Browsing by Author "Raphael, Robert M"
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Item A study of the C-terminus in prestin by designing cleavable constructs(2016-12-01) Guo, Jing; Raphael, Robert MThe membrane protein prestin plays a central role in the mammalian auditory system by enabling Outer Hair Cells (OHCs) in the cochlea to actively respond to electrical signals. Prestin belongs to the SLC26A family of membrane proteins, which all have a large cytosolic terminus containing a conserved region known as the STAS domain (Sulfate Transporters and Anti-Sigma factor antagonist), whose function re- mains unknown. In this work, we engineered prestin by inserting three types of short peptides, flexible, rigid and cleavable linkers, into different positions in the C- terminus and tested prestin’s funtionality, self-interactions, and lateral mobility in the membrane. First, we inserted short peptides between the last trans-membrane domain and the STAS domain. We found that the prestin’s functionality is inhibited, and the self-interaction is significantly decreased. The FRAP data revealed that the mobility of prestin is altered by inserting different types of linkers before the STAS domain. Second, we inserted cleavable linkers in the disordered region within the STAS domain. The data revealed that prestins inserted with linkers between 596aa and 597aa, and between 620aa and 621aa, can successfully locate to the membrane and retain NLC function. Next, by using advanced fluorescence microscopy and im- munoprecipitation, we discovered that prestin is confined by a binding site lcoated between the 597aa and the 620aa. Together, these data suggest that the interface between the C-terminus of prestin, STAS domain, and the last TMD is crucial to prestin’s function, self-interactions, and mobility, and that a binding site locates in the disordered region of STAS domain that influence prestin’s lateral mobility.Item Embargo Computational Insights Into the Generation of Endocochlear Potential: Role of Potassium Channels and Tight Junctions(2024-04-19) Garcia Camargo, Lucas; Raphael, Robert MIon transport and homeostasis is vital to all organ systems in the body. In the auditory system, mechanotransduction by sensory hair cells constantly drains potassium ions from the endolymphatic fluid that bathes hair cell stereocilia. Proper hearing function depends on efficient resupply of these ions through several cochlear spaces that are delineated by tight junctions as well as maintenance of the endocochlear potential in the endolymph. Mutations in the genes responsible for encoding ion channels, transporters and tight junctions are responsible for many cases of hearing loss and auditory dysfunction. The main goal of this study is to understand how the network of ion channels and transporters maintains cochlear ion homeostasis and establishes the endocochlear potential. The model developed in this thesis is able to predict the effect of genetic mutations on the system and for its validation I compare predicted results to previous experimental data in the literature. To accomplish this, I expanded upon the framework of a computational model previously generated by a former student from our group, Dr. Imram Qurashi. The resulting model incorporates biophysical properties of the ion channels, transporters and tight junctions in the various cochlear compartments. The model predicted steady state electrical potentials and ion concentrations consistent with experimental data for both healthy conditions and results from loss-of-function knockout experiments. The sensitivity analysis, demonstrated that the concentrations and potentials were most sensitive to variations in ATP drive from marginal cells. We also found that the endocochlear potential is established by both inward and outward rectifying ion channels (Kir4.1 and Kv) present in the intermediate cells in the syncytium, supporting recent experimental results. The model predicts that the absence of Claudin-11, a tight junction protein between the intrastrial space and perilymph, and the endolymph and perilymph leads to a drop in EP while maintaining a high $K^+$ concentration in the endolymph, as observed in recent experimental results. Notably, the model allowed users to study the pathological state of endolymphatic hydrops, a key feature of Meniere’s disease, by integrating a variable endolymph volume through an osmolar constraint. This computational model serves as a significant step forward in our understanding of cochlear ion homeostasis, the development of auditory pathologies, and the potential for targeted therapeutic interventions. Its ability to simulate the effects of specific ion channel blockades and predict changes in endolymph volume highlights its value as a tool for future research in the realm of inner ear diseases.Item Computational Model of Synaptic Transmission at the Vestibular Hair Cell Calyx Synapse(2020-08-13) Govindaraju, Aravind Chenrayan; Raphael, Robert M"In the sensory neuroepithelia of the vestibular system, the organ which detects head orientation and acceleration, Type I sensory hair cells are enveloped by a cup-like process (calyx) of the afferent neuron and possess a characteristic low voltage activated potassium conductance (gKL) on their basolateral surface. The presence of the calyx creates a unique synapse morphology which is thought to limit the diffusion of ions and support two modes of neurotransmission between the hair cell and afferent neuron: Quantal (Q) – through the release of neurotransmitters and Non-Quantal (NQ) – through non-neurotransmitter mediated effects such as ephaptic coupling and potassium accumulation in the synaptic cleft. The importance and necessity of NQ transmission has been unclear. Direct experimental measurement of electric potentials and ion concentrations in the hair cell and afferent, let alone the synaptic cleft, is difficult. We have developed a computational model to probe the dynamic behavior of the Vestibular Hair Cell Calyx (VHCC) synapse and understand the role of non-quantal transmission. The VHCC model uses expressions for K+ and Na+ electro-diffusion in the cleft, Hodgkin-Huxley-like ion currents based on whole-cell recordings, stochastic vesicle release, and the cable equation to calculate potentials in the hair cell, cleft, afferent calyx and afferent fiber. Model simulations suggest that ephaptic coupling at the VHCC synapse is active at all frequencies, does not exhibit high-pass behavior as previously thought and may be an indefatigable method of communication between the type I hair cell and calyx."Item The Vestibular Hair Cell-Calyx Model: Insights into ion channels, experimental data, and ephaptic transmission in the vestibular epithelium(2023-04-21) Govindaraju, Aravind Chenrayan; Raphael, Robert MA system of bony canals and membranous ducts in the inner ear, collectively called the vestibular system, contains sensory cells that rapidly transduce head motion. Information from these cells drives some of the fastest reflexes in the body such as the vestibulo-ocular reflex and vestibulo-collic reflex which are vital for maintaining gaze, balance and orientation. Of these cells, the Type I sensory hair cells are surrounded by a cup-like terminal (calyx) of the afferent neuron and possess a characteristic low voltage activated potassium conductance (gKL) on their basolateral surface. The apposition of the presynaptic hair cell membrane and the postsynaptic calyx creates a unique synapse morphology that limits the diffusion of ions entering the intermediate cleft space. Transmission between the hair cell and afferent neuron across this cleft space occurs through the release of neurotransmitters (quantal) or without conventional neurotransmitters (nonquantal). I developed a computational model of the Vestibular Hair Cell-Calyx (VHCC) to investigate the mechanism of nonquantal transmission. The VHCC model uses expressions for K+ and Na+ electrodiffusion in the cleft, Hodgkin-Huxley-like ion currents based on whole-cell recordings, and the cable equation to calculate potentials in the hair cell, cleft, afferent calyx and afferent fiber. Using the VHCC model I show that nonquantal transmission occurs through changes in cleft electrical and K+ potential and that it is a case of ephaptic (due to proximity) transmission. I have identified features in existing experimental data that support the idea of ephaptic transmission between the hair cell and afferent calyx: 1) phase of synaptic transmission, 2) retrograde transmission, 3) fast post synaptic currents. Based upon this work, I advance the hypothesis that electrical and K+ potentials change throughout the intercellular spaces of the tightly packed vestibular epithelium, not just within the VHCC synaptic cleft. If true, this may explain the speed with which vestibular afferents encode even small head motions.