- 學位論文
Kir2.1離子通道的內向整流特性及藥理調節之分子機制
Molecular Mechanisms Underlying the Inward Rectification and Pharmacological Modulation of the Kir2.1 Channel
摘要
內向整流性鉀離子通道(Kir通道)最主要特徵就是在生理情況下，會使往細胞膜內通過的鉀離子電導遠大於往細胞膜外通過的鉀離子電導，因此呈現向內整流的特性。從生理學的角度而言，這類型的內向整流性鉀離子通道所具有的離子電導特性，主要是既能維持細胞膜的靜止膜電位，將細胞膜的靜止膜電位接近於鉀離子的平衡電位。又能在進行去極化的過程中，避免細胞內過多的鉀離子排出細胞膜外。目前為止，認為此種內向整流性的特性，起因於細胞膜內的內生性多聚合胺(例如：精胺)阻塞Kir通道，因而阻塞鉀離子的外向電流。不過細胞膜內的內生性精胺造成阻塞的效果，並不能完全以電壓依賴特性來解釋，精胺阻塞Kir通道造成內向整流特性，很有可能起因於精胺本身和該離子通道內的可通透離子之間存在著流向偶合的特性。 本論文利用分子生物學突變技術、電生理記錄方式與分子動態模擬方式，探討與瞭解精胺在內向整流性Kir 2.1通道的結合位置、精胺和Kir 2.1通道內可通透性離子的作用、及Kir 2.1通道本身門閥開關機制三者之間的作用關係。研究結果發現精胺阻塞Kir 2.1的親和力曲線明顯會隨著鉀離子平衡電位的移動。精胺結合於Kir 2.1通道的動力學分析，發現當突變Kir 2.1通道的細胞膜內區域中E224和E299氨基酸後，明顯減小精胺的進入孔洞的結合速率，這現象反應出突變此兩個胺基酸後，會破壞精胺和可通透離子之間的流向依賴及流向偶合的性質。實驗指出無論是野生型Kir 2.1通道抑或是突變兩個胺基酸(E224及E299)後，精胺的結合速率和脫離速率具有少許的電壓依賴特性。藉由精胺在Kir 2.1通道內的動力學分析以及電壓依賴特性。我們發現精胺阻塞Kir 2.1的結合位置位於從該通道的細胞質區域算起約0.5電距離，並且精胺與通道內可通透的鉀離子流向偶合位置在於四個次單位蛋白螺旋交會區域。當鉀離子往細胞膜外流出後，精胺受鉀離子外向電流推往Kir 2.1通道中四個次單位蛋白螺旋交會區域最外端的位置(即D172)。另一方面，隨著鉀離子流向的改變(例如：向內電流)，則可以將精胺推往該區域較內端的位置(大約在M183到A184)。實驗也發現雙突變E224及A178(或是M183)胺基酸後，將會改變精胺在Kir 2.1通道中細胞質內結構和結合位置之間不對稱障礙的高度。而且在Kir 2.1通道的細胞質內區域結構中E224和E299極有可能和其他正電的胺基酸(例如：R228和R260)，透過異位性作用方式開啟該通道的四個次單位蛋白螺旋交會區域，讓精胺可以透過和鉀離子流向偶合作用阻塞該通道，進而造成內向整流特性。 Kir 2.1通道的四個次單位蛋白螺旋交會區域同時也是該通道門閥開關機制中極為重要區域。我們發現突變氨基酸A184R後，不僅會改變原有精胺阻塞該通道所造成的內向整流特性，更可以使得該通道孔洞趨向於關閉狀態，並且也發現細胞內的陽離子可以作用在該通道的細胞質內結構使A184R突變通道孔洞趨向開啟狀態。我們也進一步發現Kir 2.1通道孔洞可以同時結合兩個以上的精胺。會嚴重阻塞Kir 2.1通道的是位於較深位置的精胺，其位置與阻塞效果是具有流向依賴性特性。結合在該通道內淺層區域(較近通道內口區域)的精胺，則可以打開該通道孔洞之門閥，加速深層位置精胺的脫離速率。因此精胺在內向整流性鉀離子通道不僅是扮演孔洞的阻塞物質，同時也扮演Kir 2.1的孔洞內門閥開關的物質。 除此以外，我們也進一步進行藥物阻塞Kir 2.1通道的分子藥理機轉。Ethusuximide (ETX)藥物在臨床上經常選擇性使用於治療癲癇小發作(petil mal)的病患。在癲癇動物模式的研究發現，Kir通道對於癲癇的致病機轉有其關聯性。我們利用分子生物學突變技術、電生理記錄方式與分子動態模擬方式，進行ETX結合於Kir 2.1通道的親和力以及動力學分析。實驗結果證明ETX會抑制Kir 2.1通道的外向鉀離子電流，且具有明顯的濃度依賴特性；而另一種Valproic acid (VPA)藥物則不具有此抑制作用。我們的結果更可以進一步推論出ETX是透過與鉀離子偶合作用(在細胞內外有對稱性100 mM K+的溶液情況下，ETX應是與1.2個鉀離子相互偶合)，在主要是外向電流的情況下，被推往Kir 2.1通道中四個次單位蛋白螺旋交會區域最外端的位置(即S165–T141附近)。另一方面，隨著鉀離子流向改變為主要係內向電流時，則可以將ETX推往該通道中四個次單位蛋白螺旋交會區域最內端的位置。綜合實驗結果以上，我們結論Kir 2.1通道中四個次單位蛋白螺旋交會區域，對於通道之運作而言，兼具有重要的生理與藥理意義。不僅與內生性的精胺及抗癲癇藥物ETX阻塞Kir 2.1通道造成內向整流的特性密切相關，同時也是造成離子通透與通道門閥開關相互作用的關鍵區域。
並列摘要
Inward rectifier K+ channels (Kir channels) conduct K+ ions across the cell membrane inwardly much larger than outwardly in physiological conditions. This intriguing conduction property is essential for the physiological function of these channels, including the maintenance of the resting membrane potential close to the K+ equilibrium potential without excessively losing the intracellular K+ to the extracellular compartment during membrane depolarization. The mechanism underlying the intriguing inward rectification phenomenon in the Kir channel has been ascribed mostly to intracellular polyamines (e.g., spermine, SPM) block of the pore. Voltage–dependent and flow–dependent block of outward K+ currents by intracellular SPM has been proposed as the major mechanisms underlying inward rectification. In this study, we show that the SPM blocking affinity curve is shifted according to the shift in K+ reversal potential. The inhibition of the outward currents by the SPM has been shown dependent on the driving force (Vm–EK+) which is equivalent to the different of the electrochemical potentials. Moreover, the kinetics of SPM entry to and exit from the binding site are correlatively slowed by specific E224 and E299 mutations, which always also disrupt the flux–coupling feature of SPM block. The entry rates carry little voltage dependence, whereas the exit rates are e–fold decelerated per ~15 mV depolarization. Interestingly, the voltage dependence remains rather constant among WT and quite a few different mutant channels. This