RESUMO
BACKGROUND: Members of the ß-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.
Assuntos
Conexinas , Junções Comunicantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conexinas/metabolismo , Conexinas/genética , Conexinas/química , Junções Comunicantes/metabolismo , Junções Comunicantes/fisiologia , Humanos , Animais , Mutação , Comunicação Celular/fisiologiaRESUMO
Under normal conditions, almost all cell types communicate with their neighboring cells through gap junction channels (GJC), facilitating cellular and tissue homeostasis. A GJC is formed by the interaction of two hemichannels; each one of these hemichannels in turn is formed by six subunits of transmembrane proteins called connexins (Cx). For many years, it was believed that the loss of GJC-mediated intercellular communication was a hallmark in cancer development. However, nowadays this paradigm is changing. The connexin 46 (Cx46), which is almost exclusively expressed in the eye lens, is upregulated in human breast cancer, and is correlated with tumor growth in a Xenograft mouse model. On the other hand, extracellular vesicles (EVs) have an important role in long-distance communication under physiological conditions. In the last decade, EVs also have been recognized as key players in cancer aggressiveness. The aim of this work was to explore the involvement of Cx46 in EV-mediated intercellular communication. Here, we demonstrated for the first time, that Cx46 is contained in EVs released from breast cancer cells overexpressing Cx46 (EVs-Cx46). This EV-Cx46 facilitates the interaction between EVs and the recipient cell resulting in an increase in their migration and invasion properties. Our results suggest that EV-Cx46 could be a marker of cancer malignancy and open the possibility to consider Cx46 as a new therapeutic target in cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Conexinas/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Celular , Conexinas/genética , Feminino , Células HeLa , Humanos , Células MCF-7RESUMO
To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.
Assuntos
Conexinas/química , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Linhagem Celular Tumoral , Conexina 26 , Conexinas/genética , Células HeLa , Humanos , Canais Iônicos/metabolismo , Mutação , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Gap junction hemichannels and cell-cell channels have roles in coordinating numerous cellular processes, due to their permeability to extra and intracellular signaling molecules. Another mechanism of cellular coordination is provided by a vast array of growth factors that interact with relatively selective cell membrane receptors. These receptors can affect cellular transduction pathways, including alteration of intracellular concentration of free Ca(2+) and free radicals and activation of protein kinases or phosphatases. Connexin and pannexin based channels constitute recently described targets of growth factor signal transduction pathways, but little is known regarding the effects of growth factor signaling on pannexin based channels. The effects of growth factors on these two channel types seem to depend on the cell type, cell stage and connexin and pannexin isoform expressed. The functional state of hemichannels and gap junction channels are affected in opposite directions by FGF-1 via protein kinase-dependent mechanisms. These changes are largely explained by channels insertion in or withdrawal from the cell membrane, but changes in open probability might also occur due to changes in phosphorylation and redox state of channel subunits. The functional consequence of variation in cell-cell communication via these membrane channels is implicated in disease as well as normal cellular responses.
Assuntos
Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Animais , Comunicação Celular , Conexinas/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de SinaisRESUMO
Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.
Assuntos
Conexina 43/química , Conexina 43/metabolismo , Conexinas/química , Conexinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Conexina 26 , Conexina 43/genética , Conexinas/genética , Imunofluorescência , Células HeLa , Humanos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Connexins and pannexins comprise 2 families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than 1 connexin or pannexin. Members of the same protein family form homo- or hetero-hexamers termed hemichannels. Hemichannels are pathways for the transmembrane diffusional exchange of ions and small molecules. Several human genetic diseases are associated with connexin mutants that may form hemichannels with increased or reduced activity. Pro-inflammatory conditions of different duration and/or intensity can lead to acute or chronic increase in hemichannel activity. Non-lethal stimuli can lead to transient increases in hemichannel activity (required for normal autocrine and/or paracrine cell signaling that might lead to preconditioning responses) whereas lethal stimuli induce long lasting hemichannel-mediated membrane permeabilization that accelerate cell death. Thus, in addition to transporters that mediate active and facilitated transport, the plasma membrane of most cells contains diffusional transporters (hemichannels) that are essential for normal cell functioning; their malfunctioning can cause or worsen a pathological condition.
Assuntos
Membrana Celular/fisiologia , Conexinas/fisiologia , Doenças Genéticas Inatas/metabolismo , Animais , Doenças Transmissíveis/metabolismo , Difusão , Humanos , Inflamação/metabolismo , Isquemia/metabolismo , Multimerização Proteica , Transdução de SinaisRESUMO
Extracellular signal-regulated kinases (ERKs) are widely implicated in multiple physiological processes. Although ERK1/2 has been proposed as a common mediator of antidepressant action in naive rodents, it remains to be determined whether the ERK1/2 pathway plays a role in depressive disorder. Here, we investigated whether chronic restraint stress (14 days) and antidepressant treatment [desipramine (DMI), 10 mg/kg intraperitoneally] induce changes in animal behavior and hippocampal levels of phospho-ERK1/2 and its substrate phospho-cAMP response element-binding protein (CREB). The results indicated that stress-induced depressive-like behaviors were correlated with an increase in P-ERK1/2 and P-CREB in the hippocampus evaluated by immunoblot analysis. As an indication of CREB activity, we evaluated changes in mRNA levels of its target genes. Brain-derived neurotrophic factor (BDNF) mRNA was reduced by stress, an effect prevented by DMI only in the CA3 area of hippocampus. Bcl-2 mRNA was reduced in all hippocampal regions by stress, an effect independent of DMI treatment. However, immunoblot from hippocampal extracts revealed that stress increased BCL-2 levels, an effect prevented by chronic DMI. These results suggest that ERKs and BDNF may be altered in depressive disorder, modifications that are sensitive to DMI action. In contrast, the stress-induced increase in BCL-2 may correspond to a neuroprotective response.
Assuntos
Antidepressivos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Desipramina/farmacologia , Hipocampo/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Psicológico/prevenção & controle , Animais , Antidepressivos/uso terapêutico , Comportamento Animal , Biomarcadores/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Desipramina/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Fármacos Neuroprotetores/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologiaRESUMO
Members of the connexin gene family are integral membrane proteins that form hexamers called connexons. Most cells express two or more connexins. Open connexons found at the nonjunctional plasma membrane connect the cell interior with the extracellular milieu. They have been implicated in physiological functions including paracrine intercellular signaling and in induction of cell death under pathological conditions. Gap junction channels are formed by docking of two connexons and are found at cell-cell appositions. Gap junction channels are responsible for direct intercellular transfer of ions and small molecules including propagation of inositol trisphosphate-dependent calcium waves. They are involved in coordinating the electrical and metabolic responses of heterogeneous cells. New approaches have expanded our knowledge of channel structure and connexin biochemistry (e.g., protein trafficking/assembly, phosphorylation, and interactions with other connexins or other proteins). The physiological role of gap junctions in several tissues has been elucidated by the discovery of mutant connexins associated with genetic diseases and by the generation of mice with targeted ablation of specific connexin genes. The observed phenotypes range from specific tissue dysfunction to embryonic lethality.