Expression of Ion Channels in the Auditory System

摘要 脊髓小脑型共济失调13型(SCA13)是由KCNC3基因突变引起的，KCNC3基因编码KV3.1 电压依赖性钾通道。这种情况会导致运动异常和无法定位 太空中的声音。Kv3.3在小脑和听性脑干核团中高表达，包括 位于斜方体内侧核(MNTB)的突触前终末的胞体。Kv3.3不同于 具有扩展的C-末端细胞质结构域的其他密切相关的通道 胞质信号分子，包括Tank Binding Kinase 1(TBK1)。这种酶使Kv3.3与 HAX-1，一种细胞存活蛋白，当与通道结合时，触发致密肌动蛋白的形成 质膜下的细胞骨架。这一过程在Kv3.3突变体(G592R Kv3.3)中受损 导致迟发性SCA13。这种突变过度刺激了TBK1的活性，但阻止了通道触发 肌动蛋白成核。该方案中的实验将验证正常所需的TBK1的假设 突触小泡的突触传递和内吞作用，因为它是 Kv3.3与底层肌动蛋白细胞骨架之间的相互作用。膜片钳研究将结合 用图像和EM免疫显微镜检测TBK1抑制或操纵对TBK1活性的影响 Kv3.3-/-和G592R野生型Kv3.3-/-和G592R的细胞系和持有的末端的花萼中的遗传学方法 Kv3.3动物。将使用磷酸特异性抗体来确定TBK1的活性是否因刺激而改变 通过在活体内对完整动物进行声刺激，在脑切片制备中对听神经元进行了研究。最后， 将通过突变研究确定将TBK1偶联到Kv3.3/HAX-1复合体所需的特定结构域 并通过蛋白质组学方法确定通道复合体中TBK1的磷酸化位点。我们的发现 将为正常和病理条件下突触传递的调节提供新的见解 并将为脊髓小脑性共济失调13型等疾病的治疗创造新的靶点 影响中枢听觉处理的条件。

ABSTRACT Spinocerebellar Ataxia Type 13 (SCA13) is caused by mutations in KCNC3, the gene that encodes Kv3.1 voltage-dependent potassium channels. This condition results in motor abnormalities and the inability to locate sounds in space. Kv3.3 is highly expressed in the cerebellum and in auditory brainstem nuclei, including the calyx of Held presynaptic terminals in the medial nucleus of the trapezoid body (MNTB). Kv3.3 differs from other closely-related channels in having an extended C-terminal cytoplasmic domain that recruits several cytoplasmic signaling molecules, including Tank Binding Kinase 1 (TBK1). This enzyme keeps Kv3.3 bound to Hax-1, a cell survival protein that, when bound to the channel, triggers the formation of a dense actin cytoskeleton under the plasma membrane. This process is impaired in a Kv3.3 mutant (G592R Kv3.3) that causes late-onset SCA13. This mutation overstimulates TBK1 activity but prevents the channel from triggering actin nucleation. The experiments in this proposal will test the hypothesis that TBK1 is required for normal synaptic transmission and endocytosis of synaptic vesicles because it is a physiological regulator of the interactions between the Kv3.3 and the underlying actin cytoskeleton. Patch clamp studies will be combined with imaging and EM immunomicroscopy to test the effects of TBK1 inhibition or manipulation of TBK1 activity with genetic approaches in cell lines and in the calyx of Held terminals from wild type, Kv3.3-/- and G592R Kv3.3 animals . Phospho-specific antibodies will be used to determine if TBK1 activity is altered by stimulation of auditory neurons in brain slice preparations and by acoustic stimulation of intact animals in vivo. Finally, the specific domains required to couple TBK1 to the Kv3.3/Hax-1 complex will be defined by mutagenesis studies and by proteomic approaches that identify phosphorylation sites for TBK1 in the channel complex. Our findings will provide novel insights into the regulation of synaptic transmission in normal and pathological conditions and will generate new targets for the treatment of diseases such as Spinocerebellar Ataxia Type 13 and other conditions that affect central auditory processing.