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Gap junctions are Membrane channel between adjacent cells that allow the direct exchange of cytoplasmic substances, such as small molecules, substrates, and metabolites.
Gap junctions were first described as close appositions alongside tight junctions, however, electron microscopy studies in 1967 led to gap junctions being named as such to be distinguished from tight junctions. They bridge a 2-4 nm gap between cell membranes.
Gap junctions use protein complexes known as , composed of connexin proteins to connect one cell to another. Gap junction proteins include the more than 26 types of connexin, as well as at least 12 non-connexin components that make up the gap junction complex or nexus, including the tight junction protein ZO-1—a protein that holds membrane content together and adds structural clarity to a cell, , and aquaporin.
More gap junction proteins have become known due to the development of next-generation sequencing. Connexins were found to be structurally homologous between vertebrates and invertebrates but different in sequence. As a result, the term innexin is used to differentiate invertebrate connexins. There are more than 20 known innexins, along with unnexins in parasites and in viruses.
An electrical synapse is a gap junction that can transmit between . Such synapses create bidirectional continuous-time electrical coupling between neurons. Connexon pairs act as generalized regulated gates for ions and smaller molecules between cells. Hemichannel connexons form channels to the extracellular environment.
A gap junction or macula communicans is different from an ephaptic coupling that involves electrical signals external to the cells.
Invertebrate gap junctions comprise from the innexin protein family. Innexins have no significant sequence homology with connexins. Though differing in sequence to connexins, innexins are similar enough to connexins to form gap junctions in vivo in the same way connexins do.
The more recently characterized pannexin family, which was originally thought to form intercellular channels (with an amino acid sequence similar to innexins) in fact functions as a single-membrane channel that communicates with the extracellular environment and has been shown to pass calcium and ATP. This has led to the idea that pannexins may not form intercellular junctions in the same way connexins and innexins do and therefore should not use the same hemi-channel/channel naming. Others have presented evidence based on genetic sequencing and overall functioning in tissues, that pannexins should still be considered part of the gap junction family of proteins despite structural differences. These researchers also note that there are still more groups of connexin orthologs to be discovered.
Gap junction channels formed from two identical hemichannels are called homotypic, while those with differing hemichannels are heterotypic. In turn, hemichannels of uniform protein composition are called homomeric, while those with differing proteins are heteromeric. Channel composition influences the function of gap junction channels, and different connexins will not necessarily form heterotypic with all others.
Before innexins and connexins were well characterized, the genes coding for the connexin gap junction channels were classified in one of three groups (A, B and C; for example, , ), based on gene mapping and sequence similarity. However, connexin genes do not code directly for the expression of gap junction channels; genes can produce only the proteins that make up gap junction channels. An alternative naming system based on the protein's molecular weight is the most widely used (for example, connexin43=GJA1, connexin30.3=GJB4).
Gap junctions have continued to be found in nearly all healthy animal cells that touch each other. Techniques such as confocal microscopy allow more rapid surveys of large areas of tissue. Tissues that were traditionally considered to have isolated cells such as in bone were shown to have cells that were still connected with gap junctions, however tenuously. Exceptions to this are cells not normally in contact with neighboring cells, such as blood cells suspended in blood plasma. Adult skeletal muscle is a possible exception to the rule though their large size makes it difficult to be certain of this. An argument used against skeletal muscle gap junctions is that if they were present gap junctions may propagate contractions in an arbitrary way through cells making up the muscle. However, other muscle types do have gap junctions which do not cause arbitrary contractions. Sometimes the number of gap junctions are reduced or absent in diseased tissues such as cancers or the aging process.
the discovery of innexins, pannexins and unnexins, gaps in our knowledge of intercellular communication are becoming more defined. Innexins look and behave similarly to connexins and can be seen to fill a similar role to connexins in invertebrates. Pannexins also look individually similar to connexins though they do not appear to easily form gap junctions. Of the over 20 metazoan groups connexins have been found only in vertebrata and tunicata. Innexins and pannexins are far more widespread including innexin homologues in vertebrates. The unicellular parasites presumably have unnexin genes to aid in their infection of animals including humans. The even smaller adenovirus has its own vinnexin, apparently derived from an innexin, to aid its transmission between the virus's insect hosts.
