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.