User:Dovesmarx/Gap junction modulation

In biology, gap junction modulation describes the functional manipulation of gap junctions, specialized channels that allow direct electrical and chemical communication without exporting materials from the cytoplasm. This manipulation is achieved by endogenous chemicals, growth factors, hormones and proteins that affect gap junction expression, structure, degradation and permeability.

This modulation can be divided into two types: fast and slow regulation.

Fast regulation is voltage-gating that utilizes chemical material such as Ca2+, resulting in change in permeability via altering the size of the channel. This kind of rapid modulation is found in mammalian cells such as cardiac cells, lacrimal gland cells, etc. Although the effect of voltage gating is small, it is important in regulating other modes of gap junction modulation, such as phosphorylation, assembly and synthesis of gap junction.

Slow modulation depends on the assembly of the gap junction on cell membrane. Although the removal of channels remains unclear, the synthesis of channels is known to be regulated by a chain of phosphorylation steps and connexin binding partners.

Mechanism
Due to its molecular structure, gap junctions are sensitive to electricity. This sensitivity allows the channel to alter its size and structure according to electrical signals. The signals result in release of Ca2+, which yields a positive feedback on voltage gating. This gap junction modulation via calcium is modulated by, and closely related to, pH and Calmodulin(CaM).

Vj gating
The first type of voltage gating is Vj gating. This gating method governs the size of the gap junction, being able to reduce the channel size to 5~40% of the fully open state. The sensitivity towards voltage comes from the cytoplasmic NH2-terminal part and two locations in the NH2-terminal domain of the first extracellular loop, being sensitive even to small voltage (2-3mV). Voltage gating modulation is associated with the charge of connexins; positive charged connexins close with hyperpolarization and negative charged connexins close with depolarization. With opposite charge, the regulation of Vj gating is regulated under different concentrations of Ca2+, H+ and the mechanism of Calmodulin (CaM).

Slow voltage gating
The second type of voltage gating is hypothesized to be similar to Vj gating in terms of mechanism, but unlike Vj gating, fully closes the channel to non-conducting state. This modulation is slower than the prior gating method, as this gating method occurs in response to Vj gating. The temporal voltage regulation is also subject to higher voltage (10-30mV), chemical factors–such as lipophiles, low pH–and the docking of two hemichannels. The exact mechanisms of both Vj gating and slow voltage gating are not known yet, but it is hypothesized that the change in charge causes the cytoplasmic NH2-terminal domain to move toward the cytoplasm to reduce the pore size.

Calcium
Calcium exists in an organism as a form of ion, Ca­2+. This ion is found to be effective in modulating gap junctions. With an increase in [Ca2+] above 5x10-5, the permeability of plasma membrane decreases rapidly. This modulation via calcium is known to be protective, as it prevents dead cells from affecting neighboring cells. Yet, high Ca2+ concentration is rarely seen, as this gating method is self-inhibiting. It has been found that Ca2+ modulation and voltage gating are closely related, as the entry of Ca2+ is more effective in modulating gap junction than release of Ca2+ from internal store and increase in Ca2+ concentration via Ca2+ entry results in depolarization.

pH
The pH sensitivity depends on the type of connexin the gap junction is composed of, but the channels generally close at pH 6.4-6.2. Under weak acidic conditions, the channels are observed to not open at all with voltage changes, while under strong acidic conditions, the channels do open, but close immediately.

In some studies, the synergic effects of [Ca2+]i and pH were found. Cardiac cells, for example, EGTA releases Ca2+ at low pH. Yet, other studies contradict the synergic theory; for example, in ischemic patients, application of calcium ionophore reduced the permeability of gap junctions, but no sign of potentiation was found from the cytosolic acidification (pH 6.4 or 6.0).

Calmodulin (CaM)
Calmodulin is a protein composed of 148 amino acids that has two Ca2+ binding sites. With Ca2+, calmodulin goes through conformational change that releases two hydrophobic pockets. This protein is found in certain connexin, and with the binding of Ca2+, it blocks the channel either physically or structurally.

While the inhibition of CaM expression increases the probability of cell-to-cell uncoupling, CaM antagonist and CaM blockers, on the other hand, are found to prevent the uncoupling of cells.

Phosphorylation
Phosphorylation, the addition of a phosphate group, plays an important role in regulating gap junctions and the subunits that form them. The gap junction protein connexin generally possess a number of phosphorylation sites (Cx43 has 21). This addition can bring about various effects that influence the protein’s lifecycle and the gap junction itself. For example, phosphorylation of Cx43 promotes its trafficking from the Golgi apparatus to the plasma membrane. The oligomerization of this protein into hemichannels and the hemichannels into gap junctions is also induced by phosphorylation. Likewise, degradation can be initiated by phosphorylation as well as changes in gating, which determines the permeability of gap junctions.

