Connexin

Connexins (Cx), or gap junction proteins, are a family of structurally related transmembrane proteins that assemble to form vertebrate gap junctions. Each gap junction is comprised of 2 hemichannels, called connexons, which are constructed out of 6 connexin molecules. Gap junctions can transmit small regulatory and signaling molecules between neurons. Thus they are essential for many physiological processes, such as direct synaptic transmission, heart muscle coordination, proper embryonic development and differentiation, growth control and others. For this reason, mutations in connexin-encoding genes can lead to functional and/or developmental abnormalities.

Structure

Connexins are 7.5 nm long proteins, having four membrane-spanning regions with both C and N cytoplasmic termini, a cytoplasmic loop and two extracellular loops. Connexins may be identical or slightly different from one another. Connexins are assembled together in groups of six to form hemichannels, or connexons, and two hemichannels then combine to form a gap junction.

The various connexins have been observed to combine to form different gap junctions, each of which may exhibit different functional properties including pore conductance, size and charge selectivity, voltage and chemical gating.

More than 20 connexin genes have been found in the mouse and human genome. They usually weigh between 26 and 60 kDa, and have an average length of 380 amino acids.

Connexins are most commonly named according to their molecular weight, e.g. Cx26 is the connexin protein of 26 kDa. This can lead to confusion when connexin genes from different species are compared, because they can have different molecular weight. E.g. human Cx36 is homologous to zebrafish Cx35.

Distribution

Electrical transmission firstly was discovered in 1959 by Furshpan and Potter in the crayfish giant motor synapse [1]. Later, this type of pathway was found in other excitable as well as non-excitable tissues. Today gap junctions are known to be present in cardiac muscle [2], vascular smooth muscle [3], liver [4], epithelium [5], pancreas [6], central nervous system [7] and other tissues. In vertebrates, only a few cell types (red blood cells, spermatozoa, and skeletal muscle) in their fully differentiated state do not form gap junctions. Nevertheless, the progenitors of these cells do express gap junctions [8]. Thus gap junctions and their structural proteins connexins are widely distributed across all vertebrate tissues.

Connexin genes

The screening of genomic databases has revealed 19 and 20 connexin genes in the mouse and human genome, respectively. The structure of the connexin genes is quite conserved among vertebrates. The sequence similarities suggest that they have arisen by gene duplication. In humans, a Cx43 pseudogene has also been identified.

The genes for various connexins have been localized to human and mouse chromosomes. Connexin genes are found distributed in different chromosomes, but there is a cluster of connexins in a region of mouse chromosome 4 and in human chromosome 1. Many connexin genes have a similar organization, and practically all have only single copies in the haploid genome. Most connexin genes contain two exons and an intron of variable length; some of the genes contain three exons.

Connexin biosynthesis, assembly and degradation

Biosynthesis

The biosynthetic studies have been most extensive for Cx43, which serves as a model for all other Cxs. Cx43 is initially synthesized as a 40- to 42-kDa polypeptide that is later posttranslationally modified, adding phosphate groups. Phosphorylation of other connexins also occur after their synthesis.

The location for connexin assembly into connexons is connexin-type dependent. It appears that Cx32 assembles in the endoplasmic reticulum (ER) (or ER/Golgi intermediate place) while Cx43 assembles in the Golgi network. In cells expressing two or more connexin types, homomeric (formed by only one protein type) or heteromeric (constituted by different connexins) hemichannels can be found. Multiple Cx types expressed in the same cell can localize to the same or distinct membrane domains, suggesting the regulation of their topographic fate. Sequence motifs, responsible for the specific Cx localization, have not been identified yet. Insertion mechanism is not known as well.

Gap Junction Assembly

The level of connexin protein depends on transcriptional regulation and posttranslational mRNA modification. Both mechanisms can be regulated by various intercellular messengers, such as cAMP and others.

Connexons are inserted into the plasma membrane in a closed configuration. Formation of gap junction plaques requires clustering of connexons. Increased levels of cAMP can induce clustering of gap junction channels.

After being inserted into the plasma membrane of the cell, the hemichannels freely diffuse within the lipid bilayer. Through the aid of specific proteins, mainly cadherins, the hemichannels are able to dock with hemichannels of adjacent cells forming gap junctions. Recent studies have shown the existence of communication between various other proteins within the cell. Colocalization of connexins with a wide variety of them, including aquaporins [9], cytoplasmic proteins [10] and cytoskeletal proteins [11] has been demonstrated.

