Protein Interaction Analysis by Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is an optical technique that is utilized for detecting molecular interactions that occur in direct protein-protein interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected, after polarized light impinges upon the surface, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon).

Analysis of Dysferlin Direct Interactions with Putative Repair Proteins Links Apoptotic Signaling to Ca Elevation via PDCD6 and FKBP8.

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases.

Analysis of Protein Interactions by Surface Plasmon Resonance.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions, such as interactions that occur between proteins or other classes of molecules. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon).

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin.

Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Inner-Ear Proteins.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered, and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon).

Calcium regulates molecular interactions of otoferlin with soluble NSF attachment protein receptor (SNARE) proteins required for hair cell exocytosis.

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (KD = 267 μM) with diminished binding in a pachanga (D1767G) C2F mouse mutation.

Cyclic nucleotide-gated channel α-3 (CNGA3) interacts with stereocilia tip-link cadherin 23 + exon 68 or alternatively with myosin VIIa, two proteins required for hair cell mechanotransduction.

Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M.

HCN1 and HCN2 proteins are expressed in cochlear hair cells: HCN1 can form a ternary complex with protocadherin 15 CD3 and F-actin-binding filamin A or can interact with HCN2.

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, N. A., Drescher, M.

The SNARE complex in neuronal and sensory cells.

Transmitter release at synapses ensures faithful chemical coding of information that is transmitted in the sub-second time frame. The brain, the central unit of information processing, depends upon fast communication for decision making. Neuronal and neurosensory cells are equipped with the molecular machinery that responds reliably, and with high fidelity, to external stimuli.

Identification of the porosome complex in the hair cell.

Porosomes are proposed to be the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel their contents to the extracellular space during cell secretion. In neurons, porosomes are manifested as cup-shaped lipoprotein structures in the presynaptic membrane, 12-17 nm in diameter and possessing a central plug. Hair cells of hearing and balance secrete transmitter from synaptic vesicles in sensory signal transduction, but it has not previously been demonstrated that these mechanosensory cells possess porosome structures that could participate in the secretory process.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1.

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.

Calcium-dependent binding of HCN1 channel protein to hair cell stereociliary tip link protein protocadherin 15 CD3.

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia.

Direct interaction of otoferlin with syntaxin 1A, SNAP-25, and the L-type voltage-gated calcium channel Cav1.3.

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf.

Surface plasmon resonance (SPR) analysis of binding interactions of proteins in inner-ear sensory epithelia.

Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon).

Immunohistochemical localization of adrenergic receptors in the rat organ of corti and spiral ganglion.

Alpha(1)-, beta(1)-, and beta(2)-adrenergic receptors (ARs), which mediate responses to adrenergic input, have been immunohistochemically identified within the organ of Corti and spiral ganglion with polyclonal antibodies of established specificity. Alpha(1)-AR was immunolocalized to sites overlapping supranuclear regions of inner hair cells as well as to nerve fibers approaching the base of inner hair cells, most evident in the basal cochlear turn. A similar preponderance across cochlear turns for alpha(1)-AR in afferent cell bodies in the spiral ganglion pointed to type I afferent dendrites as a possible neural source of alpha(1)-AR beneath the inner hair cell.

Voltage-gated Ca2+ channel Ca(V)1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: cloning and comparison across vertebrate classes.

Full-length sequence (>6.5 kb) has been determined for the Ca(V)1.3 pore-forming subunit of the voltage-gated Ca(2+) channel from the saccular hair cells of the rainbow trout (Oncorhynchus mykiss).

Expression of subunits for the cAMP-sensitive 'olfactory' cyclic nucleotide-gated ion channel in the cochlea: implications for signal transduction.

Cyclic nucleotide-gated (CNG) ion channels have been implicated as functioning in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. The olfactory, cAMP-sensitive CNG ion channel in vivo is considered to comprise the pore-forming CNG2 subunit together with CNG5 and CNG4.3 modulatory subunits.