DEFINITION
Connexins are proteins of the family pf the gap junction, a kind of cellular junction that allows passages between cells of molecular components with weight inferior than 1 KDa like IP3, cAMP, Ca2+ and K+ ions, second messengers, H20, electric pulses and various kind of metabolites. These particular kind of junction have a fundamental role in biological processes as differentiation, tissues homeostasis and cell cycle regulation; they can be also involved in pathological states as heart failure.
THE GENE
Connexins’ transcripts involved 21 human genes with a simply structure, first exon is not transcripted and the coding region is in the second exon.
GENE/PROTEIN DATABASES
STRUCTURE
Söhl, G. et Al. Gap junctions and the connexin protein family. Cancer Res. 62, 228-232; 2004.
Six connexins give rise to a macromolecular structure called “connexon” which in turn give rise to an emi-channel which assembly with the corresponding emi-channel of the second cell originating the gap junction where the two cell membranes are separated by 2-3 nm. There are still membrane connexons that don’t couple with other ones of others cells but remain as emi-channel and their role is not to put in communication two different cells but to regulate Ca2+, ATP and NAD+ levels. Connexin have two transmembrane domains, two extracellular domains and three high conserved extracellular loops enriched in cysteine that allows formation of disulfide bonds with extracellular loops of another connexon making possible a stable connection and formation of a functional gap junction; there are also a n-terminal and a c-terminal cytosolic loops.
LOCALIZATION
There are 21 different types of connexins expressed in spatial and time dependent way; gap junction have different roles depemding on cellular context; there are specific connexins for myocardium: endothelial cells (Cx37, Cx40), cardiomyocytes (Cx40, Cx43, Cx45); they are still presents in neurons (Cx36/45/57), in hepatocytes (Cx26/32) and Cx43 is present in skin, cornea and its stem cells, lens, iris, ciliary epithelium, optic nerve, retina and central nervous system.
THE ISOFORM CONNEXIN 43
Known as α1 connexin, connexin 43 has a molecula weight of 43 KDa, is the most common isoform, present in 34 tissues and in more than 46 different cellular types and is the most common myocardial isoform too. In myocardium originates gap junction in intercalated discs between cardiomyocytes where has half-life of 1-3 h allowing an electric and metabolic communication (channel has a width of 250 Amstrong and a diameter of 25 Amstrong): its features include metabolic homeostasis, cardiac contraction synchronization, bi-directional movement of intracellular ions, metabolites and second messengers and ruling cellular fate even in stress conditions.
Regulation
Among different regulations mechanism as Ca2+, pH, phosphorylation state, intercellular voltages from 15 to 90 mV, calmodulin binding, cyclic nucleotides, neurotransmitters (dopamine, acetylcholine, and g-aminobutyric acid), and hormones (isoproterenol and estrogens), pH levels are very important for connexin’s structure changes: C-terminal if fundamental for “ball and chain” model in which channel is closed because of interaction of an intracellular molecule linked to a flexible element that recognize a specific receptor on the channel itself (in Cx43 the C-terminal interacts with cytosolic loop linking its second half): if pH levels are normal the channel is open while at lower pH levels is closed: different structural conformations of C-terminal loop are responsible of this mechanism: if pH is 7.5 it has a random-coil conformation as at 5.8 has an ordered α-helix conformation due to amminoacids present that are protonated if pH level are low modifying theirs structure to α-helix conformation.
Cx43 C-terminal is its regulatory region that has two α-helix and a random coil. Cx43 interacts with different protein as ZO-1, Src, caveolin, β-catenin, p120-catenin and tubulin.
Phosporilations are responsible of different post-translational modifications: 12 of 21 serines and two tyrosines are phosphorilated by kinases as PKC, PKA, MAPK, CKI, p34cdc2, and Src.
Solan, J.L. et Al. Connexin43 phosphorylation: structural changes and biological effect. Biochem. J. 419, 261-272; 2009..
Red domains in figure are responsible of Cx43 down-regulation while green domains regulate its assembly. Furthermore Ser255, when phosphorilated by MAPK1/ERK and by p34cdc2, down-regulates the protein; tyr 265/247 phosphorilates by Src cause channel closing and Ser325/328/330 phosphorilated by CKI are responsible of gap junction assembly. Mapk phosphorylates Ser279/282 too reducing channel opening time, PKC phosphorylates Ser262, causing cell cycle progression and proliferation, and Ser368, reducing channels and, as showed later, this specific phosphorilation is associated with cardiac pathologies. Increasing in cAMP and conseguent PKA activation, a kinase involved in Cx43 phosphorilation, causes an increased exportation of Cx43 through cell membrane and gap junction formation: this specific phosphorylation, that occurs on Ser365, prevents another phosphorilation on Ser368 that is conversely up-regulated in pathologic condition as cardiac I/R damage and phorbol treatment suggesting a protective role of this particular type of phosphorylation.
