Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

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Isomerase regulation of potassium channel trafficking and function. To identify Kv4.2 binding proteins, 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 downin 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, we 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 isomeraseplays a role in regulating Kv4.2 function. To further study the physiological function of isomerase and Kv4.2 channel, we generated a knockin mousein whichthe isomerase binding site is specifically abolished using Crispr-Cas9 techniques. These mice are viable and appear normal. We are now working on learning and memory related behaviors including novel objective recognition, Morris Water Maze and fear conditioning. DPP6 deletion leads to memory impairments in mice DPP6 plays an important role as an auxiliary subunit of Kv4, and the DPP6 gene has been associatedwith neurodevelopmental disorders in human. We found that DPP6 deletion leads to behavioral impairments in recognition, spatial learning and memory with less body and brain weights in adult mice. In addition, we found synaptic structure deficits in neuronal synapses in the hippocampal CA1 region. DPP6 induces synapse formation and regulates stabilization by live image and co-culture assay in neurons. Loss of regulated Cav2.3 expression in a mouse model of Fragile X Syndrome Fragile X syndrome (FXS) is a severe form of intellectual disability in humans that arises from the loss of the fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates translation downstream of group I metabotropic glutamate receptors (GpI mGluRs). Loss of FMRP leads to enhanced calcium spiking and neuronal excitability, thus Erin Gray sought to explore the possibility that FMRP regulates expression of the dendritic voltage gated calcium channel Cav2.3. In our initial studies, we showed that loss of FMRP in mice alters Cav2.3 mRNA levels in both the cortex and hippocampus. Considering that FMRP is an mRNA binding protein, we performed an RNA immunoprecipitation (RIP) assay and found that immunoprecipitation of FMRP from brain tissue also pulls down Cav2.3 mRNA. These results suggest that FMRP directly binds Cav2.3 mRNA to regulate its abundance in neurons. FMRP-dependent regulation of Cav2.3 mRNA appears to impact Cav2.3 channel expression as our previous data showed that FMRP KO mice have enhanced expression of Cav2.3 in cortical regions and in the hippocampus. This increase in Cav2.3 expression impacts neuronal physiology; Cav2.3 currents are enhanced in cultured hippocampal neurons isolated from FMRP KO mice compared to wild-type animals. Thus, it appears that FMRP binding normally represses Cav2.3 translation under basal conditions and loss of FMRP leads to an increase of Cav2.3 protein in the membrane. We are also investigating the possibility that repression of Cav2.3 expression by FMRP can be regulated by upstream activity of GpI mGluRs. In support of this idea, our previous data showed that stimulation of GpI mGluRs increases local synaptic translation and expression of Cav2.3 in WT neurons but not in neurons lacking FMRP. To determine if Cav2.3 expression downstream of GpI mGluR activation has a role in synaptic plasticity, we induced long term synaptic depression by stimulating GpI mGluRs (mGluR-LTD) in hippocampal slices from wildtype and Cav2.3 KO mice. Strikingly, we found that hippocampal slices from Cav2.3 KO mice lacked mGluR-LTD, demonstrating the importance of Cav2.3 in mGluR-dependent synaptic plasticity. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and following activity and this may be critical for mGluR-dependent forms of plasticity. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies. FMRP interacts with Cav2.3 mRNA Ying Liu has shown that Cav2.3 mRNA levels were altered in FMRP KO neurons and Cav2.3 protein levels were significantly enhanced in the FMRP KO. To study if Cav2.3 mRNA is one of the targets of FMRP, we performed RNA immunoprecipitation. Our data showed that FMRP interacts with Cav2.3 mRNA in transfected HEK293 cells and in mouse cortex and hippocampus. Our results suggest that FMRP binds to Cav2.3 mRNA directly or indirectly to repress Cav2.3 translation and regulates neuronal excitability. In related work, Jon Murphy is examining whether the dendritic FMRP regulates mRNA trafficking and protein expression of CaV2.3 and KV4.2 in the dendrites of hippocampal neurons using the FMRP KO mouse. Recent progress has centered primarily on analysis of mRNA localization and regulation of total protein translation in neuronal dendrites of WT and FMRP knockout mouse neurons. Using fluorescence in situ hybridization to detect mRNAs for CaV2.3 and CaMKII in neurons, we have found that CaMKII mRNA, a known dendritically synthesized protein has increased abundance throughout the dendritic arbor of FMRP KO mice. FMRP is known to inhibit CaMKII translation through direct binding of CaMKII mRNA suggesting either CaMKII mRNA is more highly transcribed in FMRP knockout mice, it is no longer sequestered in mRNA-protein complexes where in situ hybridization is inhibited, or both. Conversely, CaV2.3 mRNA signals are low throughout dendrites in both WT and FMRP KO neurons. Studies of Kv4.2 mRNA localization in WT and FMRP knockout neurons are ongoing. Is Cav2.3 the source of calcium regulation of Kv4.2? 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. Jakob Gutzmann compared wild type with Cav2.3 knock-out neurons, and saw a significant reduction in somatic . Were now analyzing IA from somatic and dendritic attached patch recordings, to investigate the potential influence of a loss of Cav2.3 on the distinct functional gradient that IA shows along the apical dendrites of wild type CA1 pyramidal neurons. We continue to characterize Cav2.3 knockout animals, to better understand the interplay between calcium and potassium that shape CA1 pyramidal cell electrical behavior.