Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechanoelectrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation, TMC1 p.T422K, and have characterized the homologous mouse mutant, Tmc1 p.T416K, which caused deafness and SignificanceTransmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboring Tmc1 point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca 2+ permeability by introducing a positively-charged amino acid in the putative pore. All mutations are largely consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca 2+ permeability and virtually abolished block by dihydrostreptomycin, strongly endorsing its siting within the pore.

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

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The mariner (myo7aa−/−) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa−/− mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa−/− zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa−/− hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa−/− mutants have fewer postsynaptic densities – as assessed by MAGUK immunolabeling – compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa−/− mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa−/− mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa−/− mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse – in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta – shift swimming behavior and improve acoustic startle response.