Characterisation of DRASIC in the mouse inner ear
University of Iowa, Iowa City, Iowa, United States Hearing Research
(Impact Factor: 2.97).
05/2004; 190(1-2):149-60. DOI: 10.1016/S0378-5955(04)00015-2
Within the cochlea, the hair cells detect sound waves and transduce them into receptor potential. The molecular architecture of the highly specialised cochlea is complex and until recently little was known about the molecular interactions which underlie its function. It is now clear that the coordinated expression and interplay of hundreds of genes and the integrity of cochlear cells regulate this function. It was hypothesised that transcripts expressed highly or specifically in the cochlea are likely to have important roles in normal hearing. Microarray analyses of the Soares NMIE library, consisting of 1536 cDNA clones isolated from the mouse inner ear, suggested that the expression of the mechanoreceptor DRASIC was enriched in the cochlea compared to other tissues. This amiloride-sensitive ion channel is a member of the DEG/ENaC superfamily and a potential candidate for the unidentified mechanoelectrical transduction channel of the sensory hair cells of the cochlea. The cochlear-enriched expression of amiloride-sensitive cation channel 3 (ACCN3) was confirmed by quantitative real-time polymerase chain reaction. Using in situ hybridisation and immunofluorescence, DRASIC expression was localised to the cells and neural fibre region of the spiral ganglion. DRASIC protein was also detected in cells of the organ of Corti. DRASIC may be present in cochlear hair cells as the ACCN3 transcript was shown to be expressed in immortalised cell lines that exhibit characteristics of hair cells. The normal mouse ACCN3 cDNA and an alternatively spliced transcript were elucidated by reverse transcription polymerase chain reaction from mouse inner ear RNA. This transcript may represent a new protein isoform with an as yet unknown function. A DRASIC knockout mouse model was tested for a hearing loss phenotype and was found to have normal hearing at 2 months of age but appeared to develop hearing loss early in life. The human homologue of ACCN3, acid-sensing ion channel 3, maps to the same chromosomal region as the autosomal recessive hearing loss locus DFNB13. However, we did not detect mutations in this gene in a family with DFNB13 hearing loss.
Available from: Francisco Mercado
- "SGNs and the OC express ASIC3 (Hildebrand et al., 2004). Although ASIC3 knockout mice exhibit normal hearing, they develop hearing loss early in life (4 months of age) (Hildebrand et al., 2004). Additionally, the ASIC1b subunit was detected in SGNs and in the stereocilia bundle of mouse cochlear hair cells (Ugawa et al., 2006). "
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ABSTRACT: Acid-sensing ion channels (ASICs) are activated by an increase in the extracellular proton concentration. There are four genes (ASIC1-4) that encode six subunits, and they are involved in diverse neuronal functions, such as mechanosensation, learning and memory, nociception, and modulation of retinal function. In this study, we characterize the ASIC currents of spiral ganglion neurons (SGNs). These ASIC currents are primarily carried by Na+, exhibit fast activation and desensitization, display a pH50 of 6.2 and are blocked by amiloride, indicating that these are ASIC currents. The ASIC currents were further characterized using several pharmacological tools. Gadolinium and acetylsalicylic acid reduced these currents, and FMRFamide, zinc (at high concentrations) and N,N,N’,N’–tetrakis-(2-piridilmetil)-ethylenediamine increased them, indicating that functional ASICs are composed of the subunits ASIC1, ASIC2, and ASIC3. Neomycin and streptomycin reduced the desensitization rate of the ASIC current in SGNs, indicating that ASICs may contribute to the ototoxic action of aminoglycosides. RT-PCR of the spiral ganglion revealed significant expression of all ASIC subunits. By immunohistochemistry the expression of the ASIC1a, ASIC2a, ASIC2b, and ASIC3 subunits was detected in SGNs. Although only a few SGNs exhibited action potential firing in response to an acidic stimulus, protons in the extracellular solution modulated SGN activity during sinusoidal stimulation. Our results show that protons modulate the excitability of SGNs via ASICs.
