- 學位論文
NMDA 受體被鎂離子阻塞的分子機制：電流流向與電壓依賴性
The molecular mechanism underlying magnesium block of NMDA receptors: ionic flow and voltage dependence
摘要
細胞外鎂離子的阻塞對 NNDA 受體之神經生理功能有重要影響，但是其相關機制仍然不明，雖然電壓會影響鎂離子的阻塞，但鎂離子的效果不能完全用電壓依賴性來解釋。我們在海馬迴 CA1 神經細胞上，用不同濃度的鈉離子作為主要的電荷載體，研究胞外鎂離子阻塞 NMDA 受體的機制。有趣的是，我們發現胞外鎂離子的阻塞作用，強烈受到鈉離子電流流向的影響。鈉離子電流向細胞內流時，鎂離子的阻塞效果顯著強於當鈉離子電流向細胞外流時。若調低胞內鈉離子濃度，來最大化電流向內流的傾向，則胞外鎂離子阻塞會更加強烈。分析胞外鎂離子阻塞的動力學，當減少細胞內鈉離子濃度或增加細胞外鈉離子濃度，使得反轉膜電位向正電壓方向移動時，鎂離子的脫離速率和/或結合速率對電壓的依賴性也會隨之向同一個方向移動。胞外鎂離子阻塞具備流向依賴性，表示鎂離子結合在一個可以同時容許多個離子單排排列的區域。利用流向耦合公式，我們發現至少有一個鈉離子伴隨著鎂離子在這個區域中。我們也研究了胞內鎂離子阻塞的機制，利用 inside-out macropatch 來紀錄表現在非洲爪蟾蛙卵上的重組 NR1-NR2B 電流，實驗結果指出胞內鎂離子的阻塞同樣受到鈉離子流向的影響，顯示胞內鎂離子也是結合在一個可以同時容許多個離子單排排列的區域，不過此一區域的流向耦合趨勢較前述胞外鎂離子結合區域為低。我們提出一個可以圓滿解釋這些數據的鎂離子通透能量輪廓，從中可知細胞內側和細胞外側的鎂離子的結合區域並非同一個，不過這兩個區域就電場距離而言彼此相當接近，而且皆集中於靠近通道內口之處。
並列摘要
External Mg2+ block of the N-methyl-D-aspartate receptor (NMDAR) is essential for the neurobiological function of the receptor. However, the underlying molecular mechanism of this blocking effect is unclear. Although Mg2+ block is dependent on voltage, the blocking effect can not be solely explained by the voltage-dependence. Using different concentration of Na+ as the main charge carrier, we studied external Mg2+ block of NMDAR in hippocampus CA1 neurons. Interestingly, we found that external Mg2+ block of NMDAR was strongly dependent on the direction of Na+ flow. The blocking effect of Mg2+ in inward Na+ currents is much stronger than that in outward Na+ currents, and the blocking effect is maximized if we maximize the tendency of inward current by lowering internal [Na+]. The kinetics of external Mg2+ was also analyzed. When internal [Na+] is lowered or external [Na+] is raised to shift the reversal potential in the depolarizing direction, the voltage dependence of Mg2+ dissociation and/or association rates are also shifted in the same direction. This flow-dependence of external Mg2+ block indicated that external Mg2+ binds to a multi-ion single-file region. Using flux-coupling equation, we concluded that at least one Na+ coexist with Mg2+ within the external Mg2+ binding region. We also examined the mechanism of internal Mg2+ block in recombinant NR1-NR2B expressed on Xenopus oocytes. The NMDAR currents carried by Na+ were measured in excised inside-out macropatches. Internal Mg2+ block of NMDAR was also strongly dependent on the direction of Na+ current, suggesting that internal Mg2+ also binds to a multi-ion single-file region, but the flow dependence is slightly weaker than the aforementioned region responsible for external Mg2+ block. All of the experiment data could be well explained by a permeation energy profile in which the internal Mg2+ binding region is distinct from the external Mg2+ binding region. These two regions, however, are very close in terms of electrical distance, and are both located much closer to the internal than to the the external mouth of the pore.