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
{"title":"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。","authors":"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","doi":"10.1186/s40659-024-00501-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11112876/pdf/","citationCount":"0","resultStr":"{\"title\":\"Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.\",\"authors\":\"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\",\"doi\":\"10.1186/s40659-024-00501-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>\",\"PeriodicalId\":9084,\"journal\":{\"name\":\"Biological Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2024-05-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11112876/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biological Research\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1186/s40659-024-00501-5\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biological Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s40659-024-00501-5","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOLOGY","Score":null,"Total":0}
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.
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.