臺灣博碩士論文加值系統

我們的實驗目的是希望建立一套Single Channel reconstitution系
統,能夠有效分辨純化出之AMPA receptor是否仍具有電生理的功能. 以新
鮮豬腦為研究材料,在有或無DTE(dithioerythritol)與IAA(
iodoacetamide)的存在下,以Triton X-114/KCl選擇性的溶解出AMPA
receptors,分別命名為DTE/IAA(+)與DTE/IAA(-) AMPA receptors. 由以
前的生化實驗結果已知:DTE/IAA(+) AMPA receptor其monomer的基本組成
單元與原本仍在synaptic junction上的AMPA receptor非常相似; 而DTE/
IAA(-) AMPA receptors的組成單元則呈現兩兩subunits相連的dimer形
式. 將純化出的兩種AMPA receptors分別重組回到vesicles上,並與在
電極尖端處形成的phospholipid bilayer作融合後,發現單一DTE/IAA(+)
AMPA receptor在kainate刺激下,活化產生電流的電導有三群分佈,I-V
curve的形狀是一條過原點的直線; 而在藥理學上,此DTE/IAA(+) AMPA
receptor會被CNQX所抑制,這些特性與之前文獻報導中native AMPA
receptors之特性相似,證明此純化出的蛋白質群透過重組後,仍可保有類
似native AMPA receptor的特性. 在DTE/IAA(-) AMPA receptor的實
驗結果中,發現其電生理特性與藥理特性也和native AMPA receptor相似,
故推測重組後的DTE/IAA(-) AMPA receptor結構與未被detergent溶解,仍
在膜上的native AMPA receptor結構也相似; 而過去的生化實驗已知:此
DTE/IAA(-) AMPA receptor的組成單元為dimer和tetramer的形式,可以推
測一個具有功能的DTE/IAA(-) AMPA receptor其subunit的數目應該是2
或2的倍數,綜合以往修飾交聯與與流體力學實驗的分析,我們判斷重組後
的DTE/IAA(-) AMPA receptors之結構為tetramer(Wu et al., 1996). 故
我們推論,在膜上的functional native AMPA receptor也應該事由四個
subunits所組成.

We have developed a reconstitution procedure allowing us to
exam the single channel activities of the AMPA receptors
purified from pig brains. The AMPA receptors were selectively
solublized from the synaptic junctions of the pig cerebral
cortex by Triton X-114/KCl in the presence or absecnce of DTE/
IAA (dithioerythritol/iodoacetamide), named as the DTE(+) and
DTE/IAA(-) AMPA receptors, respectively. SDS-PAGE analyses
revealed that the resultant of both preparations consisted of a
single protein band of 106 kDa, which was recognized by
antibodies against rat GluR1 and GluR2/3 subunits, but not by
antibodies against rat NR1 or GluR6/7 subunits. Western blot
analysis performedunder the non-reducing condition revealed that
the majority of the DTE/IAA(+)AMPA receptors are monomers at 106
kDa site; and the 106 kDa proteins of the DTE/IAA(-) samples are
corss-linked into dimers or tetramers by disulfide bonds. The
purified AMPA receptors were reconstituted into vesicles made of
soybean phosphatidylcholine and then these vesicles were allowed
to fuse with the artificial lipid bilayers at the tip of a
patch-clamp electrode. In the presence of kainate, single-
channel currents of the DTE/IAA(+) AMPA receptors were recored
with three different levels of conductance. They are less than 5
pS, 7~13 pS and larger than 15 pS, respectively. These kinate-
activated currents were reversibly inhibited by CNQX. Single-
channel currents were not activated by NMDA. These observations
indicate that the reconstituted DTE/IAA(+) AMPA receptors still
retain their ability to gate currents in the response of
pharmacological activation and blockage, like the native AMPA
receptors do. Similar electrophysiological and
pharmacological properties were also observed in the DTE/IAA(-)
AMPA receptors. These results suggest that the DTE/IAA(-), DTE/
IAA(+) and native AMPA receptors may share a similar structure.
ADTE/IAA(-) AMPA receptor is likely to consist of an even number
of subunitsbecause its basic units are dimer or tetramer.
Previous biochemical analysesrevealed that the purified DTE/
IAA(-) receptors are tetrameric. These observations also suggest
that a native AMPA receptor, like a reconstituted DTE/IAA(-)
AMPA receptor, consists of four subunits.