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Arylnaphthalene lignans from Taiwania cryptomerioides as novel blockers of voltage-gated [K.sup.+] channels.


Lignans are natural phytochemicals which exhibit multiple pharmacological effects such as anti-inflammation, antivirus and anti-tumor activities. Whether they have effects on neural tissues and ion channels is still unknown. The effects of several arylnaphathalene lignans purified from Taiwania cryptomerioides on voltage-gated [K.sup.+] (Kv) channels in mouse neuroblastoma N2A cells were examined. These lignans included Taiwanin E, helioxanthin (HXT) and diphyllin. All lignans showed inhibitory effects on Kv channels and HXT was the most potent compound ([IC.sub.50] = 1.7 [mu]M). The mechanism of HXT block was further investigated. Its action was found to be extracellular but not intracellular. HXT accelerated current decay, caused a left-shift in steady-state inactivation curve but had no effect on voltage-dependence of activation. HXT block was unaffected by intracellular [K.sup.+] concentrations. Further, it did not affect ATP-sensitive [K.sup.+] channels. Our data therefore suggest that HXT is a potent and specific blocker of Kv channels, possibly with an inhibitory mechanism involving acceleration of slow inactivation.


Keywords: Voltage-gated [K.sup.+] channels Arylnaphthalene lignans Helioxanthin Block

[C] 2010 Elsevier GmbH. All rights reserved.


Lignans are secondary metabolites found in leaves, stems, roots and seeds of a wide range of plants. This category of phytochemicals is derived from oxidative dimerization of two phenylpropanoid units (Pan et al. 2009). Their general antiviral, antibacterial and antifungal properties suggest their role as natural defensive compounds (Pan et al. 2009). Lignans also have multiple pharmacological effects. For instance, Taiwanin C has anti-inflammatory function as this lignan inhibits cyclooxygenase-1 and -2, thereby suppressing prostaglandin E2 production (Hong et al. 2008). Phyllanthusmin A, isolated from Phyllanthus oligospermus, exerts cytotoxic actions against KB and P-3S8 cancer cell lines (Wu and Wu 2006). Saucerneol D and machilin D isolated from Saururus chinensis have beneficial cardiovascular effects, namely, endothelium-dependent vasorelaxation and negative inotropic actions (Oh et al. 2008).

Not much is known about the effects of lignans on ionic channels. Heteroclitin D and gomisin J have been shown to inhibit cardiac L-type voltage-gated [Ca.sup.2+] channels (Zhang et al. 2000). Nordihydroguaiaretic acid (NDGA) has been shown to inhibit voltage-gated [K.sup.+] (Kv) currents in rat type I carotid body cells (Hatton and Peers 1997). NDGA also blocks Kv 1.5 channels (Gong et al. 2008). Interestingly, NDGA has also been demonstrated to activate [Ca.sup.2+] -dependent [K.sup.+] channels in rat type I carotid body cells (Hatton and Peers 1997) and porcine coronary arterial smooth muscle cells (Yamamura et al. 1999). It would be of interest to know if arylnaphthalene lignans would modulate Kv channels. Kv channels, by permitting [K.sup.+] efflux, are responsible for repolarization and thus regulate membrane potential of excitable tissues (Hille 2001). Kv channel blockers could enhance cellular excitability and are potential drugs for augmenting nerve conductivity in multiple sclerosis (Judge and Bever 2006) and stimulating insulin release in diabetes mellitus (Herrington et al. 2006).

The Kv channel [alpha]-subunit has four polypeptides and each polypeptide has six transmembrane helices (S1-S6) (Choe et al. 1999). With a P-loop between S5 and S6, the four polypeptides tetramerize in such a manner that the 4 P-loops form the [K.sup.+] selectivity filter of the central pore (Choe 2002; Yellen 2002). Sensing depolarization, S4 moves outward and subsequently S5-S6 helices undergo conformational changes, hence opening the cytoplasmic activation gate (Choe 2002; Yellen 2002). Kv channels are broadly classified into A-type [K.sup.+] channels and delayed rectifiers. A-type [K.sup.+] channels are fast-inactivating Kv channels with low activation thresholds (Hille 2001). The fast inactivation results from the plugging of the internal cavity by the cytoplasmic N-terminus ("ball-and-chain" mechanism) (Kurata and Fedida 2006). Delayed rectifiers are slow-inactivating Kv channels with higher activation thresholds. The slow inactivation (or C-type inactivation) involves distortion of the selectivity filter (Kurata and Fedida 2006).

