Ana E. Vázquez

Null Mutation of α1D Ca2+ Channel Gene Results in Deafness but No Vestibular Defect in Mice

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate α1D Ca2+ channel-deficient mice to determine the physiological role of these Ca2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that α1D−/− mice were deaf and revealed that heterozygous (α1D+/−) mice have increased hearing thresholds. However, hearing deficits in α1D+/− mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, α1D−/− mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba2+ currents in cochlear inner and outer hair cells versus utricular hair cells in α1D+/− mice. Whereas the whole-cell Ba2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (~85%), the utricular hair cells express only ~50% of this channel subtype. Thus, differential expression of α1D channels in the cochlear and utricular hair cells confers the phenotype of the α1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, α1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions.

Age-related hearing loss: current research.

Purpose of reviewSignificant changes in population demographics with respect to age have taken place, and this pattern is expected to continue. The aging of the population underscores the importance of finding ways to improve the quality of life of the elderly. Most of the elderly population, however, suffers from progressive hearing loss: 60% of people older than 70 years have hearing loss of at least 25 dB. Age-related hearing loss affects the quality of life, not only of the elderly but also of their families and loved ones. Recent findingsThe research goal in this field is to elucidate the mechanisms involved in age-related hearing loss and the molecular basis of normal and impaired auditory function, with the aim of developing preventative therapies. During the past few years, extraordinary progress has been made in the identification of genes that contribute to deafness. Additionally, inbred strains of mice have proven to be useful models to identify specific factors relevant to age-related hearing loss. A detailed description of the pathology exhibited by inbred mice that exhibit age-related hearing loss is helping to identify the specific structures and cell types affected by age-related hearing loss. A summary of current research efforts is presented. This review focuses on studies using inbred mice. SummaryBy defining the molecular basis of normal and impaired auditory function, therapies can be developed to ameliorate the effects of aging in the auditory system.

Cochlear Function in Mice Lacking the BK Channel α, β1, or β4 Subunits

Large conductance voltage- and calcium-activated potassium (BK) channels are important for regulating many essential cellular functions, from neuronal action potential shape and firing rate to smooth muscle contractility. In amphibians, reptiles, and birds, BK channels mediate the intrinsic frequency tuning of the cochlear hair cell by an electrical resonance mechanism. In contrast, inner hair cells of the mammalian cochlea are extrinsically tuned by accessory structures of the cochlea. Nevertheless, BK channels are present in inner hair cells and encode a fast activating outward current. To understand the role of the BK channel α and β subunits in mammalian inner hair cells, we analyzed the morphology, physiology, and function of these cells from mice lacking the BK channel α (Slo-/-) and also the β1 and β4 subunits (β1/4-/-). β1/4-/- mice showed normal subcellular localization, developmental acquisition, and expression of BK channels. β1/4-/- mice showed normal cochlear function as indicated by normal auditory brainstem responses and distortion product otoacoustic emissions. Slo-/- mice also showed normal cochlear function despite the absence of the BKα subunit and the absence of fast activating outward current from the inner hair cells. Moreover, microarray analyses revealed no compensatory changes in transcripts encoding ion channels or transporters in the cochlea from Slo-/- mice. Slo-/- mice did, however, show increased resistance to noise-induced hearing loss. These findings reveal the fundamentally different contribution of BK channels to nonmammalian and mammalian hearing and suggest that BK channels should be considered a target in the prevention of noise-induced hearing loss.

Roles of Alternative Splicing in the Functional Properties of Inner Ear-specific KCNQ4 Channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1–v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9–11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by ∼20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2–5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.

Local Control of Acetylcholinesterase Gene Expression in Multinucleated Skeletal Muscle Fibers: Individual Nuclei Respond to Signals from the Overlying Plasma Membrane

Nuclei in multinucleated skeletal muscle fibers are capable of expressing different sets of muscle-specific genes depending on their locations within the fiber. Here we test the hypothesis that each nucleus can behave autonomously and responds to signals generated locally on the plasma membrane. We used acetylcholinesterase (AChE) as a marker because its transcripts and protein are concentrated at the neuromuscular and myotendenous junctions. First, we show that tetrodotoxin (TTX) reversibly suppresses accumulation of cell surface AChE clusters, whereas veratridine or scorpion venom (ScVn) increase them. AChE mRNA levels are also regulated by membrane depolarization. We then designed chambered cultures that allow application of sodium channel agonists or antagonists to restricted regions of the myotube surface. When a segment of myotube is exposed to TTX, AChE cluster formation is suppressed only on that region. Conversely, ScVn increases AChE cluster formation only where in contact with the muscle surface. Likewise, both the synthesis and secretion of AChE are shown to be locally regulated. Moreover, using in situ hybridization, we show that the perinuclear accumulation of AChE transcripts also depends on signals that each nucleus receives locally. Thus AChE can be up- and downregulated in adjacent regions of the same myotubes. These results indicate that individual nuclei are responding to locally generated signals for cues regulating gene expression.

