Kv4.2 control of firing patterns in hippocampal CA1 pyramidal neurons. Although recent molecular cloning studies have found several families of voltage-gated K channel genes expressed in the mammalian brain, at present, information regarding the relationship between the protein products of these genes and their various neuronal functions is lacking. Our lab has used a combination of molecular, electrophysiological, imaging techniques to show that the voltage gated potassium channel subunit Kv4.2 controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. More recently, we examined the role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after synapse-specific long-term potentiation (LTP) induction (potentially a cellular form of memory). In electrophysiological recordings we found that LTP induced a potentiation of excitability which was accompanied by a two-phased change in A-type K+ channel activity recorded in nucleated patches from organotypic slices of rat hippocampus. Induction of LTP resulted in an immediate but short lasting hyperpolarization of the voltage-dependence of steady-state A-type K+ channel inactivation along with a progressive, long-lasting decrease in peak A-current density. Blocking clathrin-mediated endocytosis prevented the A-current decrease and most measures of intrinsic plasticity. These results suggest that two temporally distinct but overlapping mechanisms of A-channel downregulation together contribute to the plasticity of intrinsic excitability. This change in intrinsic plasticity resulted in a global enhancement of EPSP-spike coupling. Kv4.2 trafficking in CA1 pyramidal neuron dendrites. Using a modified Sindbis virus system to overexpress EGFP-labeled Kv4.2 (Kv4.2g) in cultured hippocampal neurons, we found that the EGFP fluorescence in dendritic spines of Kv4.2g expressing neurons appeared brighter than that from the adjacent dendritic shaft. The ratio of spine head to dendritic shaft fluorescence in Kv4.2g expressing neurons was approximately two-fold greater than in neurons expressing EGFP. Kv4.2 expression in spines was further shown using electronmicroscopy in collaboration with Ron Petralia here at the NIH. We found stimulation (AMPA) to result in an activity-dependent redistribution of Kv4.2g away from spines to the dendritic shaft and a punctate accumulation of Kv4.2g within the soma. This AMPA-induced redistribution of Kv4.2g occurred within 15 min of stimulation and was reversible, indicating that the treatment was not excitotoxic. Controls showed that this activity-dependent Kv4.2 internalization occurs natively and is not an artifact of overexpression. More recently we examined the role of protein kinase A (PKA) in Kv4.2 activity-dependent trafficking. In hippocampal neurons, PKA activation with forskolin or 8-Br-cAMP induced Kv4.2 internalization from dendritic spines, whereas PKA inhibition prevented AMPA-induced internalization. Furthermore, introduction of a point mutation at the C-terminal PKA phosphorylation site of Kv4.2 (S552A) prevented the AMPA-induced internalization of Kv4.2. We continue to investigate the mechanisms of Kv4.2 expression and trafficking. Role of voltage-gated potassium channels in synaptic plasticity. Using the Sindbis virus system to infect organotypic slice cultures with Kv4.2g and Kv4.2g(W362F), we have begun investigating the role of Kv4.2 in LTP using a depolarization pairing protocol. For the first 10 min after pairing, potentiation is similar in all three groups, achieving 100% increase in EPSC size. After this period, however, Kv4.2 overexpressing neurons fail to maintain potentiation such that EPSC size is back to baseline after 25 min. Conversely, expression of Kv4.2g(W362F) results in a potentiation, which reaches a greater level 40-50 min after initiation, compared to controls. These data indicate that Kv4.2 channels modulate the degree of LTP by influencing the induction of a late phase of potentiation or by controlling the mechanisms of LTP maintenance. We are currently characterizing the mechanisms of Kv4.2's effect on LTP. The effect of changes in IA on the ability to induce subsequent synaptic plasticity (meta-plasticity) has not yet been investigated. We have found that altering functional Kv4.2 expression level leads to a rapid, bidirectional remodeling of CA1 synapses. Neurons exhibiting enhanced IA showed a decrease in relative synaptic NR2B/NR2A subunit composition and, as noted above do not exhibit LTP. Conversely, reducing IA by expression of a Kv4.2 dominant negative or through genomic knockout of Kv4.2 led to an increased fraction of synaptic NR2B/NR2A and enhanced LTP. Bidirectional synaptic remodeling was mimicked in experiments manipulating intracellular Ca2+ and dependent on spontaneous activation of NMDA receptors and active CaMKII. Our data suggest that A-type K+ channels are an integral part of a synaptic complex that regulates Ca2+ signaling through spontaneous NMDAR activation to control synaptic NMDAR expression and plasticity. Functional role of Kv4.2 auxiliary subunits Kv4 currents in heterologous cells display slower kinetics of inactivation and recovery from inactivation than that typically recorded in neurons. Reconciliation of these results came with the finding of neuron specific auxiliary subunit expression of KChIPs and DPLs. In hippocampal CA1 pyramidal neurons, DPPX (also called DPP6) is the prominent DPL family member. To investigate the physiological role of DPPX in CA1 neurons, in collaboration with Dr. Bernardo Rudy, we developed short-interfering RNAs (siRNAs) to suppress the expression of all DPPX variants. The reduction of DPPX proteins in CHO cells transfected with DPPX siRNA (siDPPX) was more than 95% complete, as quantified by immunoblotting. To investigate whether DPPX alters the kinetics of A-type currents in a native system, we conducted voltage-clamp experiments in outside-out patches from CA1 pyramidal neurons in hippocampal organotypic slices infected with siDPPX. After allowing 2-3 days post-infection for DPPX knockdown we found, in accordance with heterologous studies, that siDPPX results in a delayed recovery from inactivation, slowed time-to-peak, and rightward shifted the steady-state inactivation and activation curves. To determine the physiological effect of kinetic modifications by siDPPX, we carried out current-clamp experiments in siDPPX expressing cells. Compared to negative control siRNA neurons, siDPPX-infected neurons exhibited delayed time to AP onset, increased AP threshold, decreased firing frequency, increased AP half-width and reduced fast AHP amplitudes. Thus siDPPX had contrasting effects, decreasing excitability subthreshold and increasing excitability suprathreshold. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitably. We are currently investigating the dendritic role of DPPX in knockout mice and have developed siRNAs targeting members of the KChIP family of auxiliary subunits for similar studies.
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