The Study Of An Osmosensitive Cation Channel TMEM63B And Its Physiological Function In Hearing

It's inevitable for all the living organisms to encounter various changeable environments including both the biotic and abiotic factors.Osmotic stress is one of the abiotic factors that will influence the physiological functions and activities of the living organisms.The corresponding cell responses and shape maintenance according to the changes of osmolality is precisely developed to adapt these complicated situations in either plants or animals.Traditionally,plants will unavoidably meet the situation of changeable osmolality derived from the uncontrolled climate of drought and raining,and the salinity condition[1-3].Comparably,animals will also face the conditions of lacking water,the complicated physiological activities accompanying with the mass of metabolic products that will alter the physiological environments.Several ion channels,such as the structurally elucidated mechanosensitive channels of large and small conductance proteins(Msc L and Msc S,respectively),sense osmotic shocks and tune the cell responses to them in bacteria[4,5].In mammals,the mechanosensitive ion channels including the potassium channel TREK-1,TRAAK,TRP family channels like TRPV4 and TRPC4 play a vital role in osmosensing[6,7].These ion channels sensing the osmotic stress are activated to induce the ion flux along with the osmolytes release.Whether in plants or animals,calcium ion functions as the primary regulator of the initial osmotic stress[8,9].After the osmotic stress treatment,the markedly elevation of cytoplasmic free Ca²⁺will be observed[10,11].As a matter of course,the disruption of the function of sensing osmolality will bring catastrophe to the living organisms.The loss of regulation to osmolality in plants will result in the growth retardation even die.For animals,the disruption of the osmolality regulation will lead to the disorder of physiological functions[12,13].Hair cells are the sensory cells of auditory system,involving two anatomically and functional types of cells:inner hair cells(IHCs),which are the genuine sensory cells that release transmitters and induce action potential in afferent neurons,and outer hair cells(OHCs),which act as an amplifier of sound through the intrinsic electromotility.OHCs exhibit somatic motility which is thought to contribute to the enhancement of cochlear frequency selectivity and amplification[14].It has been documented that osmotic stress by hypoosmotic stimulation will cause a slow shape change[15,16]in accompany with the elevation of intracellular Ca²⁺concentrations in OHCs[17].The ionic environment in inner ear may therefore affect the OHCs.Any disturbance in the homeostasis of inner ear fluids may thus affect the functional properties of OHCs,thereby causing a disorder in the delivery auditory information in the auditory signaling transduction process.However,the underlying mechanism of this regulation in OHCs hitherto remains unclear.In this thesis,we identified the unreported TMEM63B in Mus musculus with unknown physiological function as an osmosensitive cation channel,while its homologous protein OSCA1 in Arabidopsis had previously been documented as hyperosmolality gated calcium permeable ion channels.Using calcium fluorescence imaging and electrophysiological techniques,we found that the unreported TMEM63B was a multiple transmembrane protein mediating the calcium influx across plasma membrane and hypoosmolality-activated currents under the hypoosmotic stimulation in the heterologous expression system.And the hypoosmolality-activated currents possessed the characteristics of nonselective cation permeability.In the further study,we explored the putative pore region of TMEM63B and found two mutations that would alter the ion permeability of TMEM63B.Those data demonstrated the fact that TMEM63B was a novel hypoosmolality-activated nonselective cation channel.In the subsequent study,we generated TMEM63B knockout and HA tag knock-in mice to depict the physiological function of TMEM63B.Through series of phenotype analyses,we found that adult TMEM63B knockout mice exhibited completely hearing loss.In detail,the deficiency of TMEM63B would lead to the gradual hearing impairment since the onset of hearing,and the ultimately complete hearing loss in adulthood.With the help of HA tag knock-in mice,we confirmed the localization of TMEM63B in hair cells of inner ear,hinting that TMEM63B might affect hair cells.The TMEM63B knockout mice expectedly showed degeneration of OHCs since the onset of hearing,which was consistent with the phenotype of hearing impairment.In addition,the deficiency of TMEM63B did not directly affect the nerve fibers of spiral ganglion neurons and did not influence neither the mechanoelectrical transduction process nor the intrinsic electromotility of OHCs.It was the lack of TMEM63B protein that contributed to the severe degeneration of OHCs,presenting the phenotype of hearing loss in mice.Experimentally verified as a hypoosmolality-activated nonselective cation channel,what kind of physiological functions TMEM63B undertook in vivo attracted us to study forward.When facing the hypoosmotic solution,OHCs of TMEM63B knockout mice could not maintain their shape,while wildtype OHCs kept constant through a manner that was dependent of the extracellular calcium flux involving the activation of TMEM63B channel.Generally,we identified a hypoosmolality-activated cation channel TMEM63B,and this ion channel participated in the shape maintenance of OHCs when facing decreased of osmolality.