Hearing is normal without connexin30

Abstract

Gjb2 and Gjb6, two contiguous genes respectively encoding the gap junction protein connexin26 (Cx26) and connexin 30 (Cx30) display overlapping expression in the inner ear. Both have been linked to the most frequent monogenic hearing impairment, the recessive isolated deafness DFNB1. Although there is robust evidence for the direct involvement of Cx26 in cochlear functions, the contribution of Cx30 is unclear since deletion of Cx30 strongly downregulates Cx26 both in human and in mouse. Thus, it is imperative that any role of Cx30 in audition be clearly evaluated. Here, we developed a new Cx30 knock-out mouse model (Cx30(Δ/Δ)) in which half of Cx26 expression was preserved. Our results show that Cx30 and Cx26 coordinated expression is dependent on the spacing of their surrounding chromosomic region, and that Cx30(Δ/Δ) mutants display normal hearing. Thus, in deaf patients with GJB6 deletion as well as in the previous Cx30 knock-out mouse model, defective Cx26 expression is the likely cause of deafness, and in contrast to current opinion, Cx30 is dispensable for cochlear functions.

Figure 1.

Conditional Cx30 inactivation model. A, Homologous recombination resulting in the Cx30^fl allele where Cx30 transcript sequence (coding sequence, dark gray box; untranslated 5′ and 3′ sequences, black boxes) is flanked by LoxP sites (arrowheads) followed by the hygromycin resistance gene (hyg) and a LoxP site. Crossing Cx30^fl/fl mice with Pgk-Cre mice generated Cx30^Δ/Δ mice. Arrows indicate genotyping primers. B, PCR genotyping: GjB6 R and F produce a 350 bp band in Cx30^+/+ mice, and a 488 bp band in Cx30^fl/fl mice. GjB6F and GjB6ΔR produce a 521 bp band in Cx30^Δ/Δ mice. C, D, Cx30 expression was quantified by qPCR (C) and immunoblot (D) in whole inner ear. Mean ± SEM; 3 independent experiments; 3 mice of each genotype per experiment; t test. *p < 0.05, ***p < 0.0001.

Figure 2.

Cx26 expression in the inner ear of Cx30^+/+, Cx30^fl/fl, Cx30^Δ/Δ, and Cx30^−/− mice. Cx26 expression quantified by qPCR (A) and quantitative immunoblot (B). Mean ± SEM; 3 independent experiments; 3 mice of each genotype per experiment; t test. *p < 0.05, **p < 0.001, ***p < 0.0001.

Figure 3.

Assessment of hearing in Cx30^Δ/Δ mice. A, ABR thresholds (mean ± SEM) recorded at 20–100 dB SPL across a frequency range of 10–32 kHz. No significant difference in threshold was found between Cx30^+/+ and Cx30^Δ/Δ or Cx30^fl/fl mice. In contrast, ABR waveforms showed up only above 80 dB in the Cx30^−/− mice. B, DPOAEs at 10 kHz for equilevel stimuli (mean ± SEM), increasing stepwise from 30 to 75 dB SPL. No significant difference in threshold or level was found between Cx30^+/+ and Cx30^Δ/Δ or Cx30^fl/fl mice. In contrast, Cx30^−/− mice had no DPOAE up to 75 dB SPL. A, B, Kruskal–Wallis nonparametric test, p > 0.1.