformation of kurarinone by Cunninghamella echinulata AS 3.3400. J. Asian Nat. Prod. Res., 14, 1002–1007 (2012).
• 8) Zhang L, Liu W, Zhang R, Wang Z, Shen Z, Chen X, Bi K. Pharmacokinetic study of matrine, oxymatrine and oxysophocarpine in rat plasma after oral administration of Sophora flavescens Ait. extract by liquid chromatography tandem mass spectrometry. J. Pharm. Biomed. Anal., 47, 892–898 (2008).
• 9) Sohn HY, Son KH, Kwon CS, Kwon GS, Kang SS. Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai. Phytomedicine, 11, 666–672 (2004).
• 10) Jin JH, Kim JS, Kang SS, Son KH, Chang HW, Kim HP. Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens. J. Ethnopharmacol., 127, 589–595 (2010).
• 11) Jung HA, Jeong DM, Chung HY, Lim HA, Kim JY, Yoon NY, Choi JS. Re-evaluation of the antioxidant prenylated flavonoids from the roots of Sophora flavescens. Biol. Pharm. Bull., 31, 908–915 (2008).
• 12) Jung HA, Yokozawa T, Kim BW, Jung JH, Choi JS. Selective inhibition of prenylated flavonoids from Sophora flavescens against BACE1 and cholinesterases. Am. J. Chin. Med., 38, 415–429 (2010).
• 13) Lee BC, Kim HJ, Park SH, Phuong TT, Kang TM, Park CS. Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BK_Ca channel modulators. J. Biotechnol., 167, 41–46 (2013).
• 14) Ha TS, Lim HH, Lee GE, Kim YC, Park CS. Electrophysiological characterization of benzofuroindoleinduced potentiation of large-conductance Ca²⁺-activated K⁺ channels. Mol. Pharmacol., 69, 1007–1014 (2006).
• 15) Patton C, Thompson S, Epel D. Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium, 35, 427–431 (2004).
• 16) Sanchez M, McManus OB. Paxilline inhibition of the alpha-subunit of the high-conductance calcium-activated potassium channel. Neuropharmacology, 35, 963–968 (1996).
• 17) Han J, Sun M, Cui Y, Wang T, Zhang W, Guo M, Zhou Y, Liu W, Zhang M, Duan J, Xiong S, Yao M, Yan X. Kushen flavonoids induce apoptosis in tumor cells by inhibition of NF-κB activation and multiple receptor tyrosine kinase activities. Phytother. Res., 21, 262–268 (2007).
• 18) Kim HY, Jeong DM, Jung HJ, Jung YJ, Yokozawa T, Choi JS. Hypolipidemic effects of Sophora flavescens and its constituents in poloxamer 407-induced hyperlipidemic and cholesterol-fed rats. Biol. Pharm. Bull., 31, 73–78 (2008).