LRRC8/VRAC volume-regulated anion channels are crucial for hearing

Abstract

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC‑K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a^-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K⁺ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Keywords: Swell1; VRCC; VSOAC; VSOR; anion transport; cochlea; hair cell; mouse genetics; osmotic swelling; physiology; volume regulation.

Figure 1

LRRC8A subunit expression in cochlea.A, LRRC8A detected by HA-labeling of Lrrc8a^{HA-lox/HA-lox} tissue is widely expressed throughout cochlea of P8 mice. B, in adult 23-weeks-old (23W) mice, LRRC8A expression appears more restricted, being found mainly in the organ of Corti (OC), stria vascularis (SV), spiral prominence and outer sulcus cells (SP), Reissner membrane (RM), and spiral ganglion (SG). C, in the organ of Corti, HA-tagged LRRC8A is expressed in inner and outer hair cells (IHC and OHC) labeled with hair cell marker myosin VII, as well as in supporting cells. The 23-weeks-old Lrrc8a^{HA-lox/HA-lox} mouse. D, LRRC8A is expressed in strial cells, labeled with marginal cell marker ClC-K. The 23-weeks-old Lrrc8a^{HA-lox/HA-lox} mouse. Images are collected from at least two different mice. WT mice served as negative controls. ClC-K, member of chloride channel family CLC; HA, hemagglutinin.

Figure 2

Expression of LRRC8B and LRRC8C subunits in cochlea.A, X-gal stained cochlear section from 6-weeks-old Lrrc8b^+/lacZ mouse. The Lrrc8b promotor is active in cells of the spiral ganglion (SG), organ of Corti (OC), Reissner`s membrane (RM), spiral limbus (SLB), and spiral prominence, and outer sulcus region (SP). No X-gal staining was observed in the stria vascularis (SV) and fibrocytes in the spiral ligament (SL). B, LRRC8C, as detected by V5 labeling of cochlea form 12 weeks-old Lrrc8c<sup>V5/V5</sup> mice, is prominently expressed in blood vessels (e.g. in the stria vascularis (SV) and spiral limbus (SLB)) and hair cells (IHC and OHC; OC–organ of Corti). Reissner`s membrane (RM), type IV fibrocytes (IV) and tympanic border cells (TBC) also express LRRC8C. WT mice served as a negative control. Images are collected from at least two different mice. IHC, inner hair cell; OHC, outer hair cell; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

Figure 3

Expression of LRRC8D subunit in cochlea.A, LRRC8D, detected by tdTomato staining of a cochlea from 16-weeks-old Lrrc8c^{lox tdTomato/lox tdTomato} mouse, is found in stria vascularis (SV), inner and outer hair cells (IHC and OHC) in organ of Corti (OC), and fibroblasts of spiral limbus (SLB), and spiral ligament (SL). B, LRRC8D expression in the lateral cochlear wall. Marginal cells of the stria vascularis are identified by staining for ClC-K Cl^- channels. Same mouse as in (A). WT mice served as negative controls. Images are collected from at least two different mice. tdTomato, tandem dimer tomato (a fluorescent protein); ClC-K, member of chloride channel family CLC.

Figure 4

Expression of LRRC8E subunit in cochlea.A, LRRC8E, detected by V5 signal 12-weeks-old Lrrc8e^{smFPV5/smFPV5} mouse, is expressed in stria vascularis (SV), spiral prominence and outer sulcus region (SP), interdental cells (ID), inner and outer hair cells (IHC and OHC) in the organ of Corti (OC), and supporting cells. B, LRRC8E expression in inner and outer hair cells (IHC and OHC), labeled with hair cell marker myosin VII. The 12-weeks-old Lrrc8e^{smFPV5/smFPV5} mouse. C, LRRC8E expression in stria vascularis. Apical membranes of marginal cells labeled with KCNQ1. 13 weeks-old Lrrc8e^{smFPV5/smFPV5} mouse. WT mice served as negative controls. Images are collected from at least two different mice.

Figure 5

Schematic depiction of VRAC subunit expression. LRRC8A is broadly expressed through the entire cochlea, while nonessential VRAC subunits LRRC8B-LRRC8E display more constrained localization pattern. VRAC, volume-regulated anion channel.