voltage dependence offers an unprecedented chance of mapping the location (electrical distance) of the SPM site in the pore, because these kinetic data were obtained along the preponderant direction of K+ current flow (outward currents for the entry rate and inward currents for the exit rate) and thus contamination from flow dependence should be negligible. Moreover, double mutations involving E224 and A178 or M183 seem to alter the height of the same asymmetrical barrier between the SPM binding site and the intracellular melieu. We conclude that the SPM site responsible for the inward rectifying block is located at electrical distance ~0.5 from the inside and is involved in a flux–coupling segment in the bundle crossing region of the pore. With preponderant outward K+ flow, SPM is “pushed” to the outmost site of this segment (~D172). On the other hand, the blocking SPM would be pushed to the inner end of this segment (~M183–A184). Moreover, E224 and E299 very likely electrostatically interact with the other residues (e.g., R228, R260) in the cytoplasmic domain, and then allosterically keep the bundle crossing region in an open conformation appropriate for the flux–coupling block of SPM. Moreover, the bundle crossing region of the Kir 2.1 channel may undergo opening/closing conformational changes mimicking channel gating. We further investigate these “gating” conformational changes at this critical segment and demonstrate that A184R mutation in the inner end of the bundle crossing region not only abolishes the inward rectifying features of SPM block but also tends to close the channel pore, which can then only be opened by intracellular but not extracellular cations. This ionic site responsible for the opening of the A184R mutant channel is located in the cytoplasmic domain, and is not selective for K+ because intracellular Na+ is as effective as K+ in this action. We also found that the WT channel pore could accommodate at least 2 SPM molecules simultaneously, and the unbinding of the blocking SPM in the deep site is facilitated rather than deterred by the presence of the other SPM in the superficial site. We conclude that the SPM in the deep site serves as a flow–dependent pore blocker. The SPM in the superficial site, on the other hand, serves both as a docking form ready for permeation to the deep site, and as a gating particle capable of opening the bundle crossing region. In addition, we also studied the action of the pharmacological agent blocking of the Kir 2.1 channel. Ethosuximide (ETX), 2–ethyl–2–methylsuccinimide, is used clinical generally common for its selective effect on absence seizures. Many evidences on the seizure animal model have yielded information that Kir channels are implicated in seizure generation. We further examined the kinetics of ETX binding to and unbinding from the Kir 2.1 channel, as well as the binding affinity of ETX to this channel. We have demonstrated that the outward currents of the Kir 2.1 channel are inhibited by intracellular ETX with accelerated decay in a dose–dependent fashion, but not by valproic acid (VPA). We also found that ETX, most likely coming from the intracellular side, is “carried” by the outward K+ flux (i.e., accompanied by 1.2 K+ in symmetrical 100 mM K+) via the flux–coupling bundle crossing region to reach its block site between S165 and T141, and thus makes a flow– and voltage–dependent block of the Kir 2.1 channel pore. All these data indicate that the bundle crossing region of the Kir 2.1 channel pore thus constitutes a pivotal segment, which, in collaboration with internal SPM, ETX, and K+ ions, closely couple channel gating to inward rectifying ion permeation.