Fig. 1：HMJ-53A 加速 N2A 細胞 Kv 電流慢性鈍化現象
N2A細胞的holding potential是-70mV，連續給予+30 mV去極化 刺激（持續3.5秒，間隔時間為20秒）。從結果中每一次刺激的電流 最高往最低點fit位置，而得到衰退 τ。
(A)典型的K+ 電流外流曲線，被+30mV去極化引起，分別於0秒和 經過600秒後，且未加任何藥物。
(B) 衰退時間常數從(A)圖得之，每個時間點被plot出 。
(C)典型的K+ 電流外流曲線，被+30 mV引起，加入HMJ-53A (30 μM) 之前後之記錄。
(D)衰退時間常數從(C)圖得之，於未加及已加HMJ-53A(30 μM)後之 每個時間點plot出。
(E)典型的K+ 電流外流曲線，被+30 mV引起，記錄於未加及已加 HMJ-53A(30 μM)，和從細胞外沖洗走HMJ-53A後，得之的曲 線。
(F)衰退時間常數從(E)圖得之，於未加、已加及沖洗走HMJ-53A (30 μM)之每個時間點plot出。
以上實驗得到的結果，可重複次數皆多於三個細胞以上。
51
52
Fig. 1
Fig. 2：HMJ-53A 抑制 Kv 電流濃度依賴曲線
細胞的 holding potential 為-70mV，連續給予+30mV 去極化刺 （持 續3.5 秒，間隔時間為 20 秒）。再加入不同濃度的 HMJ-53A （3、
6、10、18 及 30 μM），並給予同樣的刺激，記錄穩定狀態之 Kv 電流。
把標準化之穩定狀態電流（已加藥/未加藥），作 Y-軸，並把 HMJ-53A 之濃度作 X-軸。
曲線用 Hill 方程式 fit 之。結果為 mean ± SEM，從 3 至 6 個細胞 得之。
53
Fig. 2
54
Fig. 3：HMJ-53A 在轉殖 Kv2.1 後之 N2A 細胞上，加速 Kv 電流慢性鈍化現象
在已轉殖 Kv2.1 之 N2A 細胞，holding potential 為-70 mV，給予 連續+30 mV 去極化刺激（持續 3.5 秒，間隔時間為 20 秒）。得到 K v2.1 電流，再加入 HMJ-53A (30 μM)。曲線分別為未加藥及加藥 後 12 分鐘。
以上實驗得到的結果，可重複於三個細胞上。
55
56
Fig. 3
Fig. 4：從 N2A 細胞內加入 HMJ-53A 並不影響 Kv 電流之 慢性鈍化
(A) 在微細玻璃管電極之細胞內液中（細胞內）加入HMJ-53A (30
μM)。細胞的 holding potential是-70 mV，給予連續+30 mV去極 化刺激（持續3.5秒，間隔時間為20秒）。得到典型的K+ 電流外 流曲線，從0 分鐘至15 分鐘記錄。
再從細胞外，加入HMJ-53A (30 μM)，皆在同一個N2A細胞上進 行，得之曲線。
(B) 從結果中每一次刺激的電流最高往最低點fit，而得到衰退 τ。
衰退時間常數從(A)圖得之，於未加及已加HMJ-53A (30 μM)之每 個時間點plot出。
以上實驗得到的結果，可重複於三個細胞上。
57
58
Fig. 4
Fig. 5：HMJ-53A作用於N2A細胞之Kv通道之關閉階段：
(A) 細胞的holding potential為-70 mV，給予第一次+30mV去極化，
得之曲線。隨後給予此細胞HMJ-53A (30 μM)，等待4分鐘，並 此期間未給予任何去極化刺激。
(B) 此細胞給予第二次+30mV去極化，並記錄其曲線。
(C) 以灌流方式，從細胞外沖洗走HMJ-53A，並連續（持續3.5秒，
間隔時間為20秒）給予+30mV去極化並記錄其曲線。
以上實驗得到的結果，可重複在多於三個細胞以上。
59
Fig. 5
60
Fig. 6：HMJ-53A加劇電流之衰退，沒有電壓依賴性
(A) 細胞的holding potential為-70 mV，開始給予刺激，一次持續10 s，
間隔10秒，每增加10 mV刺激一次，至+100 mV為止，停止刺激。
得到典型的K+ 電流外流曲線，被不同的電壓去極化引起。
(B) 加入HMJ-53A (30 μM)，經過15分鐘後；同(A)刺激方式，測量 記錄曲線。得到典型的K+ 電流外流曲線，被不同的電壓去極化 引起。
(C) 從每一次刺激的電流之最高往最低點fit之，而得到衰退 τ。衰 退時間常數根據每個不同的去極化電壓plot出。
每一組結果的mean ± SEM，從4個細胞中得到。
61
62
Fig. 6
Fig. 7：HMJ-53A 阻斷 Kv 電流之強度不受電壓與細胞 內鉀離子濃度影響
(A) HMJ-53A (10 μM)阻斷穩定狀態 Kv 電流之百分比，根據每個不
同的電壓下 plot 出。
細胞的holding potential 為-70mV，開始給予刺激（一次持續 10 秒，間隔10 秒），每增加 10mV 刺激一次，至+100mV 為止，
停止刺激。再比較兩組其每個電壓中抑制的電流百分比（已加 藥/未加藥電流）×100％，所得到此曲線。
