SK2 channels are neuroprotective for ischemia-induced neuronal cell death.
ABSTRACT: In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca(2+)-activated K(+) channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg(2+) block that reduces Ca(2+) influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.
Project description:Small conductance calcium-activated potassium type 2 channels (SK2) control excitability and contribute to plasticity by reducing excitatory postsynaptic potentials. Recent evidence suggests that SK2 channels form a calcium-dependent negative-feedback loop with synaptic N-methyl-D-aspartate (NMDA) receptors. Addiction to alcohol and other drugs of abuse induces plastic changes in glutamatergic synapses that include the targeting of NMDA receptors to synaptic sites; however, the role of SK2 channels in alcohol-associated homeostatic plasticity is unknown.Electrophysiology, Western blot, and behavioral analyses were used to quantify changes in hippocampal small conductance calcium-activated potassium (SK) channel function and expression using well-characterized in vitro and in vivo models of chronic alcohol exposure.Chronic ethanol reduced apamin-sensitive SK currents in cornu ammonis 1 pyramidal neurons that were associated with a downregulation of surface SK2 channels. Blocking SK channels with apamin potentiated excitatory postsynaptic potentials in control but not ethanol-treated cornu ammonis 1 pyramidal neurons, suggesting that chronic ethanol disrupts the SK channel-NMDA receptor feedback loop. Alcohol reduced expression of SK2 channels and increased expression of NMDA receptors at synaptic sites in a mouse model. Positive modulation of SK function by 1-EBIO decreased alcohol withdrawal hyperexcitability and attenuated ethanol withdrawal neurotoxicity in hippocampus. The 1-EBIO also reduced seizure activity in mice undergoing withdrawal.These results provide evidence that SK2 channels contribute to alcohol-associated adaptive plasticity of glutamatergic synapses and that positive modulation of SK channels reduces the severity of withdrawal-related hyperexcitability. Therefore, SK2 channels appear to be critical regulators of alcohol-associated plasticity and may be novel therapeutic targets for the treatment of addiction.
Project description:BACKGROUND AND PURPOSE: Positive modulators of small conductance Ca(2+)-activated K(+) channels (SK1, SK2, and SK3) exert hyperpolarizing effects that influence the activity of excitable and non-excitable cells. The prototype compound 1-EBIO or the more potent compound NS309, do not distinguish between the SK subtypes and they also activate the related intermediate conductance Ca(2+)-activated K(+) channel (IK). This paper demonstrates, for the first time, subtype-selective positive modulation of SK channels. EXPERIMENTAL APPROACH: Using patch clamp and fluorescence techniques we studied the effect of the compound cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) on recombinant hSK1-3 and hIK channels expressed in HEK293 cells. CyPPA was also tested on SK3 and IK channels endogenously expressed in TE671 and HeLa cells. KEY RESULTS: CyPPA was found to be a positive modulator of hSK3 (EC(50) = 5.6 +/- 1.6 microM, efficacy 90 +/- 1.8 %) and hSK2 (EC(50) = 14 +/- 4 microM, efficacy 71 +/- 1.8 %) when measured in inside-out patch clamp experiments. CyPPA was inactive on both hSK1 and hIK channels. At hSK3 channels, CyPPA induced a concentration-dependent increase in the apparent Ca(2+)-sensitivity of channel activation, changing the EC(50)(Ca(2+)) from 429 nM to 59 nM. CONCLUSIONS AND IMPLICATIONS: As a pharmacological tool, CyPPA may be used in parallel with the IK/SK openers 1-EBIO and NS309 to distinguish SK3/SK2- from SK1/IK-mediated pharmacological responses. This is important for the SK2 and SK1 subtypes, since they have overlapping expression patterns in the neocortical and hippocampal regions, and for SK3 and IK channels, since they co-express in certain peripheral tissues.
