Long-term potentiation in an innexin-based electrical synapse.
ABSTRACT: Electrical synapses are formed by two unrelated gap junction protein families, the primordial innexins (invertebrates) or the connexins (vertebrates). Although molecularly different, innexin- and connexin-based electrical synapses are strikingly similar in their membrane topology. However, it remains unclear if this similarity extends also to more sophisticated functions such as long-term potentiation which is only known in connexin-based synapses. Here we show that this capacity is not unique to connexin-based synapses. Using a method that allowed us to quantitatively measure gap-junction conductance we provide the first and unequivocal evidence of long-term potentiation in an innexin-based electrical synapse. Our findings suggest that long-term potentiation is a property that has likely existed already in ancestral gap junctions. They therefore could provide a highly potent system to dissect shared molecular mechanisms of electrical synapse plasticity.
Project description:Gap junctions are intercellular channels that allow for the movement of small molecules and ions between the cytoplasm of adjacent cells and form electrical synapses between neurons. In invertebrates, the gap junction proteins are coded for by the innexin family of genes. The stomatogastric ganglion (STG) in the crab Cancer borealis contains a small number of identified and electrically coupled neurons. We identified Innexin 1 (Inx1), Innexin 2 (Inx2), Innexin 3 (Inx3), Innexin 4 (Inx4), Innexin 5 (Inx5), and Innexin 6 (Inx6) members of the C. borealis innexin family. We also identified six members of the innexin family from the lobster Homarus americanus transcriptome. These innexins show significant sequence similarity to other arthropod innexins. Using in situ hybridization and reverse transcriptase-quantitative PCR (RT-qPCR), we determined that all the cells in the crab STG express multiple innexin genes. Electrophysiological recordings of coupling coefficients between identified pairs of pyloric dilator (PD) cells and PD-lateral posterior gastric (LPG) neurons show that the PD-PD electrical synapse is nonrectifying while the PD-LPG synapse is apparently strongly rectifying.
Project description:Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families.
Project description:Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy. Our findings revealed that connexin and innexin families share several structural properties with regard to their monomeric and oligomeric structures, while simultaneously suggesting a diversity of gap junction channels in nature. This review summarizes cutting-edge progress toward determining an innexin gap junction channel structure, as well as essential tips for preparing cryo-electron microscopy samples for high-resolution structural analysis of an innexin gap junction channel.
Project description:Connections between neurons called synapses are the key components underlying all nervous system functions of animals and humans. However, important genetic information on the formation and plasticity of one type, the electrical (gap junction-mediated) synapse, is understudied in many invertebrates. In the present study, we set forth to identify and characterize the gap junction-encoding gene innexin in the central nervous system (CNS) of the mollusk pond snail <i>Lymnaea stagnalis</i>. With PCR, 3' and 5' RACE, and BLAST searches, we identified eight innexin genes in the <i>L. stagnalis</i> genome, named <i>Lst Inx1-Lst Inx8</i>. Phylogenetic analysis revealed that the <i>L. stagnalis</i> innexin genes originated from a single copy in the common ancestor of molluskan species by multiple gene duplication events and have been maintained in <i>L. stagnalis</i> since they were generated. The paralogous innexin genes demonstrate distinct expression patterns among tissues. In addition, one paralog, <i>Lst Inx1</i>, exhibits heterogeneity in cells and ganglia, suggesting the occurrence of functional diversification after gene duplication. These results introduce possibilities to study an intriguing potential relationship between innexin paralog expression and cell-specific functional outputs such as heterogenic ability to form channels and exhibit synapse plasticity. The <i>L. stagnalis</i> CNS contains large neurons and functionally defined networks for behaviors; with the introduction of <i>L. stagnalis</i> in the gap junction gene field, we are providing novel opportunities to combine genetic research with direct investigations of functional outcomes at the cellular, synaptic, and behavioral levels.
