Gap junctions allow the exchange of ions, second messengers, and small metabolites between adjacent cells and are formed by two unrelated protein families, the pannexins and connexins. Mutations in connexin genes cause a variety of genetic disorders, implicating a critical role in tissue homeostasis. Association of congenital skin disorders to mutations in different connexins has underscored the importance of gap junctional communication in the skin and its appendages. Here, we discuss the basic structure of gap junction channels and the function of connexin genes that have been associated with human disorders to explore the physiology of intercellular communication in skin.
Mutations in GJB2, which encodes Cx26, are one of the most common causes of inherited deafness in humans. More than 100 mutations have been identified scattered throughout the Cx26 protein, most of which cause nonsyndromic sensorineural deafness. In a subset of mutations, deafness is accompanied by hyperkeratotic skin disorders, which are typically severe and sometimes fatal. Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (E1) domains. Here, we examined two such mutations, A40V and G45E, which are positioned near the TM1/E1 boundary and are associated with keratitis ichthyosis deafness (KID) syndrome. Both of these mutants have been reported to form hemichannels that open aberrantly, leading to “leaky” cell membranes. Here, we quantified the Ca2+ sensitivities and examined the biophysical properties of these mutants at macroscopic and single-channel levels. We find that A40V hemichannels show significantly impaired regulation by extracellular Ca2+, increasing the likelihood of aberrant hemichannel opening as previously suggested. However, G45E hemichannels show only modest impairment in regulation by Ca2+ and instead exhibit a substantial increase in permeability to Ca2+. Using cysteine substitution and examination of accessibility to thiol-modifying reagents, we demonstrate that G45, but not A40, is a pore-lining residue. Both mutants function as cell–cell channels. The data suggest that G45E and A40V are hemichannel gain-of-function mutants that produce similar phenotypes, but by different underlying mechanisms. A40V produces leaky hemichannels, whereas G45E provides a route for excessive entry of Ca2+. These aberrant properties, alone or in combination, can severely compromise cell integrity and lead to increased cell death.
Dominant Cx26 mutations that cause keratitis-ichthyosis-deafness syndrome (KIDS) show increased hemichannel activity. Transgenic expression of these mutations recapitulates human skin disease in mice. Excess hemichannel activity persists in diseased epidermis from the transgenic mice. Thus hemichannel activity may be a novel therapeutic target in the treatment of KIDS.
Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I h) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I h current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K+ permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were ∼10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 ≥ Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.
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