Figure 6

LRRC8A ablation leads to cochlear degeneration.A, H&E staining of cochlear sections from Sox10-Cre; Lrrc8a^lox/lox mice. At P9, before the onset of hearing, cochlear anatomy is normal. Beginning with the onset of hearing (at P12–13), degeneration of spiral ganglion neurons (thick black arrows) and organ of Corti (white arrows) develops. Thin black arrows indicate swollen and/or detached from the spiral limbus tectorial membranes. B, degeneration of spiral ganglion (left) and organ of Corti (right) in 15-17-days-old Sox10-Cre; Lrrc8a^lox/lox mice. The tectorial membrane is swollen, and the organ of Corti and the spiral ganglion are degenerated. Images are collected from at least two different mice. Sox10, SRY-box transcription factor 10.

Figure 7

Changes in Lrrc8a KO stria vascularis. Toluidine blue-stained semithin strial sections from three 1-year-old Sox10-Cre; Lrrc8a^lox/lox and one WT (ctrl) mice. In two out of three investigated inner ears, the stria is markedly thinned (indicated by arrows). Sox10, SRY-box transcription factor 10.

Figure 8

Intact tight junctions and strial barrier in Lrrc8a KO stria vascularis.A, electron microscopy images of tight junctions (arrows) between strial marginal cells. Tight junctions are intact in Sox10-Cre; Lrrc8a^lox/lox mice. B, Sox10-Cre; Lrrc8a^lox/lox mice have intact strial barrier. Inner ears were perfused with biotin, which was later labeled with FITC-conjugated streptavidin in frozen sections. The figure depicts 3-weeks-old mice in both panels. Images are collected from at least two different mice. Sox10, SRY-box transcription factor 10.

Figure 9

Cochlear morphology upon disruption of nonessential single VRAC subunits.A, no morphological changes observed in cochleae of 1-year-old Lrrc8b^−/− (N = 4), Lrrc8c^−/− (N = 3) and Lrrc8d^−/− (N = 3) mice. B, degeneration of spiral ganglion (thick black arrows) and/or of organ of Corti (white arrows) in the middle turn is observed in 60% (7 out of 12) of investigated ears of 1-year-old Lrrc8e^−/− but not in 11-weeks-old animals. In 60% (7 out of 12) of studied ears, swollen tectorial membranes were found (thin black arrow). N = 6 for old mice, N = 5 for young mice. The 1-year-old Lrrc8d^−/−; Lrrc8e^−/− displayed significantly more severe degeneration of spiral ganglion (black arrows) and organ of Corti (white arrows) and tectorial membrane swelling (thin black arrow) than Lrrc8e^−/− animals. N = 3 for Lrrc8d^−/−; Lrrc8e^−/−. N refers to the number of investigated KO mice. Numbers on the top right side of the panels refer to the animal age in weeks. VRAC, volume-regulated anion channel.

Figure 10

Cochlear morphology upon disruption of two nonessential VRAC subunits.A, late-onset cochlear degeneration in mice constitutively lacking LRRC8D and LRRC8E. The cochlea from the 7-weeks-old mouse is either normal or displays mild degeneration, while the cochleae from the 9- and 11-weeks-old Lrrc8d^−/−; Lrrc8e^−/− mice reveal degenerated spiral ganglion neurons (thick black arrows) and organ of Corti`s (white arrows). The thin black arrow in the image of the 9-weeks-old mouse indicates a tectorial membrane that is detached from the spiral limbus. N = 6 across all ages. B, no morphological changes observed in cochleae of 12-weeks-old Lrrc8c^−/−/Lrrc8e^−/− (N = 2), 12-weeks-old Lrrc8c^−/−/Lrrc8d^−/− (N = 4), and 16-weeks-old Lrrc8b^−/−/Lrrc8e^−/− (N = 4) mice. N refers to the number of investigated KO mice. Numbers on the top right side of the panels refer to the animal age in weeks. VRAC, volume-regulated anion channel.