The term gap junction cannot be defined by a single protein or family of proteins with a specific function. For example, gap junction structures are found in porifera, despite the absence of pannexins. While we are still at the early stages of understanding the nervous system of a sponge the gap junctions of sponges may as yet indicate intercellular communications pathways.
In a more general sense, gap junctions may be seen to function at the simplest level as a direct cell to cell pathway for electrical currents, small molecules and ions. The control of this communication allows complex downstream effects on multicellular organisms.
Gap junctions were found to be responsible for the transmission of signals required for drugs to have an effect. Conversely, some drugs were shown to block gap junction channels.
The bystander effect was later researched with regard to cells damaged by radiation or mechanical injury and in turn wound healing. Disease seems to have an effect on the ability of gap junctions to fulfill their roles in wound healing. The oral administration of gap junction blockers to reduce the symptoms of disease in remote parts of the body is slowly becoming a reality.
Hemichannels are thought to play a general role in the progression and severity of many diseases; this is in part due to hemichannels being an open door to the outside of each cell.
The associated figure shows how the size, shape, and frequency of gap junction plaques change with cell growth. With growth, fiber cells are progressively isolated from more direct metabolite exchange with the Aqueous humour through the capsule and lens epithelium. The isolation correlates with the classical circular shape of larger plaques shown in the yellow zone being disrupted. Changing the fiber cells' morphology requires the movements of vesicles through the gap junction plaques at higher frequencies in this area.
There has been some observation of coupling in the locus coeruleus between weak neurons and and in the cerebellum between and Bergmann glial cells. It appears that are coupled by gap junctions, both to other astrocytes and to . Moreover, mutations in the gap junction genes Cx43 and Cx56.6 cause white matter degeneration similar to that observed in Pelizaeus–Merzbacher disease and multiple sclerosis.
Connexin proteins expressed in neuronal gap junctions include mCX36, mCX57, and mCX45, with mRNAs for at least five other connexins (mCx26, mCx30.2, mCx32, mCx43, mCx47) detected but without immunocytochemical evidence for the corresponding protein within ultrastructurally defined gap junctions. Those mRNAs appear to be downregulated or destroyed by micro interfering RNAs () that are cell-type and cell-lineage specific.
Within the brain of the fruit fly Drosophila, gap junctions are known to be critical for a variety of functions.
Astrocytes
An important feature of astrocytes is their high expression levels of the gap junction proteins connexin 30 (Cx30) and connexin 43 (Cx43). These proteins play crucial roles in regulating brain homeostasis through potassium buffering, intercellular communication, and nutrient transport. Connexins typically form gap junction channels that allow direct intercellular communication between astrocytes. However, they can also form hemichannels that facilitate the exchange of ions and molecules with the extracellular space.
Studies have highlighted channel-independent functions of connexins, involving intracellular signaling, protein interactions, and cell adhesion. Specifically, Cx30 has been shown to regulate the insertion of astroglial processes into synaptic clefts, which controls the efficacy of glutamate clearance. This, in turn, affects the synaptic strength and long-term plasticity of excitatory terminals, indicating a significant role in modulating synaptic transmission. Levels of Cx30 regulate synaptic glutamate concentration, hippocampal excitatory synaptic strength, plasticity, and memory. Astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.
Cx30 is not limited to regulating excitatory synaptic transmission but also plays a crucial role in inhibitory synaptic regulation and broader neuronal network activities. This highlights the importance of connexins in maintaining the intricate balance required for proper brain function.
On a larger scale, the one-to-many communication of cells is typically carried out by the vascular and nervous systems. This makes detecting the contribution of hemichannels to extracellular communication more difficult in whole organisms. With the eye lens, the vascular and nervous systems are absent, making reliance on hemichannels greater and their detection easier. At the interface of the lens with the aqueous humor (where the lens exchanges metabolites), both gap junction plaques and more diffused connexon distribution can be seen in the accompanying micrographs.
