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1.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807656

RESUMO

Glycine is an amino acid with unique properties because its side chain is composed of a single hydrogen atom. It confers conformational flexibility to proteins and conserved glycines are often indicative of protein domains involving tight turns or bends. All six beta-type connexins expressed in human epidermis (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx32) contain a glycine at position 12 (G12). G12 is located about halfway through the cytoplasmic amino terminus and substitutions alter connexin function in a variety of ways, in some cases altering protein interactions and leading to cell death. There is also evidence that alteration of G12 changes the structure of the amino terminus in connexin- and amino acid- specific ways. This review integrates structural, functional and physiological information about the role of G12 in connexins, focusing on beta-connexins expressed in human epidermis. The importance of G12 substitutions in these beta-connexins is revealed in two hereditary skin disorders, keratitis ichthyosis and erythrokeratodermia variabilis, both of which result from missense mutations affecting G12.


Assuntos
Conexinas/metabolismo , Epiderme/metabolismo , Eritroceratodermia Variável/metabolismo , Junções Comunicantes/metabolismo , Ictiose/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Conexinas/genética , Epiderme/patologia , Eritroceratodermia Variável/genética , Eritroceratodermia Variável/patologia , Junções Comunicantes/genética , Junções Comunicantes/patologia , Glicina/genética , Glicina/metabolismo , Humanos , Ictiose/genética , Ictiose/patologia
2.
Dev Neurobiol ; 77(5): 522-547, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27582044

RESUMO

Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre-chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue- and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure-function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin-based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522-547, 2017.


Assuntos
Conexinas , Junções Comunicantes , Canais Iônicos , Animais
3.
J Biol Chem ; 291(24): 12601-12611, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27129238

RESUMO

Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.


Assuntos
Desenvolvimento Ósseo/genética , Conexina 43/genética , Mutação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Nadadeiras de Animais/crescimento & desenvolvimento , Nadadeiras de Animais/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Conexina 43/fisiologia , Feminino , Larva/genética , Larva/crescimento & desenvolvimento , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Oócitos/metabolismo , Oócitos/fisiologia , Osteogênese/genética , Técnicas de Patch-Clamp , Fenótipo , Xenopus laevis , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia
4.
J Biol Chem ; 291(3): 1053-63, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26598520

RESUMO

The zebrafish has a striped skin pattern on its body, and Connexin41.8 (Cx41.8) and Cx39.4 are involved in striped pattern formation. Mutations in these connexins change the striped pattern to a spot or labyrinth pattern. In this study, we characterized Cx41.8 and Cx39.4 after expression in Xenopus oocytes. In addition, we analyzed Cx41.8 mutants Cx41.8I203F and Cx41.8M7, which caused spot or labyrinth skin patterns, respectively, in transgenic zebrafish. In the electrophysiological analysis, the gap junctions formed by Cx41.8 and Cx39.4 showed distinct sensitivity to transjunctional voltage. Analysis of non-junctional (hemichannel) currents revealed a large voltage-dependent current in Cx39.4-expressing oocytes that was absent in cells expressing Cx41.8. Junctional currents induced by both Cx41.8 and Cx39.4 were reduced by co-expression of Cx41.8I203F and abolished by co-expression of Cx41.8M7. In the transgenic experiment, Cx41.8I203F partially rescued the Cx41.8 null mutant phenotype, whereas Cx41.8M7 failed to rescue the null mutant, and it elicited a more severe phenotype than the Cx41.8 null mutant, as evidenced by a smaller spot pattern. Our results provide evidence that gap junctions formed by Cx41.8 play an important role in stripe/spot patterning and suggest that mutations in Cx41.8 can effect patterning by way of reduced function (I203F) and dominant negative effects (M7). Our results suggest that functional differences in Cx41.8 and Cx39.4 relate to spot or labyrinth mutant phenotypes and also provide evidence that these two connexins interact in vivo and in vitro.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/fisiologia , Pigmentação da Pele , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Conexinas/química , Conexinas/genética , Fenômenos Eletrofisiológicos , Feminino , Deleção de Genes , Técnicas de Transferência de Genes , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Dados de Sequência Molecular , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
5.
J Biol Chem ; 290(28): 17074-84, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25969535

RESUMO

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.


