| The deaf population increases gradually with the development of aging society, Sensorineural hearing loss(SNHL) has become one of the major health problems currently, also it ’ s the focus and difficulty of professional treatment of Department of Otorhinolaryngology. Recently, there are many reports about pathophysiology of sensorineural deafness, the regeneration of hair cells, the conversion treatment of supporting cells and stem cells, but it’s pathogenesis and treatment still faces enormous challenges. SNHL could be subdivided into sensorinural deafness and auditory nervous deafness. Cochlear inner hair cells(IHCs) synapses are the first station of acoustic signal to be transduced into electrical signals and transmit them to auditory center by auditory nervous, if their dysfunctions, impair audibility of sounds to varying degrees but commonly affect neural encoding of acoustic temporal cues essential for speech comprehension. Clinical features of auditory synaptopathies are similar to those accompanying auditory neuropathy, a group of genetic and acquired disorders of spiral ganglion neurons. Physiologically, the IHCs synapse features sub-millisecond precision and unprecedented high rates of release and replenishment of vesicles. Its morphological feature is the synaptic ribbon, primarily composed of Ribeye/Ct BP2, tethering approximately 70-200 synaptic vesicles close to the presynaptic membrane. It has been considered as a possible regulator of vesicle-vesicle or vesicle-plasma membrane fusion, namely an inhibitor and/or accelerator and possibly a synchronizer of the vesicle fusion process. Further research on the synaptic proteins of IHCs, ribbon synapses have many differently moleculars compared with the conventional synapses. For instance, Synaptotagmins 1-2、Compexins、Munc13-1 are essentially synaptic proteins in conventional synapses, nevertheless, absence in IHCs. But auditory hair cells hire special proteins to complete the synaptic function, such as Vglut3、otoferlin and so on.The communication between IHCs and auditory nerves is the same with mostly chemical synapses. at inner hair cells, neurotransmitters are packaged into synaptic vesicles(SVs), when an action potential opens presynaptic voltage-gated Ca2+ channels, the neurotransmitters are released by Ca2+-triggered exocytosis into the synaptic cleft to activate postsynaptic receptors of auditory nerves. Synaptic exocytosis involves 2 processes: SV docking and fusion with the presynaptic membrane. SV docking is the attachment of vesicles to the active zones, priming is the preparatory step for the SVs before undergoing fusion, fusion finishes during the Ca2+-triggering of fusion-pore opening. SVs membrane fusion in the final stage of exocytosis has become the important premise of neurotransmitter secretion. Researchs show that several evolutionarily conserved proteins participate at several steps of SVs exocytosis including the SNAP receptor(SNARE) proteins synaptobrevin, syntaxin-1, and SNAP-25, which form a tight four-helix bundle called the SNARE complex that brings the vesicle and plasma membranes together; the Sec1-Munc18(SM) protein Munc18-1, which binds to a self-inhibited “closed” conformation of syntaxin-1 and to SNARE complexes; and the Ca2+ sensor synaptotagmin-1. Munc18-1 belongs to Sec1/Munc18(SM) protein family members, about 60-70 k Da polypeptides, was the first SM protein linked to membrane fusion when it was found to bind tightly to isolated syntaxin-1, an interaction that involves the closed conformation of syntaxin-1. The crystal structure of the syntaxin-1/Munc18-1 complex showed that Munc18-1 contains three domains that form an arch shape with a central cavity, and that closed syntaxin-1 binds to the central cavity. Currently the main functions of Munc18-1 have been proposed including: molecular chaperone of syntaxin-1 allowing appropriate localization and expression of syntaxin-1; priming via promotion of SNARE complex-mediated membrane fusion; docking of large dense-core vesicles to the plasma membrane.Recently, in vitro reconstitutions liposomes have shown that fusion starts with the syntaxin-1-Munc18-1 complex, furthermore, it has been reported that the central neurons and neuromuscular synapses completely lost the ability to secrete neurotransmitters in Munc18-1 gene knockout mutant mice, in chromaffin cells the dynamics of vesicle release events during exocytosis are