(B) HMJ-53A (10 μM)阻斷穩定狀態之Kv電流百分比。
兩組：微細玻璃管電極內（表示細胞內）鉀離子濃度分別為140 mM 和70 mM，細胞的 holding potential為-70mV，給予連續+30 mV去極化刺激（一個刺激持續3.5秒，間隔時間20秒）。抑制的 電流其百分比（已加藥/未加藥電流）×100％，再比較兩組的差 異。
(C) 在細胞內鉀離子濃度分別為 70 和 140 mM 時，Kv 電流的衰退時
間常數 [以加及未加 HMJ-53A (10 μM)]。
微細玻璃管電極內（表示細胞內）鉀離子濃度分別為140 mM和 70 mM。細胞的 holding potential是-70mV，給予連續+30 mV去
63
極化刺激，（一個刺激持續3.5秒，間隔時間為20秒）。再分為 未加及已加HMJ-53A(10 μM)兩大組，從結果中的電流最高至最 低點fit之，而得到衰退 τ。
每一組結果之mean ± SEM，從4至5個細胞中得到。
64
65
Fig. 7
Fig.8：TEA對電流衰退之影響與HMJ-53A不同
(A) 細胞的holding potential為-70 mV，給予連續+30 mV去極化刺激，
（一個刺激持續3.5 秒，間隔時間為20秒）。得到典型的K+ 電 流外流曲線，再加入TEA (3 mM)（20秒和600秒後）。
(B) 衰退時間常數：比較未加及已加TEA (3 mM)的結果。於電流最 高至最低點fit之，為於每個時間點之衰退 τ。
以上實驗得到的結果，可重複於三個細胞上。
66
67
Fig.8
Fig. 9：HMJ-53A 使穩定狀態之鈍化曲線左移 但不影響活化閥門
(A) 已加及未加HMJ-53A (30 μM)之N2A細胞中，進行Kv通道電流測
量，得之穩定狀態之鈍化曲線。在此實驗使用dual-pulse流程（給 予一次pre-pulse，再給予第二次短的pulse）做測量。
細胞的holding potential是-70 mV，先給予pre-pulse刺激（持續 10 s），再給予一個+70 mV的test pulse。休息10秒後，接著下一 個dual-pulse刺激 （於pre-pulse，每次增加10 mV），刺激至 pre-pulse為+100 mV，停止刺激。
而test pulse電流與最大之test pulse電流作標準化，兩組曲線比 較其差異。因為在對照組產生‘‘U型鈍化’’現象，所以只從-80至 +50mV fit（Boltzmann方程式）。結果之mean ± SEM從每組中4 個細胞中得之。
(B) 活化電壓依賴性：Kv 電流被不同電壓去極化刺激，而這些不同
的電壓，從holding potential為-70mV開始，每增加10mV刺激一 次（一次持續0.5秒，間隔2秒），至+70mV止。
Conductances (G)（conductance計算於材料方法中說明）在對 照 組 和 加 入HMJ-53A 組 中 ， 分 別 與 各 自 最 大 的 conductance
68
(Gmax)作標準化，在不同的去極化刺激下plot出結果。而此曲線 是以Boltzmann方程式去fit之。結果之mean ± SEM從每組中6個 細胞中得之。
(C) 較 早 階 段 的 電 流 traces 代 表 活 化 之 kinetics 。 細 胞 的 holding
potential為-70mV，給予連續+30 mV去極化刺激（一個刺激持續 3.5 秒，間隔時間20秒），再加入HMJ-53A。而標準化這兩組並 重疊其電流，來比較未加及已加HMJ-53A這兩組。
(D) 同 (C) 之 刺 激 方 式 ， 活 化 時 間 常 數 乃 於 電 流 上 升 階 段 作
exponential fit，於不同的去極化電壓，再plot出結果。而得到兩 組（未加及已加HMJ-53A）的活化曲線。
每組結果的mean ± SEM，從6至8個細胞中得到。
69
70
Fig. 9
Fig. 10：HMJ-53A不影響Kv電流之恢復
電流恢復實驗於N2A細胞中進行，用dual-pulse 流程，細胞的 holding potential為-70 mV，給予第一次pulse（+30 mV，持續5秒），
然後第二次pulse（+30 mV，持續200 ms）；第一次pulse與第二次 pulse間隔時間不同。
把第二個電流 (I2)與第一個電流 (I1)的最大值作標準化（I2/I1）。
再把此標準化結果根據不同之時間間隔而plot之。而此曲線用 double-exponential功能去fit。
兩組的結果的mean ± SEM，從每組中4個細胞中得到。
71
72
Fig. 10
Fig. 11：HMJ衍生物抑制Kv電流之濃度依賴性曲線
N2A細胞holding potential為-70 mV，給予連續+30 mV去極化刺激
（持續3.5秒，間隔時間20秒）。再加入HMJ-1 (30 μM)，得之電流，
