, 2009). The delayed development of mechanotransduction in hair cells that expressed Tmc1

paralleled the expression pattern of Tmc1 in wild-type mice ( Kawashima et al., 2011). These data extend the conclusion that Tmc1 and Tmc2 are required for transduction to include cochlear inner hair cells and confirm that expression of either gene alone is sufficient for transduction. Since dominant mutations in TMC1 cause progressive hearing loss in humans and mice ( Kurima et al., 2002), we investigated hair cell Y-27632 transduction in Bth mice ( Vreugde et al., 2002) which express a Tmc1 point mutation that causes a methionine to lysine substitution at residue 412 (p.M412K). To test the hypothesis that Bth mice have normal mechanotransduction during the first postnatal week selleck chemical ( Marcotti et al., 2006) due to expression of Tmc2, we generated mice with three mutant alleles at the Tmc1 and Tmc2 loci by crossing Tmc1Bth onto a Tmc1;Tmc2-null background (Tmc1Bth and wild-type alleles are indicated in bold throughout the text and figures to emphasize the identities of the proteins encoded by each genotype). Auditory brainstem responses indicated that the Tmc1Bth/Δ;Tmc2Δ/Δ mice

had profound hearing loss (see Figure S1A available online) at 1 month of age, the earliest time point tested. Cell counts from Tmc1Bth/Δ cochleas at P30–P35 revealed significant inner hair cell loss regardless of the Tmc2 genotype Carnitine dehydrogenase ( Figure S1B). Surprisingly, Tmc1Bth/Δ;Tmc2Δ/Δ hair cells had transduction current amplitudes at P5–P6 that were significantly larger than those of Tmc1+/Δ;Tmc2Δ/Δ mice ( Figures 1A, 1B, and 1D), while Tmc1Bth/Δ;Tmc2+/Δ hair cells had normal mechanotransduction during the first postnatal week ( Figures 1A, 1B, and 1D). These data suggest that p.M412K is not a loss-of-function or dominant-negative mutation but must cause deafness due to a gain or change of function. To further explore the differences between Tmc1Δ/Δ;Tmc2+/Δ, Tmc1+/Δ;Tmc2Δ/Δ,

and Tmc1Bth/Δ;Tmc2Δ/Δ hair cells, we examined several biophysical properties of hair cell mechanotransduction, including sensitivity, calcium permeability and rate and extent of adaptation. We identified no significant differences in sensitivity ( Figure S2) but found striking differences in calcium permeability and adaptation. To assay calcium permeability we used two electrophysiological measures: Ca2+ block and reversal potential. Calcium is a permeant blocker of hair cell transduction channels and reduces the whole-cell mechanotransduction conductance by ∼30% in wild-type rat cochlear hair cells (Beurg et al., 2006). To investigate calcium block in Tmc1Bth/Δ;Tmc2Δ/Δ hair cells, we plotted peak transduction currents as a function of voltage and generated mechanotransduction current-voltage (I–V) relations in two different calcium concentrations.