Project description:Visceral hypersensitivity is a highly complex and subjective phenomenon associated with multiple levels of the nervous system and a wide range of neurotransmission. The dorsal horn (DH) in spinal cord relays the peripheral sensory information into the brain. Small conductance Ca2+-activated K+ (SK) channels regulate neuronal excitability and firing by allowing K+ to efflux in response to increase in the intracellular Ca2+ level. In this study, we examined the influence of SK2 channels in the spinal DH on the pathogenesis of visceral hypersensitivity induced by colorectal distension (CRD) in rats. Electrophysiological results showed that rats with visceral hypersensitivity presented a decrease in the SK channel-mediated afterhyperpolarization current (IAHP), and an increase in neuronal firing rates and c-Fos positive staining in the spinal DH. Western blot data revealed a decrease in the SK2 channel protein in the membrane fraction. Moreover, intrathecal administration of the SK2 channel activator 1-EBIO or CyPPA alleviated visceral hypersensitivity, reversed the decrease in IAHP and the increase in neuronal firing rates in spinal DH in rats that experienced CRD. 1-EBIO or CyPPA effect could be prevented by SK2 channel blocker apamin. CRD induced an increase in c-Fos protein expression in the spinal DH, which was prevented by 1-EBIO. Together, these data suggest that visceral hypersensitivity and pain is associated with a decrease in the number and function of membrane SK2 channels in the spinal DH. Pharmacological manipulation of SK2 channels may open a new avenue for the treatment of visceral hypersensitivity and pain. Highlights: -Neonatal colorectal distension induced visceral hypersensitivity in rats.-Visceral hypersensitivity rats presented a decrease in afterhyperpolarization current (IAHP) and membrane SK2 channel protein in the spinal dorsal horn.-Visceral hypersensitivity rats presented an increase in neuronal firing rate in the spinal dorsal horn.-Intrathecal administration of SK2 channel activator 1-EBIO or CyPPA prevented visceral hypersensitivity and decrease in IAHP.
Project description:1. The apamin-sensitive small-conductance Ca(2+)-activated K(+) channel (SK(Ca)) was characterized in porcine coronary arteries. 2. In intact arteries, 100 nM substance P and 600 microM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8 +/- 0.8 mV and 24.1 +/- 1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8 +/- 0.3 mV). 3. In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K(+) conductance of 6.8 +/- 0.04 pS. The open-probability was increased by Ca(2+) and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7 +/- 0.03 pS inhibited by d-tubocurarine was observed infrequently. 4. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SK(Ca) was detected by RT - PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6. In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SK(Ca) containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.
Project description:Small-conductance Ca2+-activated K+ (SK) channels play essential roles in the regulation of cellular excitability and have been implicated in neurological and cardiovascular diseases through both animal model studies and human genetic association studies. Over the past two decades, positive modulators of SK channels such as NS309 and 1-EBIO have been developed. Our previous structural studies have identified the binding pocket of 1-EBIO and NS309 that is located at the interface between the channel and calmodulin. In this study, we took advantage of four compounds with potencies varying over three orders of magnitude, including 1-EBIO, NS309, SKS-11 (6-bromo-5-methyl-1H-indole-2,3-dione-3-oxime) and SKS-14 (7-fluoro-3-(hydroxyimino)indolin-2-one). A combination of x-ray crystallographic, computational and electrophysiological approaches was utilized to investigate the interactions between the positive modulators and their binding pocket. A strong trend exists between the interaction energy of the compounds within their binding site calculated from the crystal structures, and the potency of these compounds in potentiating the SK2 channel current determined by electrophysiological recordings. Our results further reveal that the difference in potency of the positive modulators in potentiating SK2 channel activity may be attributed primarily to specific electrostatic interactions between the modulators and their binding pocket.