與未加入HMJ-1最大的穩定狀態電流作標準化，再plot出此點。
同上刺激方式，分別加入不同濃度（3、6、10、18 及 30 μM）之 其它 HMJ 衍生物，得到之最大的穩定狀態電流與未加入 HMJ 衍生 物之最大的穩定狀態電流作標準化（Idrug/Icontrol），並依藥物之 濃度作 X 軸而 plot 之，曲線用 Hill 方程式 fit 之。
每組的結果 mean ± SEM，從 3 至 6 個細胞得之。
73
Fig. 11
74
100
1 10
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Idrug/Icontrol
Drug ( μ M)
HMJ-29A
HMJ-73A
HMJ-53A
HMJ-1
第六章：参考文獻
Andalib, P., Consiglio, J.F., Trapani, J.G., and Korn, S.J. (2004). The external TEA binding site and C-type inactivation in voltage-gated potassium channels. Biophys J 87, 3148-3161.
Baukrowitz, T., and Yellen, G. (1996). Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels. Proc Natl Acad Sci U S A 93, 13357-13361.
Bayliss, D.A., Talley, E.M., Sirois, J.E., and Lei, Q. (2001). TASK-1 is a highly modulated pH-sensitive 'leak' K(+) channel expressed in brainstem respiratory neurons. Respir Physiol 129, 159-174.
Bowlby, M.R., Fadool, D.A., Holmes, T.C., and Levitan, I.B. (1997).
Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases. J Gen Physiol 110, 601-610.
Berne, R.M., Levy, M.N., Koeppen B.M., and Stanton B.A., (1998).
Physiology. St. Louis, Missouri press.
75
Choe, S. (2002). Potassium channel structures. Nat Rev Neurosci 3, 115-121.
Choe, S., Kreusch, A., and Pfaffinger, P.J. (1999). Towards the three-dimensional structure of voltage-gated potassium channels.
Trends Biochem Sci 24, 345-349.
Choi, K.L., Aldrich, R.W., and Yellen, G. (1991). Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc Natl Acad Sci U S A 88, 5092-5095.
Choi, K.L., Mossman, C., Aube, J., and Yellen, G. (1993). The internal quaternary ammonium receptor site of Shaker potassium channels.
Neuron 10, 533-541.
Claydon, T.W., Vaid, M., Rezazadeh, S., Kehl, S.J., and Fedida, D.
(2007). 4-aminopyridine prevents the conformational changes associated with p/c-type inactivation in shaker channels. J Pharmacol Exp Ther 320, 162-172.
76
Cogolludo, A., Moreno, L., Bosca, L., Tamargo, J., and Perez-Vizcaino, F. (2003). Thromboxane A2-induced inhibition of voltage-gated K+
channels and pulmonary vasoconstriction: role of protein kinase Czeta.
Circ Res 93, 656-663.
