To investigate whether the difference in the number of transduction channels between Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Bth/Δ;Tmc2Δ/Δ hair cells was due to changes in gene expression, we performed a quantitative RT-PCR analysis using cochlear tissue harvested from Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Bth/Δ;Tmc2Δ/Δ mice. Neratinib mouse The analysis revealed significantly higher Tmc1 mRNA expression

in Bth mice ( Figure 4G), which may account for the larger whole-cell transduction currents in these mice. The mechanism for altered Tmc1 gene expression and eventual hair cell death in the Bth mice is unclear. However, recent evidence shows that proper calcium homeostasis is required for hair cell survival ( Esterberg et al., 2013), raising the possibility that altered calcium permeability in Bth mice may lead to hair cell degeneration and deafness ( Figure S1B). A similar mechanism may underlie dominant, progressive Trametinib supplier hearing loss in DFNA36 patients who carry a point mutation (p.G417R) in an adjacent residue in the human TMC1 ortholog ( Yang et al., 2010). Next, we measured single-channel currents

in wild-type inner hair cells at time points when both Tmc1 and Tmc2 were expressed ( Figure 5). During the first postnatal week we recorded a range of unitary current amplitudes from all regions of the cochlea that was significantly broader than that observed in Tmc mutant mice. Representative examples that span the range are shown in Figures 5A–5C. Unitary current values were divided by driving force (84 mV based on a 0 mV reversal potential in 50 μM Ca2+, see Figure S3) to calculate single-channel conductance (g). In wild-type mouse inner hair cells, g ranged between 60 and 330 pS during the first postnatal week. These values encompass the wide range of single-channel conductances reported for auditory hair cells of various species ( Crawford et al., 1991, Géléoc et al., 1997, Ricci et al., 2003 and Beurg et al., 2006). Figure 5D shows a scatter plot of 44 single-channel conductances measured from wild-type cells, along with measurements from

18 Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Δ/Δ;Tmc2+/Δ cells. The data from the Tmc mutant mice are tightly clustered relative Sitaxentan to the wild-type data which are more broadly distributed. To examine the possibility that the wild-type data form discrete groups we performed a cluster analysis using three statistical tests ( Experimental Procedures) each of which reported that the data are best described by four clusters. The mean value for each cluster is indicated by the straight lines ( Figure 5D). Together, these data reveal that both Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Δ/Δ;Tmc2+/Δ hair cells have transduction channels with relatively homogeneous single-channel properties, whereas wild-type hair cells that express both Tmc1 and Tmc2 display significant heterogeneity.