In this work we investigated if arylnaphthalene lignans from Taiwania cryptomerioides modulated the delayed rectifier type Kv channels in mouse neuroblastoma N2A cells. The lignans studied included Taiwanin E, helioxanthin (HXT) and diphyllin (Fig. 1). All lignans inhibited Kv channels, with HXT being the most potent one. While the antiviral property of HXT has been established (Li et al. 2005; Tseng et al. 2008), we here show for the first time the ion channel-blocking effects of HXT. The mechanism of HXT block of Kv channels was investigated.


Materials and methods

Chemicals and cell culture

HXT, Taiwanin E and diphylline (Fig. 1) were purified from Taiwania cryptomerioides as previously described (Kuo et al. 1985; Chang et al. 2000). Diazoxide and glibenclamide were purchased from Sigma (St. Louis, MO). N2A cells were grown at 37 [degrees]C in 5% [CO.sub.2] in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and penicillin-streptomycin (100 units/ml, 100[mu]g/ml) (Invitrogen). Rat insulinoma RIN-m5F cells were cultured at 37[degrees]C in 5% [CO.sub.2] in Roswell Park Memorial institute 1640 medium (Gibco) with the same FBS and antibiotics supplements as in N2A cells.


Electrophysiological experiments were performed as previously reported (Leung et al. 2003; Chao et al. 2008). N2A cells and RIN-m5F cells were voltage-clamped in the whole-cell configuration. Thin-walled borosilicate glass tubes (o.d. 1.5 mm, i.d. 1.10 mm, Sutter Instrument, Novato, CA) were pulled with a micropipette puller (P-87, Sutter Instrument), and then heat polished by a microforge (Narishige Instruments, Inc., Sarasota, FL). The pipettes, filled with intracellular solution, containing (mM): 140 KCl, 1 [MgCl.sub.2], 1 EGTA, 10 HEPES, and 5 MgATP (pH 7.25 adjusted with KOH), had resistance of about 4-8 M[omega]. In the measurement of ATP-sensitive [K.sup.+] currents, ATP concentration was reduced to 1 mM in the intracellular solution. The extracellular solution contained (mM): 140 NaCl, 4 KCl, 1 [MgCl.sub.2], 2 [CaCl.sub.2], 10 HEPES (pH 7.4 adjusted with NaOH). The currents were recorded using an EPC-10 amplifier with Pulse 8.60 acquisition software and analyzed by Pulsefit 8.60 software (HEKA Electronik, Lambrecht, Germany). Data were filtered at 2 kHz and sampled at 10 kHz. After a whole-cell configuration was established, the cells were held at -70 mV and experimented with various protocols as detailed in the text and the figure legends. All experiments were performed at room temperature (~23 [degrees]C).

Concentration-response curves for drug inhibition are fitted by the Hill equation:

[[l.sub.drug]/[l.control]] = [1/[1 + [([drug]/[K.sub.d]).sup.n]]]

where [I.sub.drug] is the end-of-pulse current in the presence of drug, [I.sub.control] is the end-of-pulse current in the absence of drug, [drug] is the extracellular drug concentration, [K.sub.d] is the apparent dissociation constant and n is the Hill coefficient.

For constructing curves showing voltage-dependence of activation, Kv currents were stimulated with increasing depolarization, and conductance (G) was calculated as:

G = [I/[V - Vr]], where Vr = (RT/zF) ln (Ko/Ki)

V is the applied voltage, Vr is the reversal potential of [K.sup.+], I is the peak current, R is the universal gas constant, T is the temperature, z is the ion valency (+1 in this case) and F is the Faraday constant. Ko and Ki represent extracellular and pipette [K.sup.+] concentrations, respectively.