Cordero-Morales, J.F., Jogini, V., Lewis, A., Vasquez, V., Cortes, D.M., Roux, B., and Perozo, E. (2007). Molecular driving forces determining potassium channel slow inactivation. Nat Struct Mol Biol 14, 1062-1069.
Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., and MacKinnon, R. (1998). The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69-77.
Fedida, D., Maruoka, N.D., and Lin, S. (1999). Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations. J Physiol 515 ( Pt 2), 315-329.
77
Goldstein, S.A., Price, L.A., Rosenthal, D.N., and Pausch, M.H. (1996).
ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93, 13256-13261.
Gulbis, J.M., Zhou, M., Mann, S., and MacKinnon, R. (2000). Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+
channels. Science 289, 123-127.
Holmgren, M., Jurman, M.E., and Yellen, G. (1996). N-type inactivation and the S4-S5 region of the Shaker K+ channel. J Gen Physiol 108, 195-206.
Hoshi, T., Zagotta, W.N., and Aldrich, R.W. (1990). Biophysical and molecular mechanisms of Shaker potassium channel inactivation.
Science 250, 533-538.
Hille, B., (2001). Ion channels of excitable membranes. Sunderland, Mass.: Sinauer press.
78
Hour, M.J., Huang, L.J., Teng, C.M., Kuo, S.C., (2000). Synthesis and antiplatelet activity of alkoxy derivatives of 2-phenylquinazolines. Chin.
Pharm. J. 52, 167-177.
Jahangir, A., and Terzic, A. (2005). K(ATP) channel therapeutics at the bedside. J Mol Cell Cardiol 39, 99-112.
Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B.T., and MacKinnon, R. (2003). X-ray structure of a voltage-dependent K+
channel. Nature 423, 33-41.
Kindler, C.H., Yost, C.S., and Gray, A.T. (1999). Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem. Anesthesiology 90, 1092-1102.
Kiss, L., Immke, D., LoTurco, J., and Korn, S.J. (1998). The interaction of Na+ and K+ in voltage-gated potassium channels. Evidence for cation binding sites of different affinity. J Gen Physiol 111, 195-206.
Klemic, K.G., Kirsch, G.E., and Jones, S.W. (2001). U-type inactivation of Kv3.1 and Shaker potassium channels. Biophys J 81, 814-826.
79
Kukuljan, M., Labarca, P., and Latorre, R. (1995). Molecular determinants of ion conduction and inactivation in K+ channels. Am J Physiol 268, C535-556.
Kurata, H.T., Doerksen, K.W., Eldstrom, J.R., Rezazadeh, S., and Fedida, D. (2005). Separation of P/C- and U-type inactivation pathways in Kv1.5 potassium channels. J Physiol 568, 31-46.
Kurata, H.T., and Fedida, D. (2006). A structural interpretation of voltage-gated potassium channel inactivation. Prog Biophys Mol Biol
92, 185-208.
Kurata, H.T., Soon, G.S., Eldstrom, J.R., Lu, G.W., Steele, D.F., and Fedida, D. (2002). Amino-terminal determinants of U-type inactivation of voltage-gated K+ channels. J Biol Chem 277, 29045-29053.
Kurata, H.T., Soon, G.S., and Fedida, D. (2001). Altered state dependence of c-type inactivation in the long and short forms of human Kv1.5. J Gen Physiol 118, 315-332.
80
Ledoux, J., Chartier, D., and Leblanc, N. (1999). Inhibitors of calmodulin-dependent protein kinase are nonspecific blockers of voltage-dependent K+ channels in vascular myocytes. J Pharmacol Exp Ther 290, 1165-1174.
Leung, Y.M., Ahmed, I., Sheu, L., Gao, X., Hara, M., Tsushima, R.G., Diamant, N.E., and Gaisano, H.Y. (2006). Insulin regulates islet alpha-cell function by reducing KATP channel sensitivity to adenosine 5'-triphosphate inhibition. Endocrinology 147, 2155-2162.
Leung, Y.M., Kang, Y., Gao, X., Xia, F., Xie, H., Sheu, L., Tsuk, S., Lotan, I., Tsushima, R.G., and Gaisano, H.Y. (2003). Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking. J Biol Chem 278, 17532-17538.
Lopez-Barneo, J., Hoshi, T., Heinemann, S.H., and Aldrich, R.W.
(1993). Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels
1, 61-71.
