Regulation of Surface Localization of the Small Conductance Ca2+-activated Potassium Channel, Sk2, through Direct Phosphorylation by cAMP-dependent Protein Kinase

Abstract

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...

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats

“…The half-life of apamin is ϳ2 h, which is fairly consistent with results showing that apamin elicited relatively long-lasting response in the present study (19). It is well known that phosphorylation of K ϩ channels can alter neuronal excitability, and several SK channel residues have been identified as being susceptible to kinase phosphorylation, resulting in modulation of channel kinetics and changes in cellular localizations (1,42). Additionally, receptor-mediated endocytosis of apamin in liver cells also suggests a slow degradation time of the toxin once bound to the apamin receptor (49).…”

SK Channels Provide a Novel Mechanism for the Control of Frequency Tuning in Electrosensory Neurons

“…We therefore hypothesize that, when the fish engages in low speed scanning, enhancing the detection of the weak low-frequency signals could occur through the inhibition of SK channels, which would cause an upregulation of burst firing. This could be accomplished through a number of second messenger systems (Nicoll, 1988;Grunnet et al, 2004;Ren et al, 2006). Preliminary results suggest that serotonergic (5-HT) and cholinergic (Maler et al, 1981;Phan and Maler, 1983) input to the ELL can regulate bursting (Ellis, unpublished observations), although direct links to SK channels have not yet been established.…”

Distinct Effects of Repeated Restraint Stress on Basolateral Amygdala Neuronal Membrane Properties in Resilient Adolescent and Adult Rats

“…The amygdala circuits that regulate fear and anxiety are hypothesized to be an important target of some of these genetic differences (Hariri et al, 2002;Smolka et al, 2005;Jovanovic and Ressler, 2010;Mozhui et al, 2010;Alexander et al, 2012;Hermann et al, 2012;White et al, 2012;Rogers et al, 2013;Volman et al, 2013). Many of these transmitter systems listed above directly or indirectly modulate the same signaling cascades that modify SK channel function (Pedarzani and Storm, 1995;Pedarzani et al, 1998;Lee et al, 2003;Bildl et al, 2004;Ren et al, 2006;Maingret et al, 2008), leading to changes in the excitability of amygdala neurons.…”

“…Activation of badrenergic receptors (Faber et al, 2008), activation of PKA (Ren et al, 2006), and neuronal activity (Lin et al, 2010) all modify the expression of SK channel subunits. Perhaps, the differences in the effects of repeated stress on these factors in adult and adolescent neurons between the BA and LA contribute to these age-and nucleus-specific differences.…”