controlled by protein kinase C(PKC) directly through phosphorylation of Munc18-1 on Ser-313, and in nerve terminals Munc18-1 redistributes in an activity- and PKC-dependent manner, meanwhile in the calyx of Held, a large CNS model synapse that expresses robust post-tetanic potentiation(PTP), Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release. The Munc18-1 is a key protein of SVs exocytosis, and affected by intracellular PKC phosphorylation.However the above research reports only in chromaffin cells, PC12 cells, neuronal cells, about IHCs synaptic vesicles membrane fusion studies are poor. The researchers compared the protein composition of the IHCs、retina ribbon synapses and central nervous conventional synapses found that Munc18-1、syntaxin-1 and SNARE proteins also express in IHCs, but the distribution and location of Munc18-1 in intracellular has no detailed explanation, then we don’t know whether or not Munc18-1 proteins also regulate synaptic vesicles exocytosis in IHCs, as the same in conventional synapses, and the relationship with auditory development still need further research. We know that the plasmalemma is composed of an insulating lipid bilayer structure, then the extracellular fluid-plasmalemma-intracellular fluid to form membrane capacitance(Cm), Cm=πd2/100(pF),d is the cell diameter(μm), is proportional to the surface area of cell membrane. It is necessary to vesicles fuse with the presynaptic membrane when activating IHCs exocytosis, transmitters are released into the synaptic cleft through the fusion pores, the membrane surface area is increased simultaneously, and Cm is upgraded. So the patch clamp technique to real-time monitoring of Cm has become an important means of IHCs exocytosis.These experiments firstly detect the expression、distribution of Munc18-1 proteins in adult mice cochlear IHCs, and the relationship between Munc18-1 and ribbon synapses Ribeye/CtBP2 by immunofluorescence staining and confocal laser scanning fluorescence microscopy imaging technology; In most altricial rodents, the onset of hearing occurs around postnatal day 12, So we secondly compare the diversity of Munc18-1 gene expression in postnatal day 6 、12、18(P6、P12、P18) cochlear IHCs by real time fluorescence quantitative q RT-PCR, to observe the correlation between Munc18-1 and the development of mice auditory; Finally, we use the whole cell patch clamp technique, to observe the calcium currents(ICa)、membrane capacitance(Cm) of acutely isolated inner hair cells, by inhibition and activation of endogenous PKC mediated phosphorylation of Munc18-1, in order to carry on the preliminary discussion on the mechanism of Munc18-1 regulates synaptic vesicles exocytosis in IHCs.Result:1、The laser scanning confocal microscope imaging detected that the Munc18-1 proteins express high concentration in the cytoplasm of IHCs, Ct BP2 and Munc18-1 double fluorescein detecting showed that Munc18-1 proteins accumulated close to the ribbon synapses, and compared the fluorescence intensity(IMV) of Munc18-1 showed that the nucleus hypotegum is stronger than the nucleus epitegum, P<0.05, there was statistical significance.2、Real-time fluorescence quantitative q RT-PCR analysis revealed that the expression of Munc18-1 was no difference in P6、P12、P18 three ages mice cochlea, P>0.05, there was not statistically significant.3、Detection of patch clamp whole cell voltage clamp model, the recordings of action potential frequency and voltage-evoked changes in membrane electric capacitance(△Cm) in IHCs treated with PKC inhibitor were reduced than the normal IHCs(Control groups), P<0.05(n=5); then those in IHCs treated with PKC agonists were increased than the above two kinds of treatment(Control groups and PKC inhibitor groups), P<0.05(n=5), there was statistical significance.Conclusion:This experiment indicate that: the expression of Munc18-1 proteins gather near the nucleus hypotegum, closs to the ribbon synapses in IHCs; Munc18-1 is the essential protein for synaptic vesicles exocytosis in IHCs, moreover there are existence in immature inner hair cells, the connection with the development of mouse auditory needs to be further verified; PKC inhibitor and agonists can affect the action potential frequency and membrane capacitance, probably be mediated by endogenous PKC phosphorylation of Munc18-1 to regulate synaptic vesicles exocytosis. |
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