Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na⁺, K⁺, 2Cl⁻ cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K⁺-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3’-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl⁻ influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans. Sounds are perceived by auditory sensory cells, owing to tissues surrounding them, including the cochlear lateral wall. Part of the cochlear lateral wall, the stria vascularis, is critical for production and maintenance of inner-ear fluid with a high potassium concentration, and for generating the positive voltage in the inner ear, important for sound perception, by stimulating secretion of potassium from marginal cells. The gene SLC12A2 encodes a protein involved in sodium, potassium, and chloride transport essential for proper function of specific cells in the stria vascularis; however, human variants of SLC12A2 have not previously been associated with hearing loss. By comprehensive genetic analysis of protein-coding sequences, we identified four candidate changes in SLC12A2 in four families with congenital, severe-to-profound hearing loss. Intriguingly, all four genetic variants were either within or at the 3’-splice site of the exon 21 which encodes a part of the carboxy terminal intracellular domain of SLC12A2. Experiments in cultured cells showed that skipping or mutation of exon 21 significantly decreased chloride influx mediated by the SLC12A2 protein. Overall, our results strongly indicate that mutations influencing exon 21 of SLC12A2 represent a novel mechanism underlying deafness in humans.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a “9 + 2” microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3^tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3^tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3^tm1a/tm1a, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia. Significance Cilia are composed of hundreds of proteins whose identities and functions are far from being completely understood. In this study, we determined that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) plays an important role for the function of motile cilia in multiciliated cells (MCCs). Global knockdown of CAMSAP3 protein expression in mice resulted in defects in ciliary structures, polarity, and synchronized beating in MCCs. These animals also displayed signs and symptoms reminiscent of primary ciliary dyskinesia (PCD), including a mild form of hydrocephalus, subfertility, and impaired mucociliary clearance that leads to hyposmia, anosmia, rhinosinusitis, and otitis media. Functional characterization of CAMSAP3 enriches our understanding of the molecular mechanisms underlying the generation and function of motile cilia in MCCs.

Outer hair cell elecromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized prestin (Pres^TS), complexed with chloride, sulfate, or salicylate at 3.52-3.63 Å resolutions. The central positively-charged cavity allows flexible binding of various anion species, which likely accounts for the known distinct modulations of nonlinear capacitance (NLC) by different anions. Comparisons of these Pres^TS structures with recent prestin structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insights into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.

Prestin (SLC26A5)-mediated voltage-driven elongations and contractions of sensory outer hair cells within the organ of Corti are essential for mammalian cochlear amplification. However, whether this electromotile activity directly contributes on a cycle-by-cycle basis is currently controversial. By restoring motor kinetics in a mouse model expressing a slowed prestin missense variant, this study provides experimental evidence acknowledging the importance of fast motor action to mammalian cochlear amplification. Our results also demonstrate that the point mutation in prestin disrupting anion transport in other proteins of the SLC26 family does not alter cochlear function, suggesting that the potential weak anion transport of prestin is not essential in the mammalian cochlea.

KCNQ4 encodes the homotetrameric voltage-dependent potassium ion channel Kv7.4, and is the causative gene for autosomal dominant nonsyndromic sensorineural hearing loss, DFNA2. Dominant-negative inhibition accounts for the observed dominant inheritance of many DFNA2-associated KCNQ4 variants. In addition, haploinsufficiency has been presumed as the pathological mechanism for truncated Kv7.4 variants lacking the C-terminal tetramerization region, as they are unlikely to exert a dominant-negative inhibitory effect. Such truncated Kv7.4 variants should result in relatively mild hearing loss when heterozygous; however, this is not always the case. In this study, we characterized Kv7.4Q71fs (c.211delC), Kv7.4W242X (c.725G>A) and Kv7.4A349fs (c.1044_1051del8) in heterologous expression systems and found that expression of these truncated Kv7.4 variants induced cell death. We also found similar cell death-inducing cytotoxic effects in truncated Kv7.1 (KCNQ1) variants, suggesting that the generality of our findings could account for the dominant inheritance of many, if not most, truncated Kv7 variants. Moreover, we found that the application of autophagy inducers can ameliorate the cytotoxicity, providing a novel insight for the development of alternative therapeutic strategies for Kv7.4 variants.