The gap junction proteins connexin 26 (cx26) and cx30 are widely expressed and colocalised throughout the mammalian cochlea (Lautermann et al. 1998; Forge et al. 2002). Mutations in both genes have been linked to hearing impairment, demonstrating the importance of these proteins to normal auditory function. The interplay between these two connexins in the inner ear and their role in hearing, however, is not clearly understood. In this study, the effects of four mutant cx26 proteins on wild-type (wt) cx30 have been investigated. All four mutant proteins arise from point mutations putatively responsible for either non-syndromic (W44S, R75W) or syndromic (G59A, D66H, R75W) dominantly inherited hearing loss.
Communication-deficient HeLa cells (Ohio strain 84121901; www.ecacc.org) were transiently transfected with wt and mutant cDNA by microinjection. When expressed alone, G59A and D66H have a perinuclear localisation within the cell (Forge et al. 2002). Co-expression of wt cx30 with either G59A or D66H mutants directly ‘tagged’ with GFP resulted in GFP expression at the plasma membrane, revealing that cx30 can rescue the impairment in trafficking. This result demonstrates that cx30 and cx26 can oligomerise to form heteromeric connexons.
Dye transfer studies, utilising the tracer Neurobiotin, were used to reveal intercellular communication between transfected cells. High levels of communication were observed between wt cx30-expressing cells, with 81 % of the Neurobiotin-injected cells (n = 16) transferring the tracer to more than one neighbour. A significant decrease in this communication was revealed between cells co-transfected with wt cx30 and W44S, G59A or R75W (P < 0.05; x{special}2 test) with, respectively, 31 % (n = 32), 42 % (n = 24) and 45 % (n = 33) of injected cells transferring the tracer to more than one neighbour. These results demonstrate a dominant negative effect of certain mutant cx26 proteins on cx30. The two mutants associated with profound, non-syndromic hearing loss, W44S and R75W, similarly have a dominant negative effect on wt cx26 (N.K. Marziano, unpublished observation). Heteromeric cx26/cx30 gap junction channels may exist in vivo with particular properties essential for hearing. The disruption of these heteromeric channels by certain mutations may account for the non-syndromic nature of the deafness.
This work was supported by The Wellcome Trust.