SLC26A4 targeted to the endolymphatic sac rescues hearing and balance in Slc26a4 mutant mice.

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (Δ/Δ) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (Δ/Δ) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.

cAMP-stimulated Cl- secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium.

BACKGROUND: The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under ß2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids. RESULTS: Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 µM), 8-Br-cAMP (EC50: 180 µM), 8-pCPT-cAMP (100 µM), IBMX (250 µM), and RO-20-1724 (100 µM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 µM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 µM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 µM), flufenamic acid (5 µM) and diphenylamine-2,2'-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/- mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR-/- mice had no responses. Nonetheless, CFTR-/- mice showed no difference from CFTR+/- mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 µM), prednisolone (0.3, 1 & 3 µM), hydrocortisone (0.01, 0.1 & 1 µM), and corticosterone (0.1 & 1 µM) and mineralocorticoid aldosterone (1 µM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined. CONCLUSIONS: These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.

Molecular detection of SARS-CoV-2 and differentiation of Omicron and Delta variant strains.

The SARS-CoV-2 virus is the causative agent of COVID-19 and has undergone continuous mutations throughout the pandemic. The more transmissible Omicron variant has quickly spread and is replacing the Delta variant as the most prevalent strain globally, including in the United States. A new molecular assay that can detect and differentiate both the Delta and Omicron variants was developed. A collection of 660,035 SARS-CoV-2 full- or near-full genomes, including 169,454 Delta variant and 24,202 Omicron variant strains, were used for primer and probe designs. In silico data analysis predicted an assay coverage of >99% of all strains, including >99% of the Delta and >99% of Omicron strains. The Omicron variant differential test was designed based on the Δ31-33 aa deletion in the N-gene, which is present in the original B.1.1.529 main genotype, BA.1, as well as in BA.2 and BA.3 subtypes. Therefore, the assay should detect the majority of all Omicron variant strains. Standard curves generated with human clinical samples indicated that the PCR amplification efficiencies were 104%, 90.7% and 90.4% for the Omicron, Delta, and non-Delta/non-Omicron wild-type genotypes, respectively. Correlation coefficients of the standard curves were all >0.99. The detection limit of the assay was 14.3, 32.0, and 21.5 copies per PCR reaction for Omicron, Delta, and wild-type genotypes, respectively. The assay was designed to specifically detect SAR-CoV-2 strains. Selected samples with Omicron, Delta and wild-type genotypes identified by the RT-qPCR assay were also confirmed by sequencing. The assay did not detect any animal coronavirus-positive samples that were tested. Human nasal swab samples that previously tested positive (n = 182) or negative (n = 42) for SARS-CoV-2 by the ThermoFisher TaqPath COVID-19 Combo Kit, produced the same result with the new assay. Among positive samples, 55.5% (101/182), 23.1% (42/182), and 21.4% (39/182) were identified as Omicron, Delta, and non-Omicron/non-Delta wild-type genotypes, respectively.

Expression of epithelial calcium transport system in rat cochlea and vestibular labyrinth.

BACKGROUND: The low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium. RESULTS: We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system. CONCLUSIONS: These findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.

Cerebellar Ataxia Caused by Type II Unipolar Brush Cell Dysfunction in the Asic5 Knockout Mouse.

Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K+ currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.

Slc26a7 chloride channel activity and localization in mouse Reissner's membrane epithelium.

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner's membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner's membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO3- over Cl- and inhibited by I- and NPPB. Elevated NO3- currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl- transport in Reissner's membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl- conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner's membrane cells during local perturbations of pH.

Sodium selectivity of semicircular canal duct epithelial cells.

BACKGROUND: Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, ß- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. RESULTS: We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, ß- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. CONCLUSIONS: These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αßγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.

Lack of pendrin HCO3- transport elevates vestibular endolymphatic [Ca2+] by inhibition of acid-sensitive TRPV5 and TRPV6 channels.

The low Ca(2+) concentration ([Ca(2+)]) of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported (Yamauchi et al., Biochem Biophys Res Commun 331: 1353-1357, 2005) that the epithelial Ca(2+) channels TRPV5 and TRPV6 (transient receptor potential types 5 and 6) are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D(3), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca(2+)], and transepithelial potential of the utricle were measured in Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4(-/-) mice exhibit reduced pH and utricular endolymphatic potential and increased [Ca(2+)]. Monolayers of primary cultures of rat semicircular canal duct cells were grown on permeable supports, and cellular uptake of (45)Ca(2+) was measured individually from the apical and basolateral sides. Net uptake of (45)Ca(2+) was greater after incubation with 1,25-dihydroxyvitamin D(3). Net (45)Ca(2+) absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum, and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca(2+)] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca(2+) absorption.

Loss of cochlear HCO3- secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model.

Pendred syndrome, characterized by childhood deafness and postpuberty goiter, is caused by mutations of SLC26A4, which codes for the anion exchanger pendrin. The goal of the present study was to determine how loss of pendrin leads to hair cell degeneration and deafness. We evaluated pendrin function by ratiometric microfluorometry, hearing by auditory brain stem recordings, and expression of K(+) and Ca(2+) channels by confocal immunohistochemistry. Cochlear pH and Ca(2+) concentrations and endocochlear potential (EP) were measured with double-barreled ion-selective microelectrodes. Pendrin in the cochlea was characterized as a formate-permeable and DIDS-sensitive anion exchanger that is likely to mediate HCO(3)(-) secretion into endolymph. Hence endolymph in Slc26a4(+/-) mice was more alkaline than perilymph, and the loss of pendrin in Slc26a4(-/-) mice led to an acidification of endolymph. The stria vascularis of Slc26a4(-/-) mice expressed the K(+) channel Kcnj10 and generated a small endocochlear potential before the normal onset of hearing at postnatal day 12. This small potential and the expression of Kcnj10 were lost during further development, and Slc26a4(-/-) mice did not acquire hearing. Endolymphatic acidification may be responsible for inhibition of Ca(2+) reabsorption from endolymph via the acid-sensitive epithelial Ca(2+) channels Trpv5 and Trpv6. Hence the endolymphatic Ca(2+) concentration was found elevated in Slc26a4(-/-) mice. This elevation may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells. Degeneration of the hair cells closes a window of opportunity to restore the normal development of hearing in Slc26a4(-/-) mice and possibly human patients suffering from Pendred syndrome.

Vitamin D upregulates expression of ECaC1 mRNA in semicircular canal.

The low luminal Ca2+ concentration of mammalian endolymph in the vestibular labyrinth is required for normal balance. We found transcripts in primary cultures of semicircular canal duct (SCCD) epithelial cells from neonatal rats representing a complete transport system for transepithelial absorption of Ca2+ that is comprised of the epithelial Ca2+ channels ECaC1 (CaT2, TRPV5) and ECaC2 (CaT1, TRPV6), calbindin (calbindin-D9k, calbindin-D28k), Na+/Ca2+ exchanger (NCX1, NCX2, and NCX3), and plasma membrane Ca2+-ATPase (PMCA1, PMCA3, and PMCA4) by RT-PCR. Further, vitamin D receptor was also expressed in SCCD and it was found by quantitative RT-PCR that incubation for 24 h with 1,25-dihydroxyvitamin D3 upregulated the expression of ECaC1, calbindin-D9k, and calbindin-D28k. These observations provide evidence for the first time of an ECaC-based Ca2+ transport system in SCCD that could maintain the low Ca2+ concentration in vestibular endolymph.