臺灣博碩士論文加值系統

本論文利用了全細胞膜片箝制術（whole-cell patch-clamp tech-nique），研究視丘神經元（thalamic neurons ）上T型鈣離子通道（T-type calcium channel）之門閥性質（gating properties）以及鎂離子對此通道的抑制作用（Mg2+ block）。第一部份主要在探討門閥性質（gating properties）。我們發現，T型鈣離子通道之活化速率（activation rate）以及去活化速率（deactivation rate ）都具有電壓依賴性（voltage dependence）；但不活化速率（inactivation rate）以及由不活化態回復（recovery from inactivation）的速率會達到飽和，因此，這兩步驟為非電壓依賴性（voltage independence）。另外，我們也發現，在剛開始由不活化態回復時，會有一小段沒有電流產生的時間，稱為初始遲滯現象（initial delay），且會隨著回復電壓的過極化（hyperpolarization）而縮短。此外，無論回復電壓為－120 mV 或是－80mV ，此通道由不活化態回復的過程中，幾乎沒有電流產生。基於以上幾點，我們認為T型鈣離子通道由不活化態回復時，必須先回到一個不導電的狀態，然後才能再度開啟；這和鈉離子通道（sodium channel）的性質，在定性上十分類似，都可用鉸鏈－蓋子模型（hinged--lid model）來描述；但兩者在定量上卻有顯著的差異，尤其是在不活化以及由不活化態回復等兩個反應，在大部分之電壓下，T型鈣離子通道的速率決定步驟皆牽涉到不活化球的結合或是脫落。這暗示了此兩種通道在參與不活化反應的結構上，有顯著的差異。本論文的第二部份是探討鎂離子對T型鈣離子通道之抑制作用。我們發現，鎂離子對於位在視丘神經元上之通道抑制效果較佳，而對於位在背根神經節（dorsal root ganglion）之通道的抑制效果則不顯著。鎂離子是直接結合在通道之孔洞（pore ）內以阻斷電流。其對通道結合之解離常數（dissociation constant ） 有電壓依賴性，當測試電壓為－80mV時，其解離常數約為1.3 m M ，之後每當測試電壓增加40mV時，親和力減少e 倍。由此估算，其結合的位置大約距離孔洞外口30%電距（electric distance）處。

The gating properties of T-type calcium channels and its blockade by Mg2+ in thalamic neurons were studied with the whole-cell patch-clamp technique. In the first part, we tried to find out the process of channel gating. The activation rate and deactivation rate were both voltage dependent. The inactivation rate and the rate of recovery from inactivation were saturable , so they were both voltage independent steps. We also found that recovery from inactivation started with an initial delay, a period of time without current produced. The duration of this delay was shortened with hyperpolarized membrane potential. Little current were produced during the entire process of channel recovery from inactivation, no matter the recovery voltage was -120mV or -80 mV. We proposed that recovery from inactivation of T-type calcium channel in thalamic neurons was just like sodium channel, which could be described with the hinged-lid model, but quantitatively, they are not the same, especially those steps involving inactivation. We thought that the stuctures involving inactivation were quite different between these 2 channels. Blockade of T-type calcium channels by Mg2+ in thalamic neurons was investigated then. We found that Mg2+ blocked T-type calcium channels only in thalamic neurons, but not in dorsal root ganglia. Because neither 3mM Mg2+ nor 3mM Ca2+ in 5mM Ca2+ solution could alter surface charge, Mg2+ was proposed to exert its effect by binding inside the pore to block the channel. Dissociation constant (Kd) of Mg2+ binding to the channel was 1.3mM at -80mV, and decreased e-fold per 40mV. We suggested that the Mg2+ binding site was located inside the pore around 30% electric distance from the external mouth.

圖次.................................................................................................................................iv
圖次.................................................................................................................................iv
中文摘要........................................................................................................................v
英文摘要........................................................................................................................vii
前言.................................................................................................................................1
第一部份Ｔ型鈣離子通道的門閥性質.....................................................................7
緒論...............................................................................................................................8
材料與方法..................................................................................................................13
結果...............................................................................................................................20
討論...............................................................................................................................29
第二部份鎂離子對Ｔ型鈣離子通道的抑制作用....................................................44
緒論...............................................................................................................................45
材料與方法..................................................................................................................47
結果...............................................................................................................................48
討論...............................................................................................................................51
參考文獻.........................................................................................................................57

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