Gap Junction Proteins (Connexins, Pannexins, and Innexins)
Gap Junction Proteins
Tissue distribution and effects of deficiencies of connexins in mouse and man
Phenotype of connexin-null mutant mouse
Most tissues, esp. heart
Oculodentodigital dysplasia (ODDD)
Cardiac malformation (postnatal lethality)
Zonular pulverulent cataract-3 (CZP3)
Zonular nuclear cataracts
Predisposition to arteriosclerosis
Atrial fibrillation familial 11
No human orthologue
Gja6/Cx33 in mouse
Zonular pulverulent cataract-1 (CZP1)
Microphthalmia, congenital cataracts
No mouse orthologue
Mouse orthologue is Cx57, horizontal cells not coupled
X-linked Charcot-Marie-Tooth disease (CMTX)
Late-onset neuropathy, liver carcinogenesis
Liver, skin, cochlea
Deafness (DFNA3, DFNB1A), skin diseasea
Lethality at ED 11
Deafnessb, skin diseasec
Transient placental dysmorphogenesis
Erythrokeratodermia variabilis (EKV)
Reduced vanilla scent, no hearing anomalies
Impaired placental development
Skin, brain, cochlea
Autosomal and digenic (with GJB2) deafness, hydrotic ectodermal dysplasia (Clouston syndrome)
Hearing insufficiency, no skin phenotype
No mouse orthologue
Lethality at ED 10.5
Hypomyelinating leukodystrophy 2 (HLD2)
Vacuolation of nerve fibers
Cx29 in mouse; myelinating cells
Neurons, β cells
Predisposition to juvenile myoclonic epilepsy
Mouse 30.3; decreased AV conduction time
Cx39 in mouse; –
Invertebrate gap junction channels are formed by the innexin family of proteins, of which there are 8 in Drosophila and 25 in Caenorhabiditis. In vertebrates, there are weakly homologous proteins termed pannexins, one of which (Pannexin1) has been demonstrated to form channels when expressed in Xenopus oocytes or in transfected mammalian cells; for the other isoforms, channel formation remains to be unequivocally demonstrated. Pannexin sequences contain two cysteine residues in each extracellular loop and there also are glycosolation sites within the loops suggesting that such branched sugar oligomers might serve to physically separate pannexons from one another. Although a few reports have indicated that over expressed Pannexin1 may form gap junction channels, and that deglycosylation might facilitate such gap junction formation, it seems unlikely that this occurs to any substantial extent (Sosinsky et al. 2011).
Gene Structures of Connexins and Pannexins
Most gap junction proteins are encoded by genes where the entire coding sequence is contained within a single exon. Although there are a few exceptions, where coding exons are interrupted, in no case does the coding region span more than two exons. Pannexin genes are much more complicated, with five exons for Pannexin1.
How Do Gap Junctions Form?
It was first shown in Xenopus oocytes that gap junctions would rather quickly form if oocytes were manipulated into contact. Similarly in both insect and mammalian cell lines formation of gap junctions between cells manipulated into contact is rapid. This has been interpreted as indicating that there is a large pool of connexons or so-called hemichannels in the surface membrane which are ready and able to join with partners across extracellular space to form the junctions. However, it is increasingly appreciated that most cells are highly polarized and that delivery to the site of the junctions may well be directed through cytoskeletal proteins attaching to adhesive junctions (Shaw et al. 2007) and including vesicular motors driving the directed mobility (Fort et al. 2011). It is now also increasingly appreciated that junctional proteins are added to the margins of the plaques and are destroyed following internalization of the connexons in both cells occurring at the plaque center (Fort et al. 2011; Laird 2006).
Are There Functional Hemichannels?
It has been demonstrated that certain connexins expressed exogenously readily form channels in the nonjunctional membrane; this is an especially prominent characteristic of the lens gap junction proteins, Cx46 and Cx50. The manipulation generally used to achieve opening of channels includes lowering extracellular calcium concentration. It has been argued that “hemichannels” are formed by Cx43, although in most cases the possibility that other channels (including pannexin1) may be responsible, has not been rigorously excluded. In fact, laboratories that originally attributed dye uptake and ATP release to opening of Cx43 hemichannels (Bennett et al. 2003) have now indicated that at least part of the exchange is through pannexin1 channels.
What Opens and Closes Connexin and Pannexin1 Channels?
Most of the original studies identifying agents that close gap junction channels were done using invertebrate cells because the method used (measurement of electrical coupling) was facilitated by having large cells to work with (insect salivary glands, giant axons of crayfish and earthworm). Some of the agents used for uncoupling experiments likely act through depolarizing the cells, a type of voltage dependence that is largely absent in mammalian gap junction channels. Thus, it should be appreciated that the uncoupling caused by elevation in intracellular calcium in salivary gland cells could be reversed by membrane hyperpolarization. Since pannexin1 is weakly homologous to innexins, it is somewhat surprising that the long chain alcohols (heptanol and octanol) have only minor effects on pannexin1 channels but totally block all connexin and innexin channels that have been tested.
Carbenoxolone and flufenamic acid block pannexin1 channels at concentrations somewhat lower than those needed to block connexin channels, whereas mefloquine is much more potent on pannexin1 than on any of the connexins (Bruzzone et al. 2005; Iglesias et al. 2009). There is literature indicating blockade of pannexin1 channels by agents that activate P2X7 receptors, which would be a protective effect against positive feedback on channel opening (Qiu and Dahl 2009).
