Formation of heteromeric channels by Cx26S17F syndromic deafness mutant and Cx30 and its consequences on cochlear pathology



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Universidad de Valparaíso





Facultad de Ciencias

Departamento o Escuela

Programa de Doctorado de Ciencias Mención Neurociencia




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Doctor en Ciencias con Mención en Neurociencia. Universidad de Valparaiso. 2020.


Deafness is the most common condition associated with mutations in connexin (Cxs) genes, being mutations in Cx26 gene (GJB2) the most prevalent. Specific mutations in Cx26 also produce syndromic deafness, like Keratitis-Ichthyosis-Deafness (KID) syndrome, in which patients with deafness show severe alterations in the skin. KID syndrome symptoms include deep deafness, hard skin in hands and feet (palmoplantar keratoderma), red spots of thick (erythrokeratoderma), and scaly skin (ichthyosis), alopecia, bad nail formation, autoamputation of the last phalanges and thickness in the cornea (keratitis) that in severe cases leads to blindness. In the main organs affected by KID mutations, Cx26 is co-expressed with Cx30. In the skin they are co-expressed in keratinocytes, while in the inner ear, they are co-expressed in the supporting cells of the organ of Corti. One mutation observed in KID patients is mutation Cx26S17F, which affect the N-terminal of Cx26, a Cx segment that is involved in the closure and opening of hemichannels (HC). In this doctoral thesis, we studied the functional properties of heteromeric channels formed by Cx26S17F and Cx30. Here, we describe that Cx26S17F interact with Cx30 in an exogenous expression system like transfected HeLa cells, forming mutant heteromeric hemichannels (HC). These mutant heteromeric HCs are localized mainly in intracellular compartment, but some of them reach the plasma membrane forming few and small gap junction (GJ) plaques in appositional cell membranes. On contrary, mutant HCs in non- appositional cell membranes are hyperactive and do not respond to HC blocker La3+. In addition, these mutant HCs present altered response to extracellular calcium (Ca2+e) remaining open at extracellular 1 and 1.5 mM that normally close wild type HCs, which is consistent with altered intracellular Ca2+ homeostasis in cells expressing these HCs. Finally, expression of these mutant HCs increased cell damage and death in the cultures. Ex vivo expression of Cx26S17F in cochlear explants from conditional knock-in transgenic mouse (cKI), changes the distribution of Cx26 and Cx30 in supporting cells of the organ of Corti, with more intracellular localization and reduced gap junction plaques, which contrast with the honeycomb-like GJ network observed in control explants. Also, supporting cells of cKI Cx26S17F cochlear explants show increased uptake of an extracellular tracer, suggesting that expression of mutant in supporting cells form hyperactive HC, that do not respond to extracellular Ca2+ and nor to the HC blocker carbenoxolone. To our knowledge, is the first study in cochlear Cxs using a model of syndromic deafness. Because ATP is a very relevant paracrine molecule in the cochlea, we briefly explored the ATP-induced Ca2+ response in HeLa cells expressing mutant or WT heteromeric HC, as well as in cKI Cx26S17F cochlear explants. In absence and presence of Ca2+e, HeLa cells expressing Cx26S17F/Cx30 HC showed decreased intracellular Ca2+ signal to the stimulation with ATP, suggesting purinergic receptor desensitization and/or loss of Ca+2 homeostasis in cells. Preliminary observations in cKI cochlear explants expressing Cx26S17F also indicates that expression of hyperactive heteromeric HCs produces dysregulation of intracellular Ca2+ and altered purinergic signaling in cochlear cells, which are linked to hair cell damage.


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