Laura A. Schrader

Calcium–Calmodulin-Dependent Kinase II Modulates Kv4.2 Channel Expression and Upregulates Neuronal A-Type Potassium Currents

Calcium–calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K+ channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K+ current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K+ channels.

Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase

Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 μm) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser465 (minor site) and three amino acid residues Ser568, Ser569, and Ser570 (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.

The role of calsenilin/DREAM/KChIP3 in contextual fear conditioning

Potassium channel interacting proteins (KChIPs) are members of a family of calcium binding proteins that interact with Kv4 potassium (K(+)) channel primary subunits and also act as transcription factors. The Kv4 subunit is a primary K(+) channel pore-forming subunit, which contributes to the somatic and dendritic A-type currents throughout the nervous system. These A-type currents play a key role in the regulation of neuronal excitability and dendritic processing of incoming synaptic information. KChIP3 is also known as calsenilin and as the transcription factor, downstream regulatory element antagonist modulator (DREAM), which regulates a number of genes including prodynorphin. KChIP3 and Kv4 primary channel subunits are highly expressed in hippocampus, an area of the brain important for learning and memory. Through its various functions, KChIP3 may play a role in the regulation of synaptic plasticity and learning and memory. We evaluated the role of KChIP3 in a hippocampus-dependent memory task, contextual fear conditioning. Male KChIP3 knockout (KO) mice showed significantly enhanced memory 24 hours after training as measured by percent freezing. In addition, we found that membrane association and interaction with Kv4.2 of KChIP3 protein was significantly decreased and nuclear KChIP3 expression was increased six hours after the fear conditioning training paradigm with no significant change in KChIP3 mRNA. In addition, prodynorphin mRNA expression was significantly decreased six hours after fear conditioning training in wild-type (WT) but not in KO animals. These data suggest a role for regulation of gene expression by KChIP3/DREAM/calsenilin in consolidation of contextual fear conditioning memories.

Local Glutamatergic and GABAergic Synaptic Circuits and Metabotropic Glutamate Receptors in the Hypothalamic Paraventricular and Supraoptic Nuclei

Burst generation and synchronization in the hypothalamus is controlled by a combination of the intrinsic membrane conductances and the synaptic organization of the hypothalamic neurons. The projected synaptic inputs as well as the local synaptic organization of the cells play a crucial role in generating patterned electrical activity and synchronizing activity among individual cells of a given population of hypothalamic neurons. The majority of the synaptic inputs to the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) are mediated by glutamate and γ-aminobutyric acid (GABA)1,2, and a substantial proportion of these inputs appear to derive from local sources3,4,5. Given this, and the fact that the bursting patterns of neuroendocrine cells of the hypothalamus are sensitive to subtle changes in membrane potential and excitability, we hypothesize that local synaptic circuits in the hypothalamus and neuromodulatory inputs to hypothalamic neurons may be important for hypothalamic burst generation. To test these hypotheses, we studied the intrahypothalamic glutamatergic and GABAergic projections to neurons of the PVN and SON, and the neuromodulatory actions of glutamate via metabotropic glutamate receptors in these cells.

REGULATION OF HISTONE ACETYLATION IN THE HIPPOCAMPUS OF CHRONICALLY STRESSED RATS: A POTENTIAL ROLE OF SIRTUINS

The hippocampus is a brain region that is particularly susceptible to structural and functional changes in response to chronic stress. Recent literature has focused on changes in gene transcription mediated by post-translational modifications of histones in response to stressful stimuli. Chronic variable stress (CVS) is a rodent model that mimics certain symptoms of depression in humans. Given that stress exhibits distinct effects on the cells of the sub-regions of the hippocampus, we investigated changes in histone acetylation in the CA1, CA3, and dentate gyrus (DG) of the hippocampus in response to CVS. Western blotting revealed a significant decrease in acetylation of histone 4 (H4) at Lys12 in CA3 and DG of CVS animals compared to control animals. Furthermore, phospho-acetyl H3 (Lys9/Ser10) was also decreased in the CA3 and DG regions of the hippocampus of CVS animals. In addition, since histone deacetylases (HDACs) contribute to the acetylation state of histones, we investigated the effects of two HDAC inhibitors, sodium butyrate, a class I and II global HDAC inhibitor, and sirtinol, a class III sirtuin inhibitor, on acetylation of histone 3 (H3) and H4. Application of HDAC inhibitors to hippocampus slices from control and CVS animals revealed increased histone acetylation in CVS animals, suggesting that levels of histone deacetylation by HDACs were higher in the CVS animals compared to control animals. Interestingly, histone acetylation in response to sirtinol was selectively increased in the slices from the CVS animals, with very little effect of sirtuin inhibitors in slices from control animals. In addition, sirtuin activity was increased specifically in CA3 and DG of CVS animals. These results suggest a complex and regionally-specific pattern of changes in histone acetylation within the hippocampus which may contribute to stress-induced pathology.

The other half of Hebb: K+ channels and the regulation of neuronal excitability in the hippocampus.

