The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10-6 M for trout hair cell proteins and Kd=2.7×10-7 M for organ of Corti proteins at 68 μM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and β1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.
"Forty micrograms of MDCK cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) using 5–20% gel , . Following electrophoretic resolution, the proteins were transferred onto a nitrocellulose membrane using an electrophoretic system (BioRad, Germany). "
[Show abstract][Hide abstract] ABSTRACT: Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.
PLoS ONE 04/2014; 9(4):e93056. DOI:10.1371/journal.pone.0093056 · 3.23 Impact Factor
"Proteins outside of the TRP family have been reported as possible candidates for MET channels in hair cells. These include the cyclic nucleotide-gated channel α-3(CNGA3) ,  and transmembrane channel-like (TMC) proteins . The TRP channel literature supports the idea of redundancy. "
[Show abstract][Hide abstract] ABSTRACT: Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca(2+) permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1(-/-) ) or a missense mutation in the pore domain of TRPM1 (Trpm1(tvrm27/tvrm27) ). Hearing thresholds were evaluated in adult (4-5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1(-/-) or Trpm1(tvrm27/tvrm27) mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.
PLoS ONE 10/2013; 8(10):e77213. DOI:10.1371/journal.pone.0077213 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A unique coupling between HCN1 and stereociliary tip-link protein protocadherin 15 has been described for a teleost vestibular hair-cell model and mammalian organ of Corti (OC) (Ramakrishnan et al. (2009) J. Biol. Chem. 284, 3227-3238). We now show that Ca2+- dependent interaction of organ of Corti HCN1 and protocadherin 15 CD3 is mediated by N-terminus sequence specific to HCN1, not replicated by analogous specific peptides for HCN2 or HCN4 nor by N-terminus sequence conserved across HCN isoforms utilized in channel formation. Further, the HCN1-specific peptide binds both PIP3 and PIP2 but not PIP. Singly-isolated cochlear inner and outer hair cells express HCN1 transcript, and HCN1 and HCN2 protein is immunolocalized to hair-cell stereocilia by both z-stack confocal and pre-embedding EM immunogold microscopy, with stereociliary tip-link and subcuticular plate sites. Quantitative PCR indicates HCN1: HCN2:HCN3:HCN4 = 9:9:1:89 in OC of wildtype mouse, with HCN4 protein primarily attributable to inner sulcus cells. A mutant form of HCN1 mRNA and protein is expressed in OC of an HCN1 mutant, corresponding to full-length sequence with the in-frame deletion of pore-S6 domains, predicted by construct. The mutant transcript of HCN1 is ~ 9-fold elevated relative to wildtype levels, possibly representing molecular compensation, with unsubstantial changes in HCN2, HCN3 and HCN4. Immunoprecipitation protocols indicate alternate interactions of full-length proteins: HCN1 can interact with protocadherin 15 CD3 and F-actin-binding filamin A forming a complex which does not include HCN2; or HCN1 can interact with HCN2 forming a complex without protocadherin 15 CD3 but including F-actin-binding fascin-2.
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