Degradation

A remarkable aspect of connexins is that they have a relatively short half-life of only a few hours. [12, 13]. However, at least some of the connexins are more stable, with half-lives of 2–3 days or more [14]. Both degradation pathways, proteasomal and lysosomal ones have been demonstrated for Cx43 in the heart. List of human connexins and their functions

List of human connexins and their functions

Connexin	Gene	Location and Function
Cx23
Cx25
Cx26	[1]
Cx30.2		Expressed in structures of the inner ear. Thought to have a role in ion transport for signal transduction in hair cells [15].
Cx30	[2]
Cx31.9	[3]
Cx30.3	[4]	Fonseca et al. confirmed Cx30.3 expression in thymocytes [16].
Cx31	[5]
Cx31.1	[6]
Cx32	[7]	Major component of the peripheral myelin. Its deletion causes significant developmental defects to the central nervous system.
Cx36		Pancreatic beta cell function, mediating the release of insulin.
Cx37	[8]	Induced in vascular smooth muscle during coronary arteriogenesis. Cx37 mutations are not lethal. Forms gap junctions between oocytes and granulosa cells, and are requred for oocyte survival.
Cx40.1
Cx40	[9]	Expressed selectively in atrial myocytes. Responsible for mediating the coordinated electrical activation of atria [17].
Cx43	[10]	Expressed at the surface of vasculature with atherosclerotic plaque, and up-regulated during atherosclerosis in mice. May have pathological effects. Also expressed between granulosa cells, which is required for proliferation.
Cx45	[11]	Human pancreatic ductal epithelial cells [18].
Cx46	[12]
Cx47	[13]
Cx50	[14]
Cx59	[15]
Cx62

References

1. Furshpan EE, Potter DD. (1959). Transmission at the giant motor synapses of the crayfish. Journal of Physiology, 145: 289-325.

2. Barr L, Dewey MM, and Berger W. (1965). Propagation of action potentials and the structure of the nexus in the cardiac muscle. J Gen Physiol 48: 797–823.

3. Brink PR. (1998).Gap junctions in vascular smooth muscle. Acta Physiol Scand 164: 349–356.

4. Benedetti EL and Emmelot P. (1965).Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J Cell Biol 26: 299–305.

5. Loewenstein WR and Kanno Y. (1964).Studies on an epithelial (gland) cell junction. I. Modification of cell permeability. J Cell Biol 22: 565–586.

6. Meda P. (1996).Gap junction involvement in secretion: the pancreas experience. Clin Exp Pharmacol Physiol 23: 1053–1057.

7. Dermietzel R and Spray DC. (1993).Gap junctions in the brain: where, what type, how many and why? Trends Neurosci 16: 186–192.

8. Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. (2003). Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev. 83(4):1359-1400.

9. Rash JE and Yasumura T. (1999). Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis. Cell Tissue Res 296: 307–321.

10. Sotkis A, Wang XG, Yasumura T, Peracchia LL, Persechini A, Rash JE, and Peracchia C. (2001).Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating. Cell Commun Adhes 8: 277–281.

11. Giepmans BN, Verlaan I, Hengeveld T, Janssen H, Calafat J, Falk MM, and Moolenar WH. (2001).Gap junction protein connexin-43 interacts directly with microtubules. Curr Biol 11: 1364–1368.

12. Crow DS, Beyer EC, Paul DL, Kobe SS, and Lau AF. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol 10: 1754–1763, 1990.

13. Fallon RF and Goodenough DA. (1981). Five hour half-life of mouse liver gap junction protein. J Cell Biol 90: 521–526.

14. Berthoud VM, Bassnett S, and Beyer EC. (1999). Cultured chicken embryo lens cells resemble differentiating fiber cells in vivo and contain two kinetic pools of connexin56. Exp Eye Res 68: 475–484.

15. del Castillo I, et al. (Jan 24 2002). A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N Engl J Med 346 (4): 343-9. PMID 11807148.

16. Fonseca PC, Nihei OK, Urban-Maldonado M, Abreu S, de Carvalho AC, Spray DC, Savino W, Alves LA (June 2004). Characterization of connexin 30.3 and 43 in thymocytes. Immuno lett. 94 (1-2): 65-75. PMID 15234537.

17. Gollob MH, et al. (Jun 22 2006). Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med 354 (25): 2677-88. PMID 16790700.

18. Tai M-H (2003). Characterization of Gap Junctional Intercellular Communication in Immortalized Human Pancreatic Ductal Epithelial Cells With Stem Cell Characteristics. Pancreas (1): e18-e26.