鉀離子通道的內向整流性質和不活化閥門氧化還原調控之研究

Studies on the Inward Rectification and the Redox Modulation of the Inactivation of Potassium Channels

謝其磐(Chi-Pan Hsieh)

指導教授 ： 郭鐘金

國立臺灣大學/醫學院/生理學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.01751

摘要

興奮性細胞上的各種鉀離子通道，可以調節細胞的膜電位和動作電位等電生理活性。本論文內容包括我們對Kir2.1鉀離子通道的研究，以及負責痛覺的背根神經節神經元上的A型鉀離子電流所做的電生理研究；此外，也有部份章節探討離子通透的理論機制。以下簡述本論文的三個主題：
1.我們在蛙卵上表現Kir2.1離子通道，以研究其受鋇離子阻斷時之內向整流的機制。Kir2.1離子通道具有很明顯的內向整流特性。在生理條件下，此離子通道的內向電流比外向電流大很多，一般認為，是因為外向電流受到細胞內的鎂離子和帶正電之多胺類分子阻斷所致。然而，內向整流的分子機轉尚未完全明朗。在鋇離子對此離子通道的阻斷研究中，我們發現細胞內側的鋇離子與通道的直觀親和力，會受到鉀離子電流方向的影響。動力學的分析顯示，結合速率常數(kon)受到鉀電流之驅動力影響。當電位在平衡電位(EK)以上+40 mV的範圍內，kon隨電位變大而急遽上升。解離速率常數(koff)在測試的電位範圍內則呈現單純線性的輕微電位依賴性。由此可知鋇離子阻斷的內向整流性質主要是來自kon在EK附近急遽增加所致，這可以用鉀離子在狹長孔洞中與鉀離子流之離子流耦合效應來解釋。Kir2.1通道的結構包括位於細胞內側的狹長孔洞，離子在此區域的電擴散或是擴散具有方向性，在此包含多個離子之狹窄長型孔洞內，離子流耦合效應使得鋇離子的運動受到鉀離子流的方向和大小影響。點突變研究顯示在T141附近有一高親和力之鋇離子的結合位置，很可能是內側鋇離子的結合位置。細胞外側鋇離子的阻斷研究顯示來自外側的鋇離子可能結合在另一個位置。我們也試著探討此通道孔洞內的鋇離子之結合位置和其結合能量的概廓，以及這些結合位置對應的等效電場距離。透過鋇離子的阻斷實驗，有助於了解生理情況下鎂離子和多胺類分子的阻斷機制，並進一步釐清內向整流性質的分子機轉。
2.Ussing在1949年提出了一個數學式來表示內向離子流和外向離子流在同一電位下的流量比。此巨觀的流量比在單一離子通道的層次可以視為正向的離子輸送(順著電化學梯度)與負向的離子輸送(逆著電化學梯度)的發生的機率比。1990年代，物理學家提出了漲落定理(fluctuation theorem)，此定理可以抽述微小系統在非平衡狀態下，在一小段時間內，正向熵產生和負向熵產生的機率比。我們若把離子通道和其相鄰的溶液視為一微小的非平衡系統，將擴散或電擴散視為一熵增加的非平衡過程，就可以由非平衡熱力學中的熵變化之基礎方程式，來表示出離子輸送過程中的熵變化。在此研究中，我們證實了Ussing提出的離子流量比方程式，可以由更廣義的漲落定理導出。由於過去數十年間，已有前人的實驗測量證實了Ussing離子流量比之適用性，此生理學的經驗方程式也為近年來才發展出的漲落定理提供了例證。此研究提供了一個例子，說明漲落現象如何影響奈米尺度生物分子機械的運作。
3.前人研究已知有幾種鉀離子通道(Kv1.4, Kv3.4, and Kvbeta1.1) 在體外表現系統中，其快速不活化(fast inactivation)閥門對氧化還原作用很敏感。然而，此項性質尚未在活體神經細胞中被證實過。我們研究氧化還原劑對痛覺背根神經節細胞上的A型鉀離子電流作用，發現其快速不活化過程對氧化還原劑非常敏感。半胱氨酸的氧化劑2,2'-dithio-bis(5-nitropyridine)(DTBNP)以及chloramine-T很顯著地移除了A型鉀通道的快速不活化性質。而雙硫鍵的還原劑dithiothreitol則可以恢復其不活化性質。根據前人的免疫螢光染色實驗和電生理實驗的結果，痛覺背根神經節上的A型鉀離子通道，很可能是由Kv1.4組成。前人的動物研究顯示，週邊組織的氧化還原狀態，會改變大鼠之痛覺的敏感性。我們的研究指出痛覺細胞上Kv1.4 通道可能為此種氧化還原調控的分子標的之一。在背根神經節裡，Kv1.4通道的分布具有選擇性，主要分布在負責痛覺的小型細胞；在疼痛藥理研究上，此離子通道或許可做為止痛藥的分子作用標的。