Historically, much attention has focused on the mechanisms of activity-dependent plasticity since the description of long-term potentiation by Bliss and Lomo in the early 1970s, while extrasynaptic changes have received much less interest. However, recent work has concentrated on the role of back-propagating action potentials in hippocampal dendrites in synaptic plasticity. In this review, we focus on the modulation of back-propagating action potentials by K+ currents in the dendrites of hippocampal cells. We described the primary K+-channel subunits and their interacting subunits that most likely contribute to these currents, and how these sites can be regulated by phosphorylation and other mechanisms. In conclusion, we provide a model for an alternative form of coincidence detection through K+ channels in the hippocampus.

Kv4.2 is a locus for PKC and ERK/MAPK cross-talk

Transient outward K+ currents are particularly important for the regulation of membrane excitability of neurons and repolarization of action potentials in cardiac myocytes. These currents are modulated by PKC (protein kinase C) activation, and the K+- channel subunit Kv4.2 is a major contributor to these currents. Furthermore, the current recorded from Kv4.2 channels expressed in oocytes is reduced by PKC activation. The mechanism underlying PKC regulation of Kv4.2 currents is unknown. In the present study, we determined that PKC directly phosphorylates the Kv4.2 channel protein. In vitro phosphorylation of the intracellular N- and C-termini of Kv4.2 GST (glutathione transferase) tagged fusion protein revealed that the C-terminal of Kv4.2 was phosphorylated by PKC, whereas the N-terminal was not. Amino acid mapping and site-directed mutagenesis revealed that the phosphorylated residues on the Kv4.2 C-terminal were Ser447 and Ser537. A phospho-site-specific antibody showed that phosphorylation at the Ser537 site was increased in the hippocampus in response to PKC activation. Surface biotinylation experiments revealed that mutation to alanine of both Ser447 and Ser537 in order to block phosphorylation at both of the PKC sites increased surface expression compared with wild-type Kv4.2. Electrophysiological recordings of the wild-type and both the alanine and aspartate mutant Kv4.2 channels expressed with KChIP3 (Kv4 channel-interacting protein 3) revealed no significant difference in the half-activation or half-inactivation voltage of the channel. Interestingly, Ser537 lies within a possible ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) recognition (docking) domain in the Kv4.2 C-terminal sequence. We found that phosphorylation of Kv4.2 by PKC enhanced ERK phosphorylation of the channel in vitro. These findings suggest the possibility that Kv4.2 is a locus for PKC and ERK cross-talk.

Role of gonadal hormones in anxiety and fear memory formation and inhibition in male mice

Recent research investigating Pavlovian fear conditioning and fear extinction has elucidated the neurocircuitry involved in acquisition and inhibition of fear responses. Modulatory factors that may underlie individual differences in fear acquisition and inhibition, however, are not well understood. Testosterone is known to affect anxiety-like behavior and cognitive processing. In this study, we hypothesized that castration would increase anxiety and reduce memory for contextual fear conditioning in an age-dependent manner. In addition, castration would reduce the rate of extinction to context, as high levels of testosterone correlate with reduced PTSD-like symptoms. We compared behaviors in male mice that were castrated at one of two different time points, either before puberty (at 4 weeks) or after puberty (at 10 weeks) to sham-operated control mice. The behaviors investigated included: anxiety, cued and contextual fear conditioning, and extinction of the fear memory. An interaction of hormone status and age and a significant effect of age were measured in the elevated plus maze, a measure of anxiety. Castration caused a significant reduction of contextual fear memory, but no effect on cued fear memory. There was no significant effect of castration on extinction. Interestingly, a significant effect of age of the mouse at the time of testing was observed on extinction. These results suggest that endogenous androgens during puberty are important for anxiety and fear memory formation. In addition, these results define a late post-adolescent developmental time point for changes in anxiety and fear extinction.

Cage mate separation in pair-housed male rats evokes an acute stress corticosterone response

Corticosterone (CORT) release from the adrenal glands in response to acutely stressful stimuli is well-characterized, however several non-experimental, environmental stressors can also engender a CORT response. The aim of this study was to investigate an acute activation of the HPA axis in pair-housed animals in response to separation. We observed a rapid significant increase in CORT in the animal remaining in the home cage following cage mate removal, that was not caused by cage opening and transient removal of cage mate. In addition, we examined this response in both control, non-stressed animals and in animals subjected to chronic variable stress (CVS) and found that although basal levels of CORT differed between control and CVS animals, there was no significant difference in the acute CORT levels between the control and CVS animals after separation, indicating that this environmental event is perceived as acutely stressful in both conditions. Furthermore, we examined the time course of CORT activation and found that CORT levels rapidly rise within minutes of separation peaking at 15 min and returning to baseline by 90 min. The results of this study demonstrate that separation can induce an acute stress response in the remaining cage mate measured by increased CORT and should be considered in molecular, behavioral, and electrophysiological studies.

A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node.

In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. The genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-like myocardial identity in the PV, is lacking in the SAN. Here we present evidence that in mice Shox2 antagonizes the transcriptional output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5+ domain within the SAN to determine cell fate. Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of the pacemaker program. Explanted Shox2+ cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2− cells. Shox2 deletion led to Hcn4 ablation in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid bodies. We found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome-wide co-occupancy of Shox2, Nkx2-5 and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development. Summary: Antagonism between Shox2 and Nkx2-5 in the cardiac venous pole of mouse embryos regulates cell fate, morphogenesis and the distinction between pacemaker cells and working myocardium.