Isomerase regulation of potassium channel trafficking and function. Kv4.2 channels are key determinants of dendritic excitability and integration, spike timing-dependent plasticity and long-term potentiation. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics. To identify Kv4.2 binding proteins, staff scientist Jiahua Hu employed a tandem affinity purification approach (TAP)to isolate the Kv4.2 protein complex from hippocampal neurons. Mass-spectrometry analysis identified known proteins such as KChIP family members and DPP6/10. The TAPMS assay also identified an isomerase as a binding partner of Kv4.2. The binding was confirmed by brain co-immunoprecipitation, co-expression in HEK293T cells, and peptide pull down in vitro. The isomerase binds to a specific Kv4.2 site, and the association is regulated by neuronal activity and seizure. To determine if and how the isomerase regulates the trafficking of Kv4.2, postbac Travis Tabor generated bungarotoxin binding site-tagged Kv4.2 at the second extracellular loop for visualizing Kv4.2 in live neurons. The bungarotoxin binding site-tagged Kv4.2 showed similar channel properties as WT Kv4.2 in biochemical and electrophysiological assays. The isomerizing activity may also regulate Kv4.2 binding to its auxiliary subunits. These data suggested that the isomerase plays a role in regulating Kv4.2 function. To further study the physiological function of isomerase and Kv4.2 channel, we generated a knockin (KI) mouse in which the isomerase binding site is specifically abolished using Crispr-Cas9 techniques. These mice are viable and appear normal. They showed normal initial learning and memory in Morris Water Maze. However, these Kv4.2 KI mice showed better reversal learning in Morris Water Maze than WT. In the operant reversal lever press, the KI mice displayed improved reversal learning. These data strongly support the idea that activity-dependent regulation of Kv4.2 plays an important role in cognitive flexibility. Cognitive flexibility is the ability to appropriately adjust ones behavior according to a changing environment. Cognitive flexibility is impaired in various neurodevelopmental disorders such as autism spectrum disorder (ASD). In light of these findings, postdoc Cole Malloy investigated how isomerization of Kv4.2 impacts neuronal function using whole-cell patch clamp electrophysiology in acute hippocampal slices. He utilized current-clamp recordings to detect alterations in action potential firing properties in the knock-in mice. Pharmacological manipulation of isomerase and kinase activity addressed the dependence of phosphorylation and conformation change induced by the isomerase to gain further insights into the molecular cascade impacting Kv4.2 function. Furthermore, given the behavioral results showing altered cognitive flexibility, experiments investigating synaptic function and plasticity in the KI mice are underway. Ca2+ regulation of potassium channel function. In addition to pore forming Kv4 subunits, native hippocampal A-type currents require non-conducting modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). Both KChIPs and DPLPs work in concert to enhance Kv4 function. Interestingly, in recent unpublished work we have identified a mechanism by which Kv4.2 current density is regulated by Ca2+ via R-type voltage gated Ca2+ channels (Cav2.3). Ca2+ regulation of Kv4.2 channels occurs despite an apparent lack of the structural determinants of the canonical Ca+-activated K+ channels. Proteomic and subcellular localization studies suggest, that Cav2.3-containing voltage gated calcium channels could be a potential calcium source for a modulatory effect on Kv4.2-mediated A-type K currents (IA) in CA1 hippocampal neurons. Postdoc Jakob Gutzmann established that apical dendrites from CA1 pyramidal neurons in Cav2.3 KO animals show a severe reduction in the typical somato-dendritic gradient of Kv4.2 current density, and used 2-photon calcium-imaging to investigate the functional consequence to this lack of dendritic potassium-current in the Cav2.3 KO animals. Further investigation revealed that individual action potentials showed profound changes in waveform. DPP6 plays a role in Brain Development, Function and Behavior DPP6 is well known as an auxiliary subunit of Kv4.2 which has been associated with numerous developmental and intellectual disorders and neuropsychiatric pathologies, especially ASD. We have reported previously, a novel role for DPP6 in regulating dendritic filopodia formation and stability, affecting synaptic development and function. This year we found DPP6 knockout mice are impaired in learning and memory. Results from the Morris water maze, T-maze, Objects spatial location, Novel Object Recognition and Cued and fear conditioning tasks showed that DPP6-KO mice exhibit slower learning and reduced memory performance. We continued to study DPP6-KO mice in behavioral tasks, and found that DPP6-KO mice are impaired in hippocampus dependent learning and memory and have lower brain weight (Lin et al 2018). To determine which regions effected the smaller brain size, we performed in vivo MRI to scan the live mouse brain, the results showed live DPP6-KO mice display significantly decreased volume specifically in the hippocampus and Cerebellum. Our findings indicate DPP6-loss drives microcephaly and learning and memory impairment in DPP6-KO mice, hallmarks of Alzheimers Disease. We continue to investigate DPP6 in neurodegeneration. Kv4.2 trafficking MD/PhD student Adriano Bellotti has discovered quantitative and qualitative differences in microtubule-based transport of Kv4.2 in axons versus dendrites. He characterized these differences by recording time series of over 500 neurites, and has validated an unexpected result using mathematical models of cargo transport. He developed a deterministic model that corroborates differences in cargo frequency and a stochastic model that validates differences in puncta speed, superdiffusivity, and frequency in axons vs dendrites.
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