81
Loussouarn, G., Rose, T., and Nichols, C.G. (2002). Structural basis of inward rectifying potassium channel gating. Trends Cardiovasc Med 12, 253-258.
MacKinnon, R., Aldrich, R.W., and Lee, A.W. (1993). Functional stoichiometry of Shaker potassium channel inactivation. Science 262, 757-759.
McKay, M.C., and Worley, J.F., 3rd (2001). Linoleic acid both enhances activation and blocks Kv1.5 and Kv2.1 channels by two separate mechanisms. Am J Physiol Cell Physiol 281, C1277-1284.
Molina, A., Castellano, A.G., and Lopez-Barneo, J. (1997). Pore mutations in Shaker K+ channels distinguish between the sites of tetraethylammonium blockade and C-type inactivation. J Physiol 499
( Pt 2), 361-367.
Nerbonne, J.M. (2000). Molecular basis of functional voltage-gated K+
channel diversity in the mammalian myocardium. J Physiol 525 Pt 2, 285-298.
82
Oliver, D., Lien, C.C., Soom, M., Baukrowitz, T., Jonas, P., and Fakler, B. (2004). Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids. Science 304, 265-270.
Oxford, G.S., and Wagoner, P.K. (1989). The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents. J Physiol 410, 587-612.
Poolos, N.P., and Johnston, D. (1999). Calcium-activated potassium conductances contribute to action potential repolarization at the soma but not the dendrites of hippocampal CA1 pyramidal neurons. J Neurosci 19, 5205-5212.
Rezazadeh, S., Claydon, T.W., and Fedida, D. (2006). KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-c hlorocinnamyl)-N-methylbenzylamine),a
calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. J Pharmacol Exp Ther 317, 292-299.
83
Sheng, M., Liao, Y.J., Jan, Y.N., and Jan, L.Y. (1993). Presynaptic A-current based on heteromultimeric K+ channels detected in vivo.
Nature 365, 72-75.
Sheng, M., Tsaur, M.L., Jan, Y.N., and Jan, L.Y. (1994). Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain. J Neurosci 14, 2408-2417.
Shimada, H., Uta, D., Nabekura, J., and Yoshimura, M. (2007).
Involvement of Kv channel subtypes on GABA release in mechanically dissociated neurons from the rat substantia nigra. Brain Res 1141, 74-83.
Sun, H.S., Feng, Z.P., Barber, P.A., Buchan, A.M., and French, R.J.
(2007). Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo.
Neuroscience 144, 1509-1515.
Wang, S., Morales, M.J., Qu, Y.J., Bett, G.C., Strauss, H.C., and Rasmusson, R.L. (2003). Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect. J Physiol 546, 387-401.
84
Wang, Y.J., Lin, M.W., Lin, A.A., and Wu, S.N. (2008).
Riluzole-induced block of voltage-gated Na+ current and activation of BKCa channels in cultured differentiated human skeletal muscle cells.
Life Sci 82, 11-20.
Yamada, K., and Inagaki, N. (2005). Neuroprotection by KATP channels. J Mol Cell Cardiol 38, 945-949.
Yellen, G. (2002). The voltage-gated potassium channels and their relatives. Nature 419, 35-42.
Zagotta, W.N., Hoshi, T., and Aldrich, R.W. (1990). Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science 250, 568-571.
85
86
附 錄一：
SCI 期刊之
文章發表
HMJ-53A accelerates slow inactivation gating of voltage-gated K
þ
channels in mouse neuroblastoma N2A cellsChia-Chia Chao
a, Jeffrey Shieh
b, Sheng-Chu Kuo
c, Bor-Tsang Wu
d, Mann-Jen Hour
b,**, Yuk-Man Leung
a,e,*aDepartment of Physiology, China Medical University, Taichung 404, Taiwan, ROC
bSchool of Pharmacy, China Medical University, Taichung 404, Taiwan, ROC
cGraduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 404, Taiwan, ROC
dDepartment of Physical Therapy, China Medical University, Taichung 404, Taiwan, ROC
eGraduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung 404, Taiwan, ROC
a r t i c l e i n f o
Article history:
Received 7 January 2008
Received in revised form 10 March 2008 Accepted 12 March 2008
Keywords:
HMJ-53A
Voltage-gated Kþchannels C-type inactivation Electrophysiology Nerve cells
a b s t r a c t
Voltage-gated Kþ(Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely un-derstood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding
Voltage-gated Kþ(Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely un-derstood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding