烏頭鹼是烏頭屬植物中的高毒性強鹼物質，許多研究指出烏頭鹼對於許多中樞神經系統相關的疾病具有治療的效果。過去相關的研究報告認為烏頭鹼會增加納離子電流造成動作電位延長，對動作電位的再極化時期也有影響。然而，到目前為止，烏頭鹼對於鉀離子通道的研究卻很少被提出。因此，我們利用膜片箝制技術(patch-clamp technique)去探討烏頭鹼對於已分化的NG108-15神經細胞其細胞膜上的離子電流及細胞膜電位的影響。從實驗中，我們首先發現烏頭鹼會抑制NG108-15神經細胞上的延遲修正性鉀離子電流，且此抑制作用具有濃度依賴性，在3.1微莫耳濃度之下能達到百分之五十的抑制效果。雖然烏頭鹼對延遲修正性鉀離子電流的抑制作用具有時間依賴性但卻幾乎沒有電壓依賴性。我們觀察到，烏頭鹼對於延遲修正性鉀離子電流最初的活化階段並無影響，其作用主要是會加速電流的去活化速率。從延遲修正性鉀離子電流的去活化曲線，我們也發現在烏頭鹼的作用之下，去活化曲線斜率並無改變但曲線中點平行地向過極化的電壓方向左移。進一步分析烏頭鹼抑制延遲修正性鉀離子電流後所需的回復時間，利用單一指數計算出的數值為2.6秒。烏頭鹼對於NG108-15神經細胞的鈉離子電流則有抑制作用。從反轉錄聚合酶鏈鎖反應的結果可以看出，長時間以烏頭鹼處理NG108-15神經細胞並不會影響KV3.1鉀離子通道的轉錄表現。為了瞭解烏頭鹼對於神經細胞興奮性的影響，我們利用電流箝制技術，記錄到NG108-15神經細胞的動作電位，發現烏頭鹼會增加NG108-15神經細胞動作電位產生的頻率，對於動作電位也具有延長的顯著效果。從我們一連串的實驗可以總結出烏頭鹼對於延遲修正性鉀離子電流的抑制作用具有時間、濃度及階段上的依賴性。烏頭鹼的確會影響神經細胞的動作電位且其作用機制可由其抑制了延遲修正性鉀離子電流及鈉離子電流來解釋。

Aconitine (ACO), a highly toxic alkaloid occurring in plants of the Aconitum genus, is recognized for its phytomedical effects on the central nervous system. It was previously reported that ACO increased amplitude of persistent Na+ current which underlies the prolongation of action potentials. However, little information is available regarding the actions of ACO on voltage-gated K+ channels. In this study, we used the patch-clamp technique to investigate the effects of ACO on ion currents and membrane potential in differentiated NG108-15 neuronal cells. We clearly demonstrated that ACO (0.3-30 M) suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in a concentration-dependent manner with an IC50 value of 3.1 M in these cells. The inhibitory effect of ACO on IK(DR) in NG108-15 cells showed little or no voltage dependence, although a time-dependent block could be observed. Moreover, exposure to ACO was found to enhance the rate and extent of IK(DR) inactivation, although it had no effect on the initial activation phase of IK(DR). ACO could also shift the inactivation curve of IK(DR) to a hyperpolarized potential with no change in the slope factor. Recovery from block by ACO (3 M) was fitted by a single exponential with a value of 2.6 s. ACO (10 M) had an inhibitory effect on voltage-dependent Na+ current (INa). RT-PCR results unraveled that no change in the transcriptional level of the KV3.1 channel was observed in chronic treatment with ACO (10 M). Under current-clamp recordings, we found that ACO increased the firing and widening of action potentials in NG108-15 cells. Taken together, our results clearly show that ACO can block delayed-rectifier K+ channels of neurons in a time-, concentration-, and state-dependent manner. Changes in action potentials induced by ACO or its structurally related compounds in neurons in vivo can be explained mainly by their blocking actions on IK(DR) and INa.

Ameri, A., 1998. The effects of Aconitum alkaloids on the central nervous system. Prog. Neurobiol. 56: 211-235.
Ameri, A., Shi, O., Aschoff, J., Peters, T., 1996. Electrophysiological effects of aconitine in rat hippocampal slices. Neuropharmacology 35, 13-22.
Amran, M.S., Hashimoto, K., Homma, N., 2004. Effects of sodium-calcium exchange inhibitors, KB-R7943 and SEA0400, on aconitine-induced arrhythmias in guinea pigs in vivo, in vitro, and in computer simulation studies. J. Pharmacol. Exp. Ther. 310, 83-89.
Baukrowitz, T., Yellen, G., 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15, 951-960.
Brown, D.A., Higashida, H., 1988. Voltage and calcium-activated potassium currents in mouse neuroblastoma × rat glioma hybrid cells. J. Physiol. 397, 149-165.
Decher, N., Kumar, P., Gonzalez, T., Pirard, B., Sanguinetti, M.C., 2006. Binding site of a novel KV1.5 blocker: a “foot in the door” against atrial fibrillation. Mol. Pharmacol. 70, 1204-1211.
Fernandez, F.R., Morales, E., Rashid, A.J., Dunn, R.J., Turner, R.W., 2003. Inactivation of KV3.3 potassium channels in heterologous expression systems. J. Biol. Chem. 278, 40890-40898.
Friese, J., Gleitz, J., Gutser, U.T., Heubach, J.F., Matthiesen, T., Wilffert, B., Selve, N., 1997. Aconitum sp. alkaloids: the modulation of voltage-dependent Na+ channels, toxicity and antinociceptive properties. Eur. J. Pharmacol. 337, 165-174.
Fu, M., Wu, M., Qiao, Y., Wang, Z., 2006. Toxicological mechanisms of Aconitum alkaloids. Pharmazie 61, 735-741.
Hardick, D.J., Cooper, G., Scott-Ward, T., Blagbrough, I.S., Potter, B.V., Wonnacott, S., 1995. Conversion of the sodium channel activator aconitine into a potent 7-selective nicotinic ligand. FEBS Lett. 365, 79-82.
Hernandez-Pineda, R., Chow, A., Amarillo, Y., Moreno, H., Saganich, M., de Miera, E.V., Hernandez-Cruz, A., Rudy, B., 1999. KV3.1-KV3.2 channels underlie a high-voltage-activating component of the delayed-rectifier K+ current in projecting neurons from the globus pallidus. J. Neurophysiol. 82, 1512-1528.
Kawaguchi, A., Asano, H., Matsushima, K., Wada, T., Yoshida, S., Ichida, S., 2007. Enhancement of sodium current in NG108-15 during neural differentiation is mainly due to an increase in Nav1.7 expression. Neurochem. Res. 32, 1469-1475.
Klemic, K.G., Kirsch, G.E., Jones, S.W., 2001. U-type inactivation of KV3.1 and Shaker potassium channels. Biophys. J. 81, 814-826.
Lien, C.C., Jonas, P., 2003. KV3 potassium conductance is necessary and kinetically optimized for high-frequency action potential generation in hippocampal interneurons. J. Neurosci. 23, 2058-2068.
Ling, K.K., Siow, N.L., Choi, R.C., Tsim, K.W., 2005. ATP potentiates the formation of AChR aggregate in the co-culture of NG108-15 cells with C2C12 myotubes. FEBS Lett. 579, 2469-2474.
Lo, Y.K., Chiang, H.T., Wu, S.N., 2003. Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid, on ion currents in NG108-15 neuronal cells. Biochem. Pharmacol. 65, 581-591.
Lowe, L., Matteucci, M.J., Schneir, A.B., 2005. Herbal aconite tea and refractory ventricular tachycardia. New. Engl. J. Med. 353, 1532.
Marom, S.,1998. Slow changes in the availability of voltage-gated ion channels: effects on the dynamics of excitable membranes. J. Membr. Biol. 161, 105-113.
Marom, S., Goldstein, S.A., Kupper, J., Levitan, I.B., 1993. Mechanism and modulation of inactivation of the KV3 potassium channels. Receptors Channels 1, 81-88.
McGehee, D.S., Role, L.W., 1995. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57, 521-546.
Meves, H., Schwarz, J.R., Wulfsen, I., 1999. Separation of M-like current and ERG current in NG108-15 cells. Br. J. Pharmacol. 127, 1213-1223.
Pubill, D., Vergaguer, E., Canudas, A.M., Sureda, F.X., Escubedo, E., Camarasa, J., Pallàs, M., Camins, A., 2001. Orphenadrine prevents 3-nitropropionic acid-induced neurotoxicity in vitro and in vivo. Br. J. Pharmacol. 132, 693-702.
Rasmusson, R.L., Morales, M.J., Wang, S., Liu, S., Campbell, D.L., Brahmajothi, M.V., Strauss, H.C. 1998. Inactivation of voltage-gated cardiac K+ channels. Circulation Research 82, 739-750.
Rudy, B., McBain, C.J., 2001. KV3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing. Trends Neurosci. 24,
517-526.
Sacco T, De Luca A, Tempia F., 2006. Properties and expression of KV3 channels in cerebellar Purkinje cells. Mol Cell Neurosci. 33, 170-179.
Tateno, T., Robinson, H.P., 2007. Quantifying noise-induced stability of a cortical fast-spiking cell model with KV3-channel-like current. Biosystems 89, 110-116.
Tojima, T., Kobayashi, S., Ito, E., 2003. Dual role of cyclic AMP-dependent protein kinase in neuritogenesis and synaptogenesis during neuronal differentiation. J. Neurosci. Res. 74, 829-837.
Tsai, T.Y., Tsai, Y.C., Wu, S.N., Liu, Y.C., 2006. Tramadol-induced blockade of delayed rectifier potassium current in NG108-15 neuronal cells. Eur J Pain 10, 597-601.
Wang, S.Y., Wang, G.K., 2003. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cell. Signalling 15, 151-159.
Wright, S.N., 2001. Irreversible block of human heart (hH1) sodium channels by the plant alkaloid lappaconitine. Mol. Pharmacol. 59, 183-192.
Wu, S.N., Chang H.D., 2006. Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH3 cells. Biochem. Pharmacol. 71, 615-623.
Wu, S.N., Lo, Y.K., Chen, H., Li, H.F., Chiang, H.T., 2001. Rutaecarpine-induced block of delayed rectifier K+ current in NG108-15 neuronal cells. Neuropharmacology 41, 834-843.
Xu, H., Arita, H., Hayashida, M., Zhang, L., Sekiyama, H., Hanaoka, K., 2006. Pain-relieving effects of processed Aconiti tuber in CCI-neuropathic rats. J. Ethnopharmacol. 103, 392-397.
Yamanaka, H., Doi, A., Ishibashi, H., Akaike, N., 2002. Aconitine facilitates spontaneous transmitter release at rat ventromedial hypothalamic neurons. Br. J. Pharmacol. 135, 816-822.
Yokoyama, S., Imoto, K., Kawamura, T., Higashida, H., Iwabe, N., Miyata, T., Numa, S., 1989. Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. FEBS Lett. 259, 37-42.