Project description:Long-term potentiation (LTP) of synaptic strength at Schaffer collateral synapses has largely been attributed to changes in the number and biophysical properties of AMPA receptors (AMPARs). Small-conductance Ca(2+)-activated K(+) channels (SK2 channels) are functionally coupled with NMDA receptors (NMDARs) in CA1 spines such that their activity modulates the shape of excitatory postsynaptic potentials (EPSPs) and increases the threshold for induction of LTP. Here we show that LTP induction in mouse hippocampus abolishes SK2 channel activity in the potentiated synapses. This effect is due to SK2 channel internalization from the postsynaptic density (PSD) into the spine. Blocking PKA or cell dialysis with a peptide representing the C-terminal domain of SK2 that contains three known PKA phosphorylation sites blocks the internalization of SK2 channels after LTP induction. Thus the increase in AMPARs and the decrease in SK2 channels combine to produce the increased EPSP underlying LTP.
Project description:Hippocampal long-term potentiation (LTP) is impaired following repeated morphine administration paired with a novel context. This procedure produces locomotor sensitization that can be abolished by blocking calcium (Ca(2+))-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the hippocampus. However, the mechanisms underlying LTP impairment remain unclear. Here, we investigate the role of N-methyl-D-aspartate receptors (NMDARs), AMPARs, and small conductance Ca(2+)-activated potassium type 2 (SK2) channels in LTP induction after context-dependent sensitization to morphine.Mice were treated with saline or escalating doses of morphine (5, 8, 10, and 15 mg/kg) every 12 hours in a locomotor activity chamber and a challenge dose of 5 mg/kg morphine was given 1 week later. After the challenge, the hippocampi were removed to assay phosphatase 2A (PP2A) activity, NMDAR, and SK2 channel synaptic expression or to perform electrophysiological recordings.Impaired hippocampal LTP, which accompanied morphine-induced context-dependent sensitization, could not be restored by blocking Ca(2+)-permeable AMPARs. Context-dependent sensitization to morphine altered hippocampal NMDAR subunit composition and enhanced the SK2 channel-mediated negative feedback on NMDAR. Increased PP2A activity observed following context-dependent sensitization suggests that the potentiated SK2 channel effect on NMDAR was mediated by increased SK2 sensitivity to Ca(2+). Finally, inhibition of SK2 channel or PP2A activity restored LTP.Our studies demonstrate that the SK2 channel-NMDAR feedback loop plays a role in opiate-induced impairment of hippocampal plasticity and that the positive modulation of SK2 channels occurs via increases in PP2A activity. This provides further evidence that small conductance Ca(2+)-activated potassium channels play a role in drug-induced plasticity.
Project description:SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning.
Project description:Ca(2+)-activated K(+) channels are present in a wide variety of cells. We have previously reported the presence of small conductance Ca(2+)-activated K(+) (SK or K(Ca)) channels in human and mouse cardiac myocytes that contribute functionally toward the shape and duration of cardiac action potentials. Three isoforms of SK channel subunits (SK1, SK2, and SK3) are found to be expressed. Moreover, there is differential expression with more abundant SK channels in the atria and pacemaking tissues compared with the ventricles. SK channels are proposed to be assembled as tetramers similar to other K(+) channels, but the molecular determinants driving their subunit interaction and assembly are not defined in cardiac tissues.To investigate the heteromultimeric formation and the domain necessary for the assembly of 3 SK channel subunits (SK1, SK2, and SK3) into complexes in human and mouse hearts.Here, we provide evidence to support the formation of heteromultimeric complexes among different SK channel subunits in native cardiac tissues. SK1, SK2, and SK3 subunits contain coiled-coil domains (CCDs) in the C termini. In vitro interaction assay supports the direct interaction between CCDs of the channel subunits. Moreover, specific inhibitory peptides derived from CCDs block the Ca(2+)-activated K(+) current in atrial myocytes, which is important for cardiac repolarization.The data provide evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Because SK channels are predominantly expressed in atrial myocytes, specific ligands of the different isoforms of SK channel subunits may offer a unique therapeutic opportunity to directly modify atrial cells without interfering with ventricular myocytes.
Project description:For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca(2+)-activated K(+) channels (KCa2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca(2+)-dependent. Further, the Ca(2+) dependence relies on another channel-interacting protein, ?-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.