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 M) 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 carboxylterminal 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: Ser 465 (minor site) and three amino acid residues Ser 568 , Ser 569 , and Ser 570(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 small conductance, Ca 2ϩ -activated K ϩ (SK) 2 channels are found in both neuronal and non-neuronal tissue (1). Functionally, the SK channels are best characterized in the central nervous system. Three genes encode the SK channel subunits (SK1, SK2, and SK3) in mammalian brain (2). SK channels are blocked by the bee venom toxin, apamin, although SK1 is slightly less sensitive than SK2 and SK3 (2-4). In neurons throughout the nervous system, the apamin-sensitive SK channels modulate firing frequency by contribution to the afterhyperpolarization (AHP) that follows a single or a train of action potentials (5-7). SK2 is thought to specifically underlie the medium AHP current (I mAHP ) in hippocampal CA1 pyramidal cells (8). The I mAHP is Ca 2ϩ -dependent with a time constant of 100 -250 ms and sensitivity to apamin (9 -11).The apamin-sensitive I mAHP modulates instantaneous firing rates and sets the interspike duration in action potential trains to produce spike frequency adaptation (12). In addition, SK channels are localized to the dendrites of pyramidal cells in hippocampal area CA1 and pyramidal neurons of the lateral amygdala where they function to shape synaptic potentials and limit Ca 2ϩ influx through NMDA receptors (13, 14) as well as plateau potentials evoked by exogenous glutamate application (15). Other studies have linked overexpression of SK2 and enhancement of the I mAHP in hippocampus with neuroprotection (16), attenuation of hippocampal LTP, and memory deficits (16, 17). These functions have major implications for a rol...

Tobacco addiction is characterized by a negative mood state upon smoking cessation and relapse after periods of abstinence. Clinical studies indicate that negative mood states lead to craving and relapse. The partial a4/a6/b2* nicotinic acetylcholine receptor (nAChR) agonists varenicline and cytisine are widely used as smoking cessation treatments. Varenicline has been approved in the United States for smoking cessation and cytisine is used in Eastern European countries. Despite the widespread use of these compounds, very little is known about their effects on mood states. These studies investigated the effects of varenicline, cytisine, and the cytisine-derivative 3-(pyridin-3 0 -yl)-cytisine (3-pyr-Cyt) on brain reward function in nicotine-naive and nicotine-withdrawing rats. The cytisine-derivative 3-pyr-Cyt is a very weak a4b2* nAChR partial agonist and like cytisine and varenicline has antidepressant-like effects in animal models. The intracranial self-stimulation (ICSS) procedure was used to investigate the effects of these compounds on brain reward function. Elevations in ICSS thresholds reflect a dysphoric state and a lowering of thresholds is indicative of a potentiation of brain reward function. It was shown that acute administration of nicotine and varenicline lowered ICSS thresholds. Acute administration of cytisine or 3-pyr-Cyt did not affect ICSS thresholds. Discontinuation of chronic, 14 days, nicotine administration led to elevations in ICSS thresholds that lasted for about 2 days. Varenicline and cytisine, but not 3-pyr-Cyt, diminished the nicotine withdrawal-induced elevations in ICSS thresholds. In conclusion, these studies indicate that varenicline and cytisine diminish the dysphoric-like state associated with nicotine withdrawal and may thereby prevent relapse to smoking in humans.

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.Theories of Hebbian-type synaptic plasticity propose that strengthening of synaptic connections is dependent on coincident activity in pre-and postsynaptic neurons. Research has focused on regulation of transmitter release and receptor function at the synapse; however, changes in neuronal excitability during learning processes have received less attention. Modulation of postsynaptic excitability, via regulation of ion channels, is another possible mechanism for modification of the threshold for induction of synaptic plasticity. Transient outward or A-type currents are generally responsible for regulation of both neuronal excitability and the inter-spike interval in neurons in both vertebrate and invertebrate systems. A-type currents control action potential broadening, back-propagating action potentials in CA1 pyramidal cells, and are regulated by neuromodulators and long-term potentiation (LTP) (Hoffman et

Rationale-Tobacco withdrawal is characterized by a negative mood state and relatively mild somatic symptoms. Increased noradrenergic transmission has been reported to play an important role in opioid withdrawal, but little is known about the role of noradrenergic transmission in nicotine withdrawal.Objectives-The aim of these experiments was to investigate the effects of prazosin, clonidine, and propranolol on the negative mood state and somatic signs associated with nicotine withdrawal in rats.Methods-A discrete-trial intracranial self-stimulation procedure was used to assess the negative affective state of nicotine withdrawal. Elevations in brain reward thresholds are indicative of a deficit in brain reward function.Results-In all the experiments, the nicotinic acetylcholine receptor antagonist mecamylamine (3 mg/kg) elevated the brain reward thresholds of the nicotine-treated rats and did not affect those of the control rats. The α1-adrenergic receptor antagonist prazosin (0.0625 and 0.125 mg/kg) dosedependently attenuated the elevations in brain reward thresholds associated with precipitated nicotine withdrawal. The α2-adrenergic receptor agonist clonidine (10-40 μg/kg) and the nonselective β-adrenergic receptor antagonist propranolol (2.5-10 mg/kg) did not attenuate the elevations in brain reward thresholds associated with nicotine withdrawal. Mecamylamine (2 mg/ kg) induced more somatic signs in the nicotine-treated rats than in the control rats. Clonidine and propranolol, but not prazosin, decreased the total number of somatic signs associated with nicotine withdrawal.Conclusion-Blockade of α1-adrenergic receptors attenuates the deficit in brain reward function associated with nicotine withdrawal. Antagonism of β-adrenergic receptors or stimulation of α2-adrenergic receptors attenuates the somatic symptoms of nicotine withdrawal.