Implicit or explicit in most of the early studies is that the area of the gap junction was different in structure to the surrounding membranes in a way that made it look different. The gap junction had been shown to create a micro-environment between the two cells in the extracellular space or gap. This portion of extracellular space was somewhat isolated from the surrounding space and also bridged by what we now call connexon pairs, which form even more tightly sealed bridges that cross the gap junction gap between two cells. When viewed in the plane of the membrane by freeze-fracture techniques, higher-resolution distribution of connexons within the gap junction plaque is possible.
Connexin free islands are observed in some junctions. The observation was largely without explanation until vesicles were shown by Peracchia using transmission electron microscopy (TEM) thin sections to be systematically associated with gap junction plaques. Peracchia's study was probably also the first study to describe paired connexon structures, which he called a globule. Studies showing vesicles associated with gap junctions and proposing the vesicle contents may move across the junction plaques between two cells were rare, as most studies focused on connexons rather than vesicles. A later study using a combination of microscopy techniques confirmed the early evidence of a probable function for gap junctions in intercellular vesicle transfer. Areas of vesicle transfer were associated with connexin free islands within gap junction plaques. Connexin 43 has been shown to be necessary for the transfer of whole mitochondrias to neighboring cells, though whether the mitochondria is transferred directly through the membrane or within a vesicle has not been determined
The ultrastructure and biochemistry of isolated gap junctions already referenced had indicated the connexins preferentially group in gap junction plaques or domains and connexins were the best characterized constituent. It has been noted that the organisation of proteins into arrays with a gap junction plaque may be significant. It is likely this early work was already reflecting the presence of more than just connexins in gap junctions. Combining the emerging fields of freeze-fracture to see inside membranes and immunocytochemistry to label cell components (Freeze-fracture replica immunolabelling or FRIL and thin section immunolabelling) showed gap junction plaques in vivo contained the connexin protein. Later studies using immunofluorescence microscopy of larger areas of tissue clarified diversity in earlier results. Gap junction plaques were confirmed to have variable composition being home to connexon and non-connexin proteins as well making the modern usage of the terms "gap junction" and "gap junction plaque" non-interchangeable. To summarize, in early literature the term "gap junction" referred to the regular gap between membranes in vertebrates and non-vertebrates apparently bridged by "globules". The junction correlated with the cell's ability to directly couple with its neighbors through pores in their membranes. Then for a while gap junctions were only referring to a structure that contains connexins and nothing more was thought to be involved. Later, the gap junction "plaque" was also found to contain other molecules that helped define it and make it function.
Studies allowing views inside the plane of the membrane of gap junctions during formation indicated that a "formation plaque" formed between two cells prior to the connexins moving in. They were particle free areas—when observed by TEM FF, indicated very small or no transmembrane proteins were likely present. Little is known about what structures make up the formation plaque or how the formation plaque's structure changes when connexins and other components move in and out. One of the earlier studies of the formation of small gap junctions describes rows of particles and particle free halos. With larger gap junctions they were described as formation plaques with connexins moving into them. The particulate gap junctions were thought to form 4–6 hours after the formation plaques appeared. How the connexins may be transported to the plaques using tubulin is becoming clearer.
The formation of plaque and the non-connexin part of the classical gap junction plaque have been difficult for early researchers to analyse. It appears in TEM FF and thin section to be a lipid membrane domain that can somehow form a comparatively rigid barrier to other lipids and proteins. There has been indirect evidence for certain lipids being preferentially involved with the formation plaque, however this cannot be considered definitive. It is difficult to envisage breaking up the membrane to analyse membrane plaques without affecting their composition. By study of connexins still in membranes lipids associated with the connexins have been studied. It was found that specific connexins tended to associate preferentially with specific phospholipids. As formation plaques precede connexins these results still give no certainty as to what is unique about the composition of plaques themselves. Other findings show connexins associate with protein scaffolds used in another junction, the zonula occludens ZO-1. While this helps us understand how connexins may be moved into a gap junction formation plaque, the composition of the plaque itself is still somewhat sketchy. Some headway on the in vivo composition of the gap junction plaque is being made using TEM FRIL.
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