Connexin Mutations in Deafness

DESCRIPTION (provided by applicant): Mutations in the human connexin26 gene (Cx26, or GJB2) are the leading cause of nonsyndromic deafness in the United States. Mutations in other connexins, Cx30 (GJB6), Cx31 (GJB3), and Cx32 (GJB1) have also been linked to hearing loss in humans. While this illuminates a critical function for cochlear gap junctions, it is unclear how a common pathology can arise from mutations within different connexin genes that have an overlapping expression pattern in the inner ear, as is the case for Cx26, Cx30, Cx31 and Cx32. There are no gap junctions between the sensory hair cells in humans; but they are expressed in the supporting cells of the cochlea. The current hypothesis is that these junctions play a role in the re-circulation of potassium ions between the end lymph and perilymph. It is difficult to reconcile this model with the available data on potassium permeation through gap junction channels, as all connexins are readily permeated by this caution and the loss of a single cochlear connexin would still leave other functional connexins available to perform this task. Connexins do show differential permeability to a wide range of other small molecules and second messengers, and we hypothesize that these permeation differences are critical for cochlear function, and more difficult to compensate for following the functional loss of one of the several available channel subunits. The objective of this application is to precisely define which permeation properties of Cx26 are necessary for normal auditory function in humans. To achieve this goal, we first propose to screen mutant Cx26 alleles for functional activity in the paired Xenopus oocyte assay. Cx26 mutants that retain channel function will have their perm selectivity properties analyzed by dual patch clamp methods in transected mammalian cell lines. Finally, We will generate and characterize genetically engineered mice where the native Cx26 gene has been replaced by the functionally active human Cx26 disease causing mutations. We will use genetic knock-in techniques to generate mice that will allow us to evaluate the in vitro derived functional differences in an animal model. Contrasting the differences in permeation between wild type and disease causing variants of Cx26 will not only provide mechanistic insight into hearing loss, but will also provide a general model for the need for connexin diversity in other tissues where human disease results from mutations in connexin genes.