目的研究耳鸣大鼠耳蜗基底膜内毛细胞耳畸蛋白(Otoferlin)表达的变化,从而推测其在耳鸣的发生中可能的作用机制。方法将24只Wistar大鼠随机分成空白对照组、生理盐水组和耳鸣模型组3组,每组8只。通过腹腔注射水杨酸钠制造耳鸣模型并用...目的研究耳鸣大鼠耳蜗基底膜内毛细胞耳畸蛋白(Otoferlin)表达的变化,从而推测其在耳鸣的发生中可能的作用机制。方法将24只Wistar大鼠随机分成空白对照组、生理盐水组和耳鸣模型组3组,每组8只。通过腹腔注射水杨酸钠制造耳鸣模型并用饮水抑制法对耳鸣模型进行验证。造模成功后,运用实时荧光定量RT-PCR方法及Western blot方法检测Otoferlin的表达情况。结果采用水杨酸钠注射方法成功建立了大鼠耳鸣模型;与空白对照组相比,生理盐水组大鼠Otoferlin m RNA和蛋白表达无明显变化(P>0.05),而耳鸣模型组Otoferlin m RNA及蛋白表达水平显著升高(P<0.05)。结论耳鸣大鼠耳蜗基底膜内毛细胞Otoferlin m RNA及蛋白质合成改变,可能是耳鸣产生的机制之一。展开更多
Otoferlin,an integral membrane protein implicated in a late stage of exocytosis,has been reported to play a critical role in hearing although the underlying mechanisms remain elusive.However,its widespread tissue dist...Otoferlin,an integral membrane protein implicated in a late stage of exocytosis,has been reported to play a critical role in hearing although the underlying mechanisms remain elusive.However,its widespread tissue distribution infers a more ubiquitous role in synaptic vesicle trafficking.Glutamate,an excitatory neurotransmitter,is converted to its inhibitory counterpart,g-aminobutyric acid(GABA),by L-glutamic acid decarboxylase(GAD),which exists in soluble(GAD67)and membrane-bound(GAD65)forms.For the first time,we have revealed a close association between otoferlin and GAD65 in both HEK293 and neuronal cells,including SHSY5Y neuroblastoma and primary rat hippocampus cells,showing a direct interaction between GAD65 and otoferlin’s C2 domains.In primary rat hippocampus cells,otoferlin and GAD65 co-localized in a punctate pattern within the cell body,as well as in the axon along the path of vesicular traffic.Significantly,GABA is virtually abolished in otoferlin-knockdown neuronal cells whereas otoferlin overexpression markedly increases endogenous GABA.GABA attenuation in otoferlin-knockdown primary cells is correlated with diminished L-type calcium current.This previously unknown and close correlation demonstrates that otoferlin,through GAD65,modulates GABAergic activity.The discovery of otoferlin–GAD65 functional coupling provides a newavenue for understanding the molecular mechanism by which otoferlin functions in neurological pathways.展开更多
文摘目的研究耳鸣大鼠耳蜗基底膜内毛细胞耳畸蛋白(Otoferlin)表达的变化,从而推测其在耳鸣的发生中可能的作用机制。方法将24只Wistar大鼠随机分成空白对照组、生理盐水组和耳鸣模型组3组,每组8只。通过腹腔注射水杨酸钠制造耳鸣模型并用饮水抑制法对耳鸣模型进行验证。造模成功后,运用实时荧光定量RT-PCR方法及Western blot方法检测Otoferlin的表达情况。结果采用水杨酸钠注射方法成功建立了大鼠耳鸣模型;与空白对照组相比,生理盐水组大鼠Otoferlin m RNA和蛋白表达无明显变化(P>0.05),而耳鸣模型组Otoferlin m RNA及蛋白表达水平显著升高(P<0.05)。结论耳鸣大鼠耳蜗基底膜内毛细胞Otoferlin m RNA及蛋白质合成改变,可能是耳鸣产生的机制之一。
基金This work was supported by funding from Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada.Z.J.is a Canada Research Chair in Structural Biology.
文摘Otoferlin,an integral membrane protein implicated in a late stage of exocytosis,has been reported to play a critical role in hearing although the underlying mechanisms remain elusive.However,its widespread tissue distribution infers a more ubiquitous role in synaptic vesicle trafficking.Glutamate,an excitatory neurotransmitter,is converted to its inhibitory counterpart,g-aminobutyric acid(GABA),by L-glutamic acid decarboxylase(GAD),which exists in soluble(GAD67)and membrane-bound(GAD65)forms.For the first time,we have revealed a close association between otoferlin and GAD65 in both HEK293 and neuronal cells,including SHSY5Y neuroblastoma and primary rat hippocampus cells,showing a direct interaction between GAD65 and otoferlin’s C2 domains.In primary rat hippocampus cells,otoferlin and GAD65 co-localized in a punctate pattern within the cell body,as well as in the axon along the path of vesicular traffic.Significantly,GABA is virtually abolished in otoferlin-knockdown neuronal cells whereas otoferlin overexpression markedly increases endogenous GABA.GABA attenuation in otoferlin-knockdown primary cells is correlated with diminished L-type calcium current.This previously unknown and close correlation demonstrates that otoferlin,through GAD65,modulates GABAergic activity.The discovery of otoferlin–GAD65 functional coupling provides a newavenue for understanding the molecular mechanism by which otoferlin functions in neurological pathways.