Voltage dependence is a prominent feature of gap junctions. In invertebrates, the prominent voltage dependence is between the inside and outside of the cell, likely sensed across the channel wall. Depolarization of one or both cells reduces junctional conductance. In vertebrate gap junction channels, there is a minor component, if any, to voltage dependence which rather involves sensing the voltage through the gap junction channel between the cytoplasm of one cell with that of the other (see del Corsso et al. 2006). For certain connexins, the voltage dependence is rather steep, such that regenerative uncoupling can occur if there is a sustained transjunction voltage and a brief triggering event (Spray et al. 1981). Early modeling studies predicted the voltage sensitivity of the gates on each side of the gap junction and studies expressing one connexin in one cell and another in the other have confirmed such independent gates. Moreover, studies on Cx46 and Cx50 indicate that hemichannels have gating as predicted from the modeling experiments. Voltage dependence of pannexin1 is such that under normal conditions of extracellular ions, channel opening begins at about 0 mv, but when extracellular K+ is raised activation threshold voltage is lowered (Silverman et al. 2009).
Finally, there are other mechanisms by which pannexin1 channels may be activated. First of all, it appears to be coupled to P2X7 receptors forming the highly permeable pore that is opened by prolonged exposure to high concentrations of ATP or specific agonists. Pannexin1 currents are also modulated by mechanical stretch, caspases, and following activation of membrane receptors (P2X and P2Y, α1-adrenergic, glutamatergic (NMDA), and thromboxane receptors) via signaling mechanisms that lead to elevation of cytosolic calcium levels and/or to kinases activation (PKA, tyrosine kinases) (reviewed in Scemes and Velísková 2017). Voltage dependence in the invertebrate gap junction channels generally displays sensitivity to absolute potential (inside-out) such that depolarization will close the channel. Examples in which this has been quantitatively examined include drosophila and midge salivary glands and crayfish. It is noteworthy that such depolarization could have been one mechanism by which calcium produced the uncoupling action that was thought to be a major control mechanism for gap junctions; it was shown that uncoupling by calcium could be reversed by hyperpolarization (Obaid et al. 1983).
The notion that junctional conductance could be decreased by elevated intracellular calcium was initiated by the finding that high calcium levels could cause cells to seal off from their neighbors following damage. This was followed by demonstration that injection of high concentrations of calcium into cells could uncouple. However, these early experiments did not consider secondary effects on pH or voltage gating, and it is now known that the calcium levels necessary to obtain uncoupling are likely very high (see Obaid et al. 1983).
What Do Connexin and Pannexin Channels Do?
It has long been appreciated that current flow through gap junctions in working myocardium mediates synchronous cardiac contractions, that gap junctions between neurons mediates rapid electrical transmission, that gap junctions in lens fibers facilitate transparency through efflux of metabolites, and that gap junctions in exocrine and endocrine glands facilitate synchronous secretion. Results from mutagenesis studies in mice and from human genetic analysis indicate that dysfunction and disease accompanying reduced or altered gap junction function was widespread (see Table 1, modified from Dobrowolski and Willecke 2009, by inclusion of recent data obtained from OMIM [Online Mendelian Inheritance in Man]), varied and in many cases, totally unexpected. Mutation in the major gap junction protein of liver and peripheral myelinating glia (Schwann cells) results in a progressive peripheral neuropathy, X-linked Charcot Marie tooth disease. Mutations in either of the lens gap junction proteins, Cx50 or Cx46, result in cataract formation, the difference being that Cx50 mutations/deletion leads to growth defects as well. Mutations in the major gap junction protein of myelinating glia in the CNS, Cx47, also lead to demyelinating diseases as well as the recently discovered lymphedema and mutations in Cx43 are now known to underlie ODDD (Pfenniger et al. 2011).
Role of Connexin and Pannexin Channels in Calcium Wave Spread Among Astrocytes
Intercellular communication can occur as a result of direct communication of intracellular signals through gap junction channels or through the release of transmitter molecules from one cell which diffuses across intracellular space, binding to receptors on another cell that respond by opening an ion channel or initiating a second messenger cascade (Scemes and Giaume 2006). Connexins in vertebrates and innexins in invertebrates are the only proteins known to form intercellular channels. They are permeable to molecules up to a molecular weight of about 1 kDa, permitting passage of ions and signaling molecules such as cAMP, IP3, and ATP. One impressive example of how gap junctions allow signaling between cells is the phenomenon of “calcium waves” where IP3 liberated within one cell diffuses to an adjacent cell, evoking calcium release in this cell. Such waves generally have velocities on the order of 10–20 μm/s and passage can be to considerable distance from the site of initiation. An additional pathway of signaling between cells which can also play a role in calcium spread involves release of ATP from one cell and reception by a purinergic receptor in an adjacent cell. Pathways of ATP release can be pannexin1 channels or vesicular release. Based on studies evaluating the contribution of the two pathways involved in the transmission of calcium waves, the emerging concept is that connexin gap junction channels define communication compartments within a tissue, whereas pannexin channels provide amplification of signaling both within and between compartments (Scemes et al. 2007).
- Scemes E, Spray DC (2008) Connexin expression (gap junctions and hemichannels) in astrocytes. In: Parpura V, Haydon PG (eds) Astrocytes in (patho)physiology of the nervous system. Springer, New York, pp 107–150Google Scholar