Project description:Intercellular gap junction channels and single-membrane channels have been reported to regulate electrical synapse and the brain function. Innexin is known as a gap junction-related protein in invertebrates and is involved in the formation of intercellular gap junction channels and single-cell membrane channels. Multiple isoforms of innexin protein in each species enable the precise regulation of channel function. In molluscan species, sequence information of innexins is still limited and the sequences of multiple innexin isoforms have not been classified. This study examined the innexin transcripts expressed in the central nervous system of the terrestrial slug Limax valentianus and identified 16 transcripts of 12 innexin isoforms, including the splicing variants. We performed phylogenetic analysis and classified the isoforms with other molluscan innexin sequences. Next, the phosphorylation, N-glycosylation, and S-nitrosylation sites were predicted to characterize the innexin isoforms. Further, we identified 16 circular RNA sequences of nine innexin isoforms in the central nervous system of Limax. The identification and classification of molluscan innexin isoforms provided novel insights for understanding the regulatory mechanism of innexin in this phylum.
Project description:Gap junctions are physical channels that connect adjacent cells, permitting the flow of small molecules/ions between the cytoplasms of the coupled units. Innexin/innexin-like proteins are responsible for the formation of invertebrate gap junctions. Within the nervous system, gap junctions often function as electrical synapses, providing a means for coordinating activity among electrically coupled neurons. While some gap junctions allow the bidirectional flow of small molecules/ions between coupled cells, others permit flow in one direction only or preferentially. The complement of innexins present in a gap junction determines its specific properties. Thus, understanding innexin diversity is key for understanding the full potential of electrical coupling in a species/system. The decapod crustacean cardiac ganglion (CG), which controls cardiac muscle contractions, is a simple pattern-generating neural network with extensive electrical coupling among its circuit elements. In the lobster, Homarus americanus, prior work suggested that the adult neuronal innexin complement consists of six innexins (Homam-Inx1-4 and Homam-Inx6-7). Here, using a H. americanus CG-specific transcriptome, we explored innexin complement in this portion of the lobster nervous system. With the exception of Homam-Inx4, all of the previously described innexins appear to be expressed in the H. americanus CG. In addition, transcripts encoding seven novel putative innexins (Homam-Inx8-14) were identified, four (Homam-Inx8-11) having multiple splice variants, e.g., six for Homam-Inx8. Collectively, these data indicate that the innexin complement of the lobster nervous system in general, and the CG specifically, is likely significantly greater than previously reported, suggesting the possibility of expanded gap junction diversity and function in H. americanus.
Project description:We combined the Hodgkin-Huxley equations and a 36-state model of gap junction channel gating to simulate electrical signal transfer through electrical synapses. Differently from most previous studies, our model can account for dynamic modulation of junctional conductance during the spread of electrical signal between coupled neurons. The model of electrical synapse is based on electrical properties of the gap junction channel encompassing two fast and two slow gates triggered by the transjunctional voltage. We quantified the influence of a difference in input resistances of electrically coupled neurons and instantaneous conductance-voltage rectification of gap junctions on an asymmetry of cell-to-cell signaling. We demonstrated that such asymmetry strongly depends on junctional conductance and can lead to the unidirectional transfer of action potentials. The simulation results also revealed that voltage spikes, which develop between neighboring cells during the spread of action potentials, can induce a rapid decay of junctional conductance, thus demonstrating spiking activity-dependent short-term plasticity of electrical synapses. This conclusion was supported by experimental data obtained in HeLa cells transfected with connexin45, which is among connexin isoforms expressed in neurons. Moreover, the model allowed us to replicate the kinetics of junctional conductance under different levels of intracellular concentration of free magnesium ([Mg2+]i), which was experimentally recorded in cells expressing connexin36, a major neuronal connexin. We demonstrated that such [Mg2+]i-dependent long-term plasticity of the electrical synapse can be adequately reproduced through the changes of slow gate parameters of the 36-state model. This suggests that some types of chemical modulation of gap junctions can be executed through the underlying mechanisms of voltage gating. Overall, the developed model accounts for direction-dependent asymmetry, as well as for short- and long-term plasticity of electrical synapses. Our modeling results demonstrate that such complex behavior of the electrical synapse is important in shaping the response of coupled neurons.
Project description:Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.
Project description:Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dm?1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels.
Project description: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.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.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.