Figure 11

Expression of different transporters in Lrrc8a KO stria vascularis and spiral ligament. Effect of Lrrc8a disruption on expression levels of several ion channels and transporters in the stria vascularis and spiral ligament (KCC3) of the middle cochlear turn of 2-weeks-old Sox10-Cre; Lrrc8a^lox/lox mice. Strikingly, Kir4.1 cannot be detected after Lrrc8a disruption, whereas staining intensities for KCNQ1, Na,K-ATPase, NKCC1, ClC-K, its subunit barttin, and KCC3 are unchanged. Images are collected from at least two different mice. Sox10, SRY-box transcription factor 10.

Figure 12

Kir4.1 expression is significantly reduced in stria vascularis, but not in spiral ganglion of Lrrc8d^−/−Lrrc8e^−/−mice above 5-weeks-old. Stria vascularis (SV, upper rows) and spiral ganglion (SG, lower rows) of middle turn of cochlea were stained for Kir4.1 in 3- (A), 5- (B) and 7- weeks (C) old mice. Each pair of SV and SG images is from one mouse. N = 3 for W3 and W7, N = 4 for W5. N refers to the number of investigated mice.

Figure 13

Hearing loss in Lrrc8a^−/−and Lrrc8d^−/−/Lrrc8e^−/−mice.A and B, Lrrc8a^−/− mice display a significant hearing loss already at 3 weeks old, as measured by auditory brainstem response (ABR; left panels), as well as a significant decrease in endocochlear potential (right panels). ABR: N = 8 (WT), N = 7 (KO) in 3 weeks group, N = 9 (WT), N = 10 (KO) in 8 weeks old group, EP: N = 4 (WT and KO) in 3 weeks group, N = 3 (WT), N = 5 (KO) in 8 weeks group. Mean ± SD, unpaired t test (ABR 3 weeks old, EP 3 and 8 weeks old), Mann-Whitney test (ABR 8 weeks old). C, Lrrc8d^−/−Lrrc8e^−/− double KO mice display a variable tendency toward hearing loss and reduced EP at 8 weeks of age. ABR: N = 8 (Lrrc8d^+/+Lrrc8e^−/−), N = 7 (Lrrc8d^−/−Lrrc8e^−/−), EP: N= 3 (Lrrc8d^+/+Lrrc8e^−/−), N = 5 (Lrrc8d^−/−Lrrc8e^−/−). Mean ± SD, unpaired t test. D, correlation between ABR and EP values from individual ears. In Lrrc8d^−/−Lrrc8e^−/− mice higher ABR is associated with lower EP. The same data as in B and C. Only data from ears that successfully underwent ABR and EP measurements are plotted. N refers to the number of investigated mice, when possible data from two ears from the same mouse was averaged. SPL, sound pressure level, eq, equivalent. ABR, auditory brainstem response; EP, endocochlear potential.

Figure 14

Scheme of endocochlear potential generation. Potassium is secreted by KCNQ1/KCNE1 potassium channels in the apical membranes of marginal cells. This secretion requires K⁺ accumulation into marginal cells across their basolateral membrane, a process carried out by the Na⁺K⁺-ATPase and the Na⁺-gradient driven Na⁺K⁺2Cl^- NKCC1 cotransporter. Chloride taken up by NKCC1 needs to be recycled over the basolateral membrane of marginal cells by Cl^- channels, most prominently by ClC-Ka and ClC-Kb channels associated with their obligatory subunit barttin. VRAC/LRRC8 channels in marginal cells may play an additional, probably minor role in this recycling process. The positive endocochlear potential depends critically on Kir4.1 (KCNJ10) in the apical membrane of intermediate cells. They generate a large positive K⁺-diffusion potential in the cleft between intermediate and marginal cells. Kir4.1 is downregulated in the absence of the obligatory VRAC subunit LRRC8A, or when both LRRC8D and LRRC8E are missing. This downregulation may be related to VRAC’s ability to transport organic molecules such as glutathione, as suggested by the sensitivity of Kir4.1 protein levels to oxidative stress. VRAC may perform this role in several cell types because it is expressed in all stria vascularis cell types (LRRC8A, D and E) and fibrocytes (LRRC8A and D for fibrocytes I). Figure adapted from (44). VRAC, volume-regulated anion channel.