形成细胞内袋的残基上的电荷反转对连接蛋白通道和半通道的调节作用不同。

IF 4.3 2区 生物学 Q1 BIOLOGY
Felipe Villanelo, Peter J Minogue, Jaime Maripillán, Mauricio Reyna-Jeldes, Joaquin Jensen-Flores, Isaac E García, Eric C Beyer, Tomás Pérez-Acle, Viviana M Berthoud, Agustín D Martínez
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引用次数: 0

摘要

背景:连接蛋白β亚家族成员含有一个由四个跨膜螺旋的氨基酸残基包围的胞内袋。此前还没有人研究过 α-、γ-、δ- 和 ε 亚家族成员是否存在这个口袋。我们研究了作为α-亚家族代表的连接蛋白50(Cx50),因为它的结构已经确定,而且Cx50的突变是先天性白内障最常见的遗传原因之一:为了研究 Cx50 细胞内口袋的存在和功能,我们使用了分子动力学模拟、定点突变、缝隙连接示踪剂细胞间转移以及通过电生理学和带电分子渗透检测的半通道活性:利用分子动力学,我们确定了 Cx50 半通道细胞内口袋的存在,并确定了参与其形成的氨基酸。我们利用定点突变改变了支持胞内袋的盐桥相互作用,这种作用发生在附件蛋白家族中高度保守的两个残基 R33 和 E162 之间。在这两个位置替换相反的电荷会减少缝隙连接斑块的形成和细胞间的通讯,并适度降低半通道电流。在这些位置上同时进行电荷反转会产生形成斑块的无功能缝隙连接通道和高活性半通道:这些结果表明,细胞内袋的相互作用会影响缝隙连接通道和半通道的功能。这些相互作用的破坏可能是导致与这些位置的突变相关的疾病的原因。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

Background: Members of the β-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts.

Methods: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules.

Results: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels.

Conclusions: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

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来源期刊
Biological Research
Biological Research 生物-生物学
CiteScore
10.10
自引率
0.00%
发文量
33
审稿时长
>12 weeks
期刊介绍: Biological Research is an open access, peer-reviewed journal that encompasses diverse fields of experimental biology, such as biochemistry, bioinformatics, biotechnology, cell biology, cancer, chemical biology, developmental biology, evolutionary biology, genetics, genomics, immunology, marine biology, microbiology, molecular biology, neuroscience, plant biology, physiology, stem cell research, structural biology and systems biology.
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