KCNN2 mutation in autosomal-dominant tremulous myoclonus-dystonia.
Balint B,Guerreiro R,Carmona S,Dehghani N,Latorre A,Cordivari C,Bhatia K P,Bras J
European journal of neurology
BACKGROUND AND PURPOSE:Despite recent advances in neurogenetics that have facilitated the identification of a number of dystonia genes, many familial dystonia syndromes remain without known cause. The aim of the study was to identify the cause of autosomal dominant tremulous myoclonus-dystonia in a UK kindred with affected individuals in three generations. METHODS:Known genetic causes of myoclonus-dystonia were excluded. We combined clinical and electrophysiological phenotyping with whole-exome sequencing and Sanger sequencing to identify candidate causal variants in a family with tremulous myoclonus-dystonia. RESULTS:The core phenotype consisted of childhood-onset dystonia predominantly affecting hands and neck, with a fast tremor with superimposed myoclonus and, in some individuals, subtle cerebellar signs. We identified a novel missense variant in potassium calcium-activated channel subfamily N member 2 (KCNN2) [NM_021614:c.1112G>A:p.(Gly371Glu)], which was the only variant that we were able to identify as segregating with the phenotype over three generations. This variant, which is absent from the most recent version of gnomAD, was predicted to be deleterious by SIFT and PolyPhen-2 and had an overall CADD score of 29.7. CONCLUSIONS:KCNN2, a member of the KCNN family of potassium channel genes, is highly conserved across species and in humans is highly expressed in the brain, particularly the cerebellum. KCNN2 mutations have never been described as pathological in human disease, but are recognized abnormalities in two rodent models of fast, jerky tremor. Segregation, absence of the variant in the normal population and in-silico prediction of a deleterious effect together with animal models compatible with the clinical phenotype are all in line with KCNN2 mutations being a plausible cause underlying myoclonus-dystonia.
10.1111/ene.14228
KCNN2 Mutation in Pediatric Tremor Myoclonus Dystonia Syndrome with Electrophysiological Evaluation.
Tremor and other hyperkinetic movements (New York, N.Y.)
BACKGROUND:Here we combine clinical, electrophysiological, and genetic findings to phenotype an unusual childhood movement disorder in a patient with a rare form of KCNN2 mutation. CASE REPORT:A 10-year-old male presented with a clinical syndrome of tremor and myoclonus. Electrophysiology demonstrated muscle activity indicative of myoclonus dystonia, an observation that was not appreciated clinically. Genetic testing revealed an abnormality in the KCNN 2 gene, not present in the parents, known to cause dystonia, as the etiology. DISCUSSION:The value of utilizing noninvasive, electrophysiological recording in pediatric movement disorders expands the precision of diagnosis, potentially informing treatment when correlated with clinical and genetic findings.
10.5334/tohm.668
Muscarinic Modulation of SK2-Type K Channels Promotes Intrinsic Plasticity in L2/3 Pyramidal Neurons of the Mouse Primary Somatosensory Cortex.
eNeuro
Muscarinic acetylcholine receptors (mAChRs) inhibit small-conductance calcium-activated K channels (SK channels) and enhance synaptic weight via this mechanism. SK channels are also involved in activity-dependent plasticity of membrane excitability ("intrinsic plasticity"). Here, we investigate whether mAChR activation can drive SK channel-dependent intrinsic plasticity in L2/3 cortical pyramidal neurons. Using whole-cell patch-clamp recordings from these neurons in slices prepared from mouse primary somatosensory cortex (S1), we find that brief bath application of the mAChR agonist oxotremorine-m (oxo-m) causes long-term enhancement of excitability in wild-type mice that is not observed in mice deficient of SK channels of the SK2 isoform. Similarly, repeated injection of depolarizing current pulses into the soma triggers intrinsic plasticity that is absent from SK2 null mice. Intrinsic plasticity lowers spike frequency adaptation and attenuation of spike firing upon prolonged activation, consistent with SK channel modulation. Depolarization-induced plasticity is prevented by bath application of the protein kinase A (PKA) inhibitor H89, and the casein kinase 2 (CK2) inhibitor TBB, respectively. These findings point toward a recruitment of two known signaling pathways in SK2 regulation: SK channel trafficking (PKA) and reduction of the calcium sensitivity (CK2). Using mice with an inactivation of CaMKII (T305D mice), we show that intrinsic plasticity does not require CaMKII. Finally, we demonstrate that repeated injection of depolarizing pulses in the presence of oxo-m causes intrinsic plasticity that surpasses the plasticity amplitude reached by either manipulation alone. Our findings show that muscarinic activation enhances membrane excitability in L2/3 pyramidal neurons via a downregulation of SK2 channels.