Conservation of Hearing by Simultaneous Mutation of Na,K-ATPase and NKCC1

Although drug-induced and age-related hearing losses are frequent otologic problems affecting millions of people, their underlying mechanisms remain uncertain. The inner ear is exclusively endowed with a positive endocochlear potential (EP) that serves as the main driving force for the generation of receptor potential in hair cells to confer hearing. Deterioration of EP leads to hearing loss or deafness. The generation of EP relies on the activity of many ion transporters to establish active potassium (K+) cycling within the inner ear, including K+ channels, the Na–K–2Cl co-transporter (NKCC1), and the α1 and α2 isoforms of Na,K–ATPase. We show that heterozygous deletion of either NKCC1, α1-Na,K–ATPase, or α2–Na,K–ATPase independently results in progressive, age-dependent hearing loss with minimal alteration in cochlear morphology. Double heterozygote deletion of NKCC1 with α1–Na,K–ATPase also shows a progressive, though delayed, age-dependent hearing loss. Remarkably, double heterozygote deletion of NKCC1 with α2–Na,K–ATPase demonstrates a striking preservation of hearing threshold both initially and with age. Measurements of the EP confirm the anticipated drop in potential for genotypes that demonstrate age-dependent hearing loss. The EP generated by the NKCC1 + α2-Na,K–ATPase double heterozygote, however, is maintained at a level comparable to that of the control condition, suggesting a potential advantage in this combination of ion transporter modification. These observations provide insight into the detailed mechanisms of EP generation, and results of combination-knockout experiments may have important implications in the future treatment of drug-induced and age-related hearing losses.

Distinct Roles of Molecular Chaperones HSP90α and HSP90β in the Biogenesis of KCNQ4 Channels

Loss-of-function mutations in the KCNQ4 channel cause DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss. Previous studies have demonstrated that the majority of the pathogenic KCNQ4 mutations lead to trafficking deficiency and loss of KCNQ4 currents. Over the last two decades, various strategies have been developed to rescue trafficking deficiency of pathogenic mutants; the most exciting advances have been made by manipulating activities of molecular chaperones involved in the biogenesis and quality control of the target protein. However, such strategies have not been established for KCNQ4 mutants and little is known about the molecular chaperones governing the KCNQ4 biogenesis. To identify KCNQ4-associated molecular chaperones, a proteomic approach was used in this study. As a result, two major molecular chaperones, HSP70 and HSP90, were identified and then confirmed by reciprocal co-immunoprecipitation assays, suggesting that the HSP90 chaperone pathway might be involved in the KCNQ4 biogenesis. Manipulating chaperone expression further revealed that two different isoforms of HSP90, the inducible HSP90α and the constitutive HSP90β, had opposite effects on the cellular level of the KCNQ4 channel; that HSP40, HSP70, and HOP, three key components of the HSP90 chaperone pathway, were crucial in facilitating KCNQ4 biogenesis. In contrast, CHIP, a major E3 ubiquitin ligase, had an opposite effect. Collectively, our data suggest that HSP90α and HSP90β play key roles in controlling KCNQ4 homeostasis via the HSP40-HSP70-HOP-HSP90 chaperone pathway and the ubiquitin-proteasome pathway. Most importantly, we found that over-expression of HSP90β significantly improved cell surface expression of the trafficking-deficient, pathogenic KCNQ4 mutants L274H and W276S. KCNQ4 surface expression was restored by HSP90β in cells mimicking heterozygous conditions of the DFNA2 patients, even though it was not sufficient to rescue the function of KCNQ4 channels.

Impaired surface expression and conductance of the KCNQ4 channel lead to sensorineural hearing loss.

KCNQ4, a voltage‐gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non‐syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant‐negative effects on the wild‐type (WT) channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.

Comparison of distortion product otoacoustic emissions in 28 inbred strains of mice

Cochlear function was evaluated in a longitudinal study of 28 inbred strains of mice at 3 and 5 mo of age using measures of distortion product otoacoustic emissions (DPOAEs) in response to a federal initiative to develop rapid mouse phenotyping methodologies. DP-grams at f(2) frequencies ranging from 6.3 to 54.2kHz were obtained in about 3min/ear by eliciting 2f(1)-f(2) DPOAEs in 0.1-octave steps of f(2) with primary tones at L(1)=L(2) =55, 65, and 75dB SPL. CBA/CaJ mice exhibited average levels of approximately 26dB SPL and this strain was selected as the normal reference strain against which the others were compared. Based upon the configurations of their DP-grams, the 28 mouse strains could be categorized into four distinct groups. That is, nine of the strains including the CBA were designated as the CBA-like group because these mice displayed robust DPOAE levels across frequency. In contrast, the remaining three groups all exhibited irregular DP-gram patterns. Specifically, eight of the remaining 19 strains showed a progressive high- to low-frequency reduction in DPOAE levels that was typical of age-related hearing loss (AHL) associated with mouse strains homozygous for the ahl allele and were labeled as AHL-like strains. Seven strains demonstrating relatively even patterns of reduced DPOAE levels across the frequency-test range were designated as Flat-loss strains. Finally, the remaining four strains exhibited no measurable DPOAEs at either 3 or 5 mo of age and thus were classified as Absent strains. Extending the f(2) test frequencies up to approximately 54kHz led to the detection of very early-onset reductions in cochlear function in non-CBA-like groups so that all strains could be categorized by 3 mo of age. Predictably, the AHL-like strains showed more pronounced DPOAE losses at 5 mo than at 3 mo. A similar deterioration in DPOAE levels was not apparent for the Flat-loss strains. Both the AHL-like and Flat-loss strains showed considerably more variability in DPOAE levels than did the CBA-like strains. Together, these findings indicate that DP-grams adequately reveal both frequency-specific loss patterns and details of inbred strain variability.

Identification of transcription factor-DNA interactions using chromatin immunoprecipitation assays.

Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study transcription factor-DNA interactions, the advantage of the chromatin immunoprecipitation (ChIP) assay is that the interaction of a transcription factor with its target genes is captured in the native context of chromatin in living cells. Therefore, ChIP base assays are powerful tools to identify the direct interaction of transcription factors and their target genes in vivo. More importantly, ChIP assays have been used in combination with molecular biology techniques, such as PCR and real time PCR, gene cloning, and DNA microarrays, to determine the interaction of transcription factor-DNA from a few potential individual targets to genome-wide surveys.