Neumann E, Weber J, Schürholz T
Faculty of Chemistry, University of Bielefeld, Germany.
Arch Physiol Biochem. 1996 Oct;104(6):731-44. doi: 10.1076/apab.104.6.731.12917.
The specific functional properties of the nicotinic acetylcholine receptors (AChR) and the particular oligomeric membrane organization of AChR are suggested to be the basis for the steep electrical depolarisation, required for the initiation of the postsynaptic action potentials causing muscle contraction and discharge of electric organs. The dimer (M(r) approximately 580,000) and the monomer (M(r) approximately 290,000) of the detergent-solubilized, affinity-purified AChR of Torpedo californica electrocytes exhibit different channel conductances and larger oligochannels. Patch clamp data of the dimer, reconstituted in large lipid vesicles, show that the dimer is a double-channel protein causing single-channel events of conductance G(D) = 84 +/- 6 pS at 0.11 M K+ and 0.1 mM Ca2+ at 293 K (20 degrees C). At the same ionic conditions the vesicle-reconstituted monomer, if prevented from aggregation, exhibits a channel conductance, G(M) = 42 +/- 3 pS, which is only half of that of the dimer. The dimer conductivity event thus reflects the synchronous switching of its two constituent monomeric parts. The K(+)-conductance of the reconstituted Torpedo dimer is the same, and shows the same inhibitory Ca(2+)-dependence, as that of the Torpedo AChR expressed in Xenopus laevis oocytes (Imoto et al., Nature, 324, 670-674, 1986). In terms of Ca(2+)-binding, reducing K(+)-transport, the equilibrium constant is KCa = 0.48 mM at 0.11 m K+, 20 degrees C; G0([Ca]-->0) = 98 +/- 6 pS and G infinity ([Ca]-->infinity) = 27 +/- 6 pS. The ratio G0/G infinity and an estimate of the lateral surface area of the channel vestibule yields about 16 negatively charged groups in an average distance of 1.8 nm. These negative charges cause an accumulation of K+ ions in the channel vestibule by a factor of about 4. Our results and the comparison with the oocyte data reveal that it is also the dimer which is the physiological opening-closing unit of the AChR in the oocyte membrane. The larger macrochannel events are multiples of the dimer or of the monomer conductances. The occurrence of such oligochannels from AChR protein oligomers could guarantee the steep electrical depolarisation necessary to generate the action potential by the Na(+)-channel system.
烟碱型乙酰胆碱受体（AChR）的特定功能特性以及AChR独特的寡聚体膜组织被认为是引发突触后动作电位（导致肌肉收缩和电器官放电）所需的陡峭电去极化的基础。加州电鳐电细胞经去污剂溶解、亲和纯化的AChR的二聚体（相对分子质量约580,000）和单体（相对分子质量约290,000）表现出不同的通道电导和更大的寡聚通道。在大脂质囊泡中重构的二聚体的膜片钳数据表明，该二聚体是一种双通道蛋白，在293K（20℃）、0.11M K⁺和0.1mM Ca²⁺条件下，其单通道事件的电导G(D)=84±6 pS。在相同离子条件下，若防止囊泡重构的单体聚集，其通道电导G(M)=42±3 pS，仅为二聚体的一半。因此，二聚体的导电事件反映了其两个组成单体部分的同步开关。重构的电鳐二聚体的K⁺电导与非洲爪蟾卵母细胞中表达的电鳐AChR相同，且表现出相同的抑制性Ca²⁺依赖性（井本等，《自然》，324卷，670 - 674页，1986年）。就Ca²⁺结合、降低K⁺转运而言，在0.11m K⁺、20℃时平衡常数KCa = 0.48mM；G0（[Ca]→0）= 98±6 pS，G∞（[Ca]→∞）= 27±6 pS。G0/G∞的比值以及通道前庭横向表面积的估计得出，平均距离为1.8nm的范围内约有16个带负电荷的基团。这些负电荷使K⁺离子在通道前庭中的积累增加约4倍。我们的结果以及与卵母细胞数据的比较表明，二聚体也是卵母细胞膜中AChR的生理开闭单元。较大的大通道事件是二聚体或单体电导的倍数。由AChR蛋白寡聚体产生的这种寡聚通道的出现可以保证由Na⁺通道系统产生动作电位所需的陡峭电去极化。