Available from: Daniel D Rubens
- "This would distinguish it from a balance stimulus and precipitate an 'unadjusted' body movement during sleep that would allow the animal to escape suffocation. In fact, evidence supports anoxic and ischemic insult precipitating a depolarization response in specialized sensory neurons, including auditory and vestibular pathways (Burke, 1993; Fern and Ransom, 1997; Kiernan and Bostock, 2000; Lin et al., 2001; Hildebrand et al., 2004; Krishnan et al., 2005; Mercado et al., 2006; Ugawa et al., 2006; Zhang et al., 2008a,b). "
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Sudden Infant Death Syndrome (SIDS) remains the leading cause of infant mortality in Western societies. A prior study identified an association between hearing suppression on the newborn hearing test and subsequent death from SIDS. This is the first finding of an abnormality in SIDS cases prior to death. A following study identified that inner ear dysfunction precipitates a marked suppression of the hypercapnic ventilatory response (HCVR). Failure of arousal has been proposed to be a key component in SIDS. The objective of the present study was to assess whether inner ear dysfunction not only weakens the hypercapnic response, but also plays a role in suppressing the arousal response to suffocating gas mixtures.
Wild-type mice (n=28) received intra-tympanic gentamicin (IT-Gent) injections bilaterally or unilaterally to precipitate inner ear hair cell dysfunction. Three control groups (n=22) received intra-tympanic saline (IT-Saline) bilaterally or unilaterally (right or left), or intra-peritoneal gentamicin (IP-Gent). The body movement arousal responses to severe hypoxia-hypercarbia combined (5% CO2 in nitrogen) were tested under light anesthesia 8 days following the administration of gentamicin or saline.
After injections, the bilateral and unilateral IT-Gent-treated animals behaved similarly to controls, however the HCVR as well as the arousal movements in response to severe hypoxia-hypercarbia were suppressed in IT-Gent-treated animals compared to control animals (P<0.05). Thus the HCVR was significantly decreased in the bilateral (n=9) and unilateral IT-Gent-treated mice (n=19) compared to bilateral (n=7) and unilateral IT-Saline (n=9) control groups (p<0.05). Arousal movements were suppressed in the bilateral IT-Gent group (n=9) compared to bilateral IT-Saline controls (n=7, P<0.0001) and in the unilateral IT-Gent group (n=19) compared to unilateral IT-Saline controls (n=10, P<0.0001).
The findings support the theory that inner ear dysfunction could be relevant in the pathophysiology of SIDS. The inner ear appears to play a key role in arousal from suffocating gas mixtures that has not been previously identified.
Available from: Chun-Ho Cheng
- "In our study, recombinant APETx2 expressed in P. pastoris inhibited ASIC3-mediated current in CHO-expressing mouse ASIC3 with an IC 50 of 37.3 nM. Because the sequence of the mouse ASIC3 channel shares over 92% similarity with that of human and rat proteins, it is not surprising that the efficacy of recombinant APETx2 is similar to that of native toxin towards rat ASIC3 (Diochot et al., 2004; Hildebrand et al., 2004). The E. coli expression system is not suitable for producing venom toxins with the correct fold, because most of them are highly disulfide-bonded (Anangi et al., 2007). "
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ABSTRACT: Acid sensing ion channels (ASICs) are family of proteins predominantly present in the central and peripheral nervous system. They are known to play important roles in the pathophysiology of pain and ischemic stroke. APETx2 is a potent and selective inhibitor of ASIC3-containing channels and was isolated from sea anemone Anthopleura elegantissima. To facilitate the study on the molecular determinants of ASIC3-ligand interactions, we expressed recombinant APETx2 in the Pichia pastoris (P. pastoris) expression system and purified it to homogeneity. Recombinant APETx2 produced in P. pastoris inhibited the acid-evoked ASIC3 current with the IC(50) value of 37.3 nM. The potency of recombinant toxin is similar to that of native APETx2. The sequential assignment and structure analysis of APETx2 were obtained by 2D and 3D (15)N-edited NMR spectra. Our NMR data suggests that APETx2 produced in P. pastoris retained its native fold. The results presented here provide the first direct evidence that highly disulfide bonded peptide inhibitor of ASIC3, APETx2, can be expressed in P. pastoris with correct fold and high yield. We also showed that the R17A mutant exhibited a decrease in activity, suggesting the feasibility of the use of this expression system to study the interactions between APETx2 and ASIC3. These evidences may serve as the basis for understanding the selectivity and activity of APETx2.
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