Phosphorylation of gap junctions and their subunits is typically achieved through protein kinases, enzymes that add phosphates to the amino acids of proteins. Serine/threonine kinases, which phosphorylate the hydroxyl group of either serine or threonine residues, form the bulk of the Connexin phosphorylation kinases. These include protein kinase C (PKC), protein kinase G (PKG), Ca2+/calmodulin-dependent kinase II (CaMKII), cAMP-dependent protein kinase A (PKA), MAP kinase (MAPK) and casein kinase (CK). Kinase Src is the lone Tyrosine kinase that has been observed to phosphorylate connexins. Protein kinases vary in their targeted connections, specific sites of phosphorylation and phosphorylation effect.

For example, PKA phosphorylation affects both hemichannel and connexin activity. Neuronal hemichannel activity is suppressed by reducing permeability. Connexins are affected by an increased trafficking and assembly into gap junctions. PKA activity is largely associated with an increased cAMP concentration. PKB phosphorylation can prevents the binding of ZO-1, resulting in an increased gap junction size and increase hemichannel permeability. Its activity is usually in response to physiological changes such as wounding or hypoxia.

Ubiquitination
Ubiquitin is a small, long lived, globular protein that covalently bonds to lysine resides of target proteins in a process known as ubiquitination. Much like phosphorylation, it acts as a post translational regulator for many proteins including connexin. Ubiquitination has been observed to be most involved in the Connexin lifecycle’s final stages, regulating both Gap junction endocytosis and Connexin degradation. However, details of specific pathways and involved proteins are still being studied.

The distinct effects of ubiquitination tend to vary widely, depending on the type of ubiquitin and the tissues and subcellular location where it occurs. For example, newly synthesized Cx43 in the endoplasmic reticulum can undergo polyubiquitintation, resulting in recognition by proteasome for endoplasmic reticulum associated protein degradation (ERAD). Ubiquitination of Cx43 that is at the plasma membrane and organized into gap junctions will result in internalization, or endocytois, followed by degradation of Cx43 by lysosomes.

Nitrosylation
Nitrosylation, the addition of a Nitric oxide (NO) group, has been demonstrated to have a substantial role in causing post translational modifications of both gap junction proteins and hemichannels. This is achieved by Nitrosylation, which is either induced on connexin proteins or proteins that further regulate connexins such as kinases. The type of Nitrosylation involved is S-nitrosylaton, the addition of a nitric oxide group to a cysteine thiol of a protein.

Experiments regarding S-nitrosylation and the lifecycle of gap junctions suggest it has a role in regulating hemichannel trafficking and gap junction formation; addition of NO rapidly increased the level of Cx40 and Cx43 at the plasma membrane as well as the formation of gap junctions in endothelial cells. The mechanism behind this phenomena is still unknown but the pro oxidant conditions induced by NO is thought to be modulating the properties of the Golgi apparatus.

Related diseases
The pathogenesis of gap junction on various diseases is studied thoroughly. Studies nominate gap junction as a drug target for the significance of these junctions in diseases.

Arrhythmogenic cardiomyopathy (ARVD/C)
Electrical coupling among cardiac cells is a crucial aspect of healthy heart, allowing cells to contract ordinally. The coupling is done by gap junctions. Gap junctions enable passive diffusion of materials–such as ions–across cytoplasm of a cell to another; this junction enables proper propagation of electrical impulse along cardiac cells.

ARVD/C, therefore, affects the gap junction functions by reducing its numbers. This cardiac disease results in change in expression of numerous substances, including N-Cadherin and PKP2, which subsequently decreases gap junction. Decrease in N-Cadherin is found to decrease the expression of connexin 43 (Cx43), major protein composition of gap junction, leading to reduction in conduction in velocity. PKP2 also yields reduced Cx43, yet via reduction in N-Cadherin reduction.

Liver diseases
Liver failure has many causes, including abnormal expression of gap junction. Taking cirrhosis and ACLF for examples, the increased expression of hepatic connexin 43 is one of their cause. The change in rate of gap junction expression is found to yield inflammation in liver. Increased expression of Cx43 propagates death signals to neighboring cells.

Gastrointestinal diseases
Just as in the heart, gap junctions play significant role in mediating electrical signal among intestines. The signals allow synchronization of smooth muscles, buffering substrate concentrations, and mediating inflammation. Dysfunction in gap junctions lead to numerous symptoms such as gastrointestinal infections, autistic spectrum disorder, and inflammatory bowel disease.

The pathogenesis in which gap junction yields disease is different for each disease. For inflammatory bowel disease, the decrease in gap junction expression disrupts junctional complexes among intestinal cells. Little is known about the mechanism of pathogenesis of gap junction on gastrointestinal infection, but the correlation is clear: increased Cx43 level during infection, abnormal localization of Cx43, and unpaired functional Cx43.