關鍵字

Kir2.1離子通道 ； 內向整流性質 ； Ussing流量比 ； 漲落定理 ； Kv1.4離子通道 ； 氧化還原調控

並列摘要

K+ channels play important roles in regulating the electrical activity in excitable cells. The thesis contains the electrophysiological studies on the cloned Kir2.1 channel in Xenopus oocytes expressing systems and studies on the native A-type K+ currents in pain-sensing dorsal root ganglion neurons. Theoretical studies on the mechanisms of ion conduction are also discussed. The three main topics are briefly introduced as the following.
1. The mechanisms of inward rectification in Ba2+ blockage were studied on the Kir2.1 channel expressed in Xenopus oocytes. The Kir2.1 channel is characterized by the strong inward rectification. The channel conducts much larger inward currents than outward currents in physiological conditions. The inward rectification has been attributed to the blockage of outward K+ currents by intracellular Mg2+ and polyamine. However, the molecular mechanisms of the inward rectification remain to be elucidated. By studies of the Ba2+ block of the channel, we found that the direction of K+ ion fluxes significantly influenced the apparent affinity of the block by internal Ba2+. The kinetic analysis revealed that the binding rate constants (kon) depended on the driving force. kon values increased steeply with voltage at the membrane potentials within +40 mV positive to the equilibrium potential (EK). The unbinding rate constants (koff) showed mild monotonic voltage dependence over the test voltages. The steep voltage dependence of kon near EK may result from the flux-coupling effect by the K+ ions moving in a narrow long pore region. The cytoplasmic pore of the Kir2.1 channel may serve as an anisotropic region for electrodiffusion and facilitate the flux-coupling effect. Mutagenesis studies revealed that the high-affinity binding site for internal Ba2+ was located near T141 at the internal entrance of the selectivity filter. External Ba2+ block was also studied. The energy profile of Ba2+ binding sites in the channel pore was constructed. The study of Ba2+ block would shed light on the mechanisms of inward rectification for physiological blockers such as Mg2+ and polyamines in inward rectifier K+ channels.
2. Ussing proposed the flux ratio equation in 1949 to describe the ratio of unidirectional outward and inward ion fluxes through membranes. The macroscopic flux ratio can be regarded as the ratio of the forward transport cycles (down the electrochemical gradient) and backward transport cycles (against the electrochemical gradient) in a single channel. The fluctuation theorem, which was developed by theoretical physicists in the early 1990s, relates the probabilities of positive entropy trajectories to negative entropy trajectories in a small system over a short period of time. We demonstrate that the Ussing flux ratio can be interpreted from the fluctuation theorem, without specifying the internal kinetic parameters of the flux system, if we consider the ion channel and the contacting solutions as a small nonequilibrium system. The entropy production of ion fluxes due to electrodiffusion can be expressed from the fundamental equation of the entropy change. Thus, the empirical flux ratio equation can be interpreted from the more general fluctuation theorem, and serves as a verification of the theorem. The study connects a classical formula in membrane physiology to a modern theorem in nonequilibrium thermodynamics of small systems. This is an example of how the thermal fluctuation plays a role in the operation of a nanometer-sized molecular machine.
3. Redox modulation of fast inactivation has been described in certain cloned A-type voltage-gated K+ (Kv) channels (Kv1.4, Kv3.4, and Kvbeta1.1) in expressing systems, but the effects remain to be demonstrated in native neurons. Previous immunofluorescence studies revealed high density of Kv1.4 in pain-sensing (small-diameter) dorsal root ganglion (DRG) neurons. In this study, we examined the effects of cysteine-specific redox agents on the A-type K+ currents in acutely dissociated pain-sensing DRG neurons from rats. The fast inactivation of most A-type currents was markedly removed or slowed by the oxidizing agents 2,2'-dithio-bis(5-nitropyridine) (DTBNP) and chloramine-T. Dithiothreitol, a reducing agent for the disulfide bond, restored the inactivation. These results demonstrated that native A-type K+ channels, probably Kv1.4, could switch the roles between inactivating and non-inactivating K+ channels via redox regulation in pain-sensing DRG neurons. The A-type channels may play a role in adjusting pain sensitivity in response to peripheral redox conditions. The Kv1.4 channel, which is selectively expressed in pain-sensing neurons, could be an interesting target for the physiology and pharmacology of pain.

並列關鍵字

Kir2.1 channel ； inward rectification ； Ussing flux ratio ； fluctuation theorem ； Kv1.4 channel ； redox modulation

參考文獻

Abrams, C.J., N.W. Davies, P.A. Shelton, and P.R. Stanfield. 1996. The role of a single aspartate residue in ionic selectivity and block of a murine inward rectifier K+ channel Kir2.1. J. Physiol. (London). 493 (Pt 3):643-649.

Alagem, N., M. Dvir, and E. Reuveny. 2001. Mechanism of Ba2+ block of a mouse inwardly rectifying K+ channel: differential contribution by two discrete residues. J. Physiol. (London). 534:381-393.

Baukrowitz, T., and G. Yellen. 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron. 15:951-960.

Begenisich, T., and D. Busath. 1981. Sodium flux ratio in voltage-clamped squid giant axons. J. Gen. Physiol. 77:489-502.

Begenisich, T., and P. De Weer. 1980. Potassium flux ratio in voltage-clamped squid giant axons. J. Gen. Physiol. 76:83-98.