Data for voltage-dependence of activation and steady-state inactivation are fitted by the Boltzmann equation: [G/G.sub.max] = l/{l + exp[([V.sub.1/2]-V)/k]} (for fitting voltage-dependence of activation), or I/[I.sub.max] = 1/{1 + exp[(V - [V.sub.1/2])/k]} (for fitting steady-state inactivation), where [V.sub.1/2] is the half-maximal activation potential (for voltage-dependence of activation) or the half-maximal inactivation potential (for steady-state inactivation), and k the slope factor.

Statistical analysis

Data are presented as means [+ or -] SEM. The unpaired or paired Student's f-test was used where appropriate to compare two groups, and a value of p<0.05 was considered to represent a significant difference.


Inhibition of Kv currents by arylnaphthalene lignans

In this report we studied the effects of three arylnaphthalene lignans (Fig. 1) on Kv currents in N2A cells. Upon depolarization to +30 mV, outward Kv currents are triggered. These currents decay very slowly by a process called slow (or C-type) inactivation (Fig. 2; Chao et al. 2008). There is a wide range of sensitivity of Kv currents to arylnaphthalene lignans: Fig. 2 left panels show representative current traces in the absence and presence of different concentrations of Taiwanin E, diphyllin or HXT. Blockade was quantified as the inhibition of end-of-pulse currents by drugs. Concentration-inhibition curves are thus constructed and shown in the right panels. Taiwanin E and diphyllin have [IC.sub.50] values of 37 [mu]M and 7.1 [mu]M, respectively, and their Hill coefficients are 1.0 and 2.4, respectively. HXT was the most potent blocker with an [IC.sub.50] value of 1.7 [mu]M with a remarkably high Hill coefficient of 3.3, suggesting that there may be multiple binding sites for this compound. A]so of note is that HXT (3 [mu]M) inhibited Kv currents with a prominent acceleration of current decay (Fig. 2C left panel). A classical Kv channel blocker, tetraethylam-monium (TEA), was used as a positive control and at 10 mM this drug blocked 81.7 [+ or -]2.6% of end-of-pulse Kv currents in N2A cells.


HXT acted extracellularly to accelerate current decay

As HXT was shown to be the most potent compound, we proceeded to examine its mechanism of channel inhibition in more details. Whether HXT acted extracellularly or intracellulary was then investigated. A 6-min intracellular dialysis of 3 [mu]M HXT did not significantly affect the currents (inactivation time constants at time 0 and 6 min = 2.48 [+ or -]0.16s and 2.99[+ or -]0.49s, respectively; n = 3; p > 0.05), while a subsequent extracellular application of 3 [mu]M HXT caused a substantial inhibition by accelerating current decay (inactivation time constant at equilibrium = 0.51 [+ or -] 0.17s; n = 3; p < 0.05) (Fig. 3A and B). These data suggest that HXT acted extracellularly.


Inhibition of Kv channel by HXT is not via a direct pore block mechanism

To investigate if HXT blocked by directly occluding the outer channel pore, the effect of intracellular [K.sup.+] concentration on HXT action was examined. If HXT directly occludes the pore, then this compound and intracellular [K.sup.+] would collide within the ion conduction pathway. Thus, lowering intracellular [K.sup.+] concentration is expected to cause greater block. Reducing the intracellular [K.sup.+] concentration to 70 mM (with 140 mM sucrose added to maintain isoosmolarity) would be expected to enhance the effects of HXT. This, however, did not significantly affect the % block by 3 [mu]M HXT (Fig. 4), suggesting that HXT is unlikely to be a direct pore blocker.


Inactivation gating but not activation gating was affected by HXT

The results above are incompatible with the proposal that HXT directly blocks at the outer pore mouth. This compound may indeed inhibit Kv channels by enhancing the closing of the inactivation gate. We then investigated if it could affect the steady-state inactivation of the Kv currents (Fig. 5A). In the presence of 3 [mu]M HXT, the steady-state inactivation curve was significantly shifted to the left ([V.sub.1/2] = -24.1 [+ or -]3.1 mV in the absence of HXT but became -34.8[+ or -]1.4mV in the presence of HXT; n = 5-6: p < 0.05). HXT, however, did not affect the voltage-dependence of steady-state inactivation (slope factor = 6.2 [+ or -] 0.7 and 7.2 [+ or -] 1.3 in the absence and presence of HXT, respectively; p > 0.05).

It was next investigated if HXT affected the activation gating of the Kv currents. This drug (3 [mu]M) did not affect the voltage-dependence of activation (Fig. 5B).


HXT did not affect ATP-sensitive [K.sup.+] channel

To examine the specificity of HXT, we examined if HXT blocked [K.sup.+] channels devoid of C-type inactivation ([K.sub.ATP] channels). As shown in Fig. 6A, B and E, when RIN-m5F cells were dialyzed with 1 mM ATP via the recording pipette and stimulated by extracellular addition of 100 [mu]M diazoxide ([K.sub.ATP] channel opener), hyperpolarization-triggered inward [K.sup.+] currents gradually developed. The [K.sub.ATP] currents were insensitive to 10 [mu]MHXT(Fig. 6C and E), but could be rapidly inhibited by the potent and selective [K.sub.ATP] channel blocker, glibenclamide (Fig. 6D and E). These data suggest that HXT did not affect [K.sub.ATP] channels.



Present in many plant species, lignans are phytochemicals believed to be natural weapons against invasion by insects and microbes (Pan et al. 2009). In addition, these chemicals display a wide array of pharmacological activities (Wu and Wu 2006; Oh et al. 2008; Hong et al. 2008; Pan et al. 2009). Not much is known about the effects of lignans on ionic channels. The only plant lignan known to affect [K.sup.+] channels is NDGA. This lignan has been shown to inhibit Kv channels (Hatton and Peers 1997; Gong et al. 2008) and activate [Ca.sup.2+]-dependent [K.sup.+] channels (Hatton and Peers 1997; Yamamura et at. 1999). Some plant lignans, after being ingested into the human body, are metabolized by intestinal bacteria into mammalian lignans such as enterodiol and enterolactone (Lampe 2003). The effects of these mammalian lignans on [K.sup.+] channels are hitherto unknown.

In this work we examined whether arylnaphthalene lignans isolated from Taiwania cryptomerioides would affect neuronal ionic channels. For the first time we reported that they could inhibit Kv channels (Fig. 2). We investigated in more mechanistic details about the block by the most potent compound, HXT. HXT acted extracellularly, but not intracellular, to accelerate current decay (Fig. 3). This implies that a direct occlusion at the channel internal cavity is unlikely. Does HXT directly block the channel outer mouth, or inhibit by destabilizing the selectivity filter? If HXT directly occludes the channel pore, this drug and [K.sup.+] ions would collide in the ion conduction pathway. Thus, lowering the intracellular [K.sup.+] concentration would be expected to enhance HXT block of [K.sup.+] efflux. The % block by 3 [mu]M HXT was, however, not significantly affected by drastically reducing the intracellular [K.sup.+] concentration (Fig. 4). This observation is inconsistent with the notion of HXT being a direct channel pore blocker. The fact that HXT caused a left-shift in the steady-state inactivation curve (Fig. 5A) lends further support to the concept that this compound enhances the closing of the C-type inactivation gate. Hence, the manner by which HXT blocked Kv channels, namely, enhancing current decay, could be interpreted as HXT-induced acceleration of C-type inactivation gate closing.

Besides being the most potent Kv channel blocker of the aryl-naphthalene lignans we studied, HXT also displayed the highest Hill coefficient of block (3.3). This remarkably high Hill coefficient may imply multiple HXT binding sites exhibiting positive cooperativity.

[K.sub.ATP] channels are not voltage-activated, but are gated by intracellular ATP, intracellular pH, [Mg.sup.2+] and spermine (Bryan et al. 2004; Nichols 2006). These channels are mainly operative for metabolism-excitation coupling in neurons, cardiac myocytes and islet cells. Our results show that HXT action was selective, as it only inhibited Kv channels but did not affect KATP channels (Fig. 6). Our proposal above that HXT targets At the C-type inactivation gate is in agreement with the finding that HXT did not inhibit KATP channels, which are devoid of C-type inactivation.

We recently reported that 6[beta]-acetoxy-7[alpha]-hydroxyroyleanone (AHR), a diterpenoid compound isolated from Taiwania cryptomerioides, specifically acts on (accelerates) C-type inactivation gate of Kv channels without affecting the activation gate (Leung et al. 2010). This suggests AHR could be a useful pharmacological probe for the Kv channel inactivation gate. AHR inhibits Kv currents with an [IC.sub.50] of 18 [mu]M (Leung et al. 2010). In this report we showed that HXT, without affecting activation gating, could also be a selective probe for the C-type inactivation gate. More arylnaphthalene lignans will await screening in the future for the discovery of potent and selective Kv channel probes.

Specific Kv channel blockers are instrumental in elucidating the physiological roles of Kv channels. Of equal importance, pharmacological blockers of Kv channels may provide therapeutic remedy by virtue of their capability to enhance cellular excitability. Block of Kv channels by 4-aminopyridine (4-AP) offers opportunities in treating multiple sclerosis by augmenting conductivity of demyelinated axons (Judge and Bever 2006). Kv channel blockers also increase glucose-stimulated insulin secretion in [beta]-cells and are therefore anti-diabetic drug candidates (Herrington et al. 2006). Although much has been known of the antiviral properties of HXT and its analogues (Li et al. 2005; Tseng et al. 2008), whether HXT could be useful clinically as a Kv channel blocker warrants future exploration.

Neurons undergoing apoptosis have enhanced [K.sup.+] efflux due to Kv channel overexpression (Yu et al. 1997; Yu 2003). Kv channel blockers such as 4-AP and tetraethylammonium have been demonstrated to rescue neuronal apoptosis by preventing excessive [K.sup.+] efflux (Yu et al. 1997; Yu 2003; Hu et al. 2006). Upon depolarization and Kv channel opening, AHR and HXT begin destabilizing the selectivity filter (that is, hastening C-type inactivation) and thus suppress [K.sup.+] efflux (Leung et al. 2010; the present work). At optimal concentrations, these C-type inactivation-dependent blockers have relatively minor effects on peak currents whilst substantially suppress sustained currents (Leung et al. 2010; Figs. 2 and 3). C-type inactivation-dependent block is also observed in inhibition of Kv currents by KN-93, HMJ-53A, rhychophylline and verapamil (Rezazadeh et al. 2006; Chao et al. 2008; Chou et al. 2009; Kuras and Grissmer, 2009). A major merit of this fashion of blockage is that it allows an initial [K.sup.+] efflux but curbs [K.sup.+] outflow during prolonged depolarization. These C-type inactivation-dependent blockers could presumably suppress massive and sustained [K.sup.+] loss but would only minimally interfere with the fast repolarizing effect of Kv channels. The potential neuroprotective effects of these compounds await investigation.


Y.M.L. would like to thank China Medical University, Taiwan, and the Taiwan National Science Council for providing research funds (CMU97-340; CMU98-S-29; NSC 97-2320-B-039-029-MY3).


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Yuk-Man Leung (a), *, (1), Yi-Huan Tsoub (b), Chang-Shin Kuo (c), Shang-Ying Lin (b), Pau-Yen Wu (b), Mann-Jen Hour (b), Yueh-Hsiung Kuod (d), **, (1)

(a) Graduate Institute of Neural and Cognitive Sciences, China Medical University, 91 Hsueh Shih Road, Taichung 40402. Taiwan

(b) Department of Pharmacy, China Medical University. Taichung 40402, Taiwan

(c) Department of Nutrition, China Medical University, Taichung 40402, Taiwan

(d) Tsuzuki Institute for Traditional Medicine, China Medical University. Taichung 40402, Taiwan

* Corresponding author, Tel.: +886 4 22053366x2185; fax: +886 4 22076853.

** Corresponding author. Tel.: +886 4 220.53366x5701; fax: +886 4 22071693.

E-mail addresses: (Y.-M. Leung), (Y.-H. Kuo).

(1) Equally contributing corresponding authors.
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Author:Leung, Yuk-Man; Tsoub, Yi-Huan; Kuo, Chang-Shin; Lin, Shang-Ying; Wu, Pau-Yen; Hour, Mann-Jen; Kuo,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9TAIW
Date:Dec 15, 2010
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