Assuntos
Conexinas/química , Junções Comunicantes/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Feminino , Junções Comunicantes/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triptofano/química , Xenopus laevis , Proteína beta-1 de Junções Comunicantes
6.
J Neurophysiol ; 111(6): 1383-95, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24381032

RESUMO

Rectifying electrical synapses are rare gap junctions that favor transmission of signals in one direction. Such synapses have been identified in neural systems, including those mediating rapid escape responses of arthropods. In the Drosophila giant fiber system, adjacent cells express and contribute different transcript variants of the innexin Shaking B, resulting in heterotypic gap junctions with rectifying properties. When expressed exogenously, variants Shaking B Lethal (ShakBL) and Shaking B neural + 16 (ShakBN16) form heterotypic junctions that gate asymmetrically in response to transjunctional voltage. To determine whether the amino terminus confers properties of gating and rectification, amino-terminal domains were exchanged between ShakBL and ShakBN16, creating chimeric proteins SBL NTN16 and SBN16 NTL. The properties were analyzed in paired Xenopus oocytes. Our results suggest that the amino terminus plays an important role in establishing rectifying properties inherent to heterotypic junctions composed of ShakBL and ShakBN16. ShakBL/SBL NTN16 junctions behaved similarly to ShakBL/ShakBN16 junctions, gating in response to transjunctional voltage of one polarity and inducing a highly asymmetric conductance-voltage relationship. However, the amino terminus did not act independently to confer sensitivity to transjunctional voltage. The complementary pairing ShakBN16/SBN16 NTL displayed little sensitivity to voltage of either polarity, and in homotypic pairings SBL NTN16 was strongly gated by transjunctional voltage. We propose a model in which the amino terminus induces gating only when matched with an accommodating innexin body.


Assuntos
Conexinas/química , Proteínas de Drosophila/química , Junções Comunicantes/metabolismo , Potenciais da Membrana , Proteínas do Tecido Nervoso/química , Potenciais Sinápticos , Sequência de Aminoácidos , Animais , Conexinas/metabolismo , Drosophila/química , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Junções Comunicantes/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Xenopus
7.
PLoS One ; 8(8): e70916, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23967136

RESUMO

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.


Assuntos
Conexinas/genética , Conexinas/metabolismo , Surdez/genética , Surdez/metabolismo , Mutação , Animais , Linhagem Celular , Membrana Celular/metabolismo , Conexina 26 , Conexinas/química , Junções Comunicantes/metabolismo , Humanos , Modelos Moleculares , Permeabilidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Células Sf9
8.
Neural Dev ; 4: 16, 2009 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-19432959

RESUMO

BACKGROUND: Approximately 10% of Caenorhabditis elegans nervous system synapses are electrical, that is, gap junctions composed of innexins. The locomotory nervous system consists of several pairs of interneurons and three major classes of motor neurons, all with stereotypical patterns of connectivity that include gap junctions. Mutations in the two innexin genes unc-7 and unc-9 result in identical uncoordinated movement phenotypes, and their respective gene products were investigated for their contribution to electrical synapse connectivity. RESULTS: unc-7 encodes three innexin isoforms. Two of these, UNC-7S and UNC-7SR, are functionally equivalent and play an essential role in coordinated locomotion. UNC-7S and UNC-7SR are widely expressed and co-localize extensively with green fluorescent protein-tagged innexin UNC-9 in the ventral and dorsal nerve cords. A subset of UNC-7S/SR expression visualizes gap junctions formed between the AVB forward command interneurons and their B class motor neuron partners. Experiments indicate that expression of UNC-7S/SR in AVB and expression of UNC-9 in B motor neurons is necessary for these gap junctions to form. In Xenopus oocyte pairs, both UNC-7S and UNC-9 form homomeric gap junctions, and together they form heterotypic channels. Xenopus oocyte studies and co-localization studies in C. elegans suggest that UNC-7S and UNC-9 do not heteromerize in the same hemichannel, leading to the model that hemichannels in AVB:B motor neuron gap junctions are homomeric and heterotypic. CONCLUSION: UNC-7S and UNC-9 are widely expressed and contribute to a large number of the gap junctions identified in the locomotory nervous system. Proper AVB:B gap junction formation requires UNC-7S expression in AVB interneurons and UNC-9 expression in B motor neurons. More broadly, this illustrates that innexin identity is critical for electrical synapse specificity, but differential (compartmentalized) innexin expression cannot account for all of the specificity seen in C. elegans, and other factors must influence the determination of synaptic partners.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Sistema Nervoso Central/citologia , Sinapses Elétricas/fisiologia , Locomoção/fisiologia , Proteínas de Membrana/metabolismo , Animais , Animais Geneticamente Modificados , Comportamento Animal , Biofísica , Caenorhabditis elegans/anatomia & histologia , Proteínas de Caenorhabditis elegans/genética , Estimulação Elétrica , Sinapses Elétricas/genética , Proteínas de Fluorescência Verde/genética , Locomoção/genética , Potenciais da Membrana/genética , Proteínas de Membrana/genética , Modelos Moleculares , Neurônios Motores/metabolismo , Mutação , Oócitos , Técnicas de Patch-Clamp , Transfecção/métodos , Xenopus
9.
Am J Physiol Cell Physiol ; 291(5): C966-76, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16738002

RESUMO

Voltage-gated K(+) channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K(+) currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At -60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K(+) channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K(+) channels.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/metabolismo , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/metabolismo , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Furões , Ativação do Canal Iônico/fisiologia , Cinética , Proteínas Interatuantes com Canais de Kv/metabolismo , Oócitos , Xenopus
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