Solan, J.L. et Al. Connexin43 phosphorylation: structural changes and biological effect. Biochem. J. 419, 261-272; 2009..
Assemblaggio e Degradazione
Cx43 degradation comes after its internalization, thanks to a structure called connexosome (a double-membraned vacuole, so that one of the membrane part of the involved gap junction comes in one of the two cells) or with a more classic way that involves endocytosis: both way are regulated by ubiquitin.
synthesis: Cx43 is co-translated in endoplasmic reticulum, transported in Golgi apparatus, assembled in connexons in trans-Golgi and finally transported through the membrane using microtubules to form functional gap junctions;
degradation: there are some hypothesis not verified yet:
1) defective subpopulation of Cx43 are degradated in a way involving endoplasmic reticulum;
2) internalization with connexosome (clatrin and proteasome are involved);
3) lisosome dependent degradation (vesicles from cellular membrane arrive at lisosome, there is a fusion between connexosome and lisosome and a proteolitic enzymes mediated degradation)
Another way of Cx43 degradation involves proteasosme, an intracellular proteic complex responsible of mono or polyubiqitinated proteins degradation. The ubiquitin ligase is the enzyme responsible of ubiquitin attachment to different proteins: there are different isoforms and each one recognize different degradation signals. Cx43 ubiquitination occurs on plasma membrane and is phosphorylation dependent, a process regulated by PKC and MAPK pathway: key role in this process is played by Ser282 located near a PY called domain enriched in prolines and tyrosines and near a tyrosines rich domain; the first domain is recognized and linked by the WW (Trp-Trp) domain of E3 Nedd4 ubiquitin lygase enzyme: phosphorylation is not necessary for ubiquitination but strongly modulate Nedd4-Cx43 linking (this occurs for Ser279 too).
The tyrosine rich domain is responsible for Cx43 internalization and lisosome-mediated degradation. It is evident how Cx43 degradation occurs under ubiqitin-dependent and ubiquitin-independent mechanisms. Cx43 hyperphosphorylation on specific amminoacids causes its disorganization, internalization and degradation under mechanisms that involve MAPK pathway and proteasome as using MEK and proteasome inhibitors these effects are blocked; the endocytosis process occurs in presence of clatrin.
Growth factors as EGF cause internalization and degrdation of CX43.
Leithe E, Rivedal E. J Cell Sci. 2004.
Cx43 and Heart
Cx43 turnover in the heart physiologically modulate pulse propagation and seems to have a key role in cardiac pathologies as it shows a role in myocardium remodeling in arythmies, in hypertrophic mechanisms, in ischemia and infarct: stressed cardiomyocites show a strong down-regulation of Cx43.
Cx43: protective or dangerous?
Ca(2+) is known as a universal messenger mediating a wide variety of cellular processes, including cell death. In fact, this ion has been proposed as the 'cell death master', not only at the intracellular but also at the intercellular level. The most direct form of cell communication is represented by gap junction that spread between cells both survival and death signals mediated by Ca(2+) that has a duouble role. Several studies pinpoint Ca(2+) or its mediator inositol triphosphate as master responsible in mediating cell death evidentiating a conseguent role of connexin as a "toxic pore": http://www.ncbi.nlm.nih.gov/pubmed/21621840 .
On the other hand, evidence shows a beneficial role of Cx43 in cardiomyocite survival: as known ischemic preconditioning protect the heart from hypoxic insults: ischemic preconditioning (PC) is due in part to: (i) disruption of cell-cell coupling (manifest as a loss in the primary gap junction protein, connexin 43 [Cx43]) and resultant impaired transmission of a 'death' messenger, or conversely, (ii) transfer of a humoral 'survival' factor via existing gap junctions. Using a gap junction uncoupler, heptanol, authors show that, while in control mice with a "wild tipe" pattern of Cx43 infarct size was reduced with preconditioning, mice treated with heptanol show no reduction of infarct size making ischemic preconditioning ineffective in eliciting protection.
Cx43 seems to have a double role but it is not clear yet if it is principally a cell-survival mediator or a cell-death mediator.
Cx43 and Cell Cycle
Cx43 has a key role in regulation of cell cycle to as it is notably up-regulated during S and M phases comparing to G0/G1 phases: cellular communications strongly increase during S phase comparing to G1/S transition, where is relatively moderate and during G2/M transition where it is incisively diminuished: the kinase that regulates phosphorylations on Cx43 durin cell cycle regulation is cdc2 (CDK1).