10.1523/ENEURO.0453-19.2020
Hypobaric Hypoxia-Induced Learning and Memory Impairment: Elucidating the Role of Small Conductance Ca-Activated K Channels.
Kushwah Neetu,Jain Vishal,Dheer Aastha,Kumar Rahul,Prasad Dipti,Khan Nilofar
Neuroscience
Hypobaric Hypoxia (HH) is well-known to cause cognitive impairment and synaptic dysfunction which results in neurodegeneration. Although the role of small conductance calcium-activated potassium channels (SK channels) has been reported in synaptic plasticity, cognition and different neurological disorders; however, the precise role of SK channels in HH-induced memory impairment remains yet to be explored. We, therefore, hypothesized the pivotal role of SK channels in HH-induced cognitive decline and investigated the SK channel expression during different duration of HH exposure (Control, 1, 3, 7 and 14 days) at mRNA and protein level in male Sprague-Dawley rats. Further the role of SK channels in spatial memory and neurodegeneration were explored by inhibiting SK channel through Apamin (a known SK channel blocker). Results from the present study revealed that acute exposure of HH for 3 days leads to significant increase in expression of SK1 and SK3 channels at mRNA and protein levels, which upon chronic exposure restored to normal. Remarkably, SK2 channel expression showed gradual increase from 3 days till 14 days. Immunohistochemical analysis revealed similar pattern in different regions of the hippocampus. Additionally, SK channel inhibition with Apamin prevented HH-induced neurodegeneration and memory impairment as evident from decreased number of Fluoro Jade-positive cells, pyknotic cells, and caspase-3 expression and improved performance in the Morris water maze task. Thus, the present study demonstrates that SK channels play a crucial role in HH-induced cognitive decline and neurodegeneration.
10.1016/j.neuroscience.2018.07.026
Functional interaction of Junctophilin 2 with small- conductance Ca -activated potassium channel subtype 2(SK2) in mouse cardiac myocytes.
Fan H K,Luo T X,Zhao W D,Mu Y H,Yang Y,Guo W J,Tu H Y,Zhang Q
Acta physiologica (Oxford, England)
AIM:Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca entry and intracellular Ca release. The small-conductance Ca -activated K channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes. METHODS:Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real-time PCR. The expression of proteins was analysed by Western blotting. Ca -activated K current (I ) in infected adult mouse cardiac myocytes was recorded using whole-cell voltage-clamp technique. The intracellular Ca transient was measured using an IonOptix photometry system. RESULTS:We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N-terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I and reduced the amplitude of the Ca transient in infected cardiomyocytes. CONCLUSION:The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.
10.1111/apha.12986
SK2 channel deletion reduces susceptibility to bupivacaine-induced cardiotoxicity in mouse.
Chen H,Jin Z,Fu Z,Xia F
Human & experimental toxicology
Bupivacaine is frequently used for regional anesthesia and postoperative analgesia. However, an inadvertent intravenous injection can cause severe cardiotoxicity, manifesting as arrhythmia, hypotension, and even cardiac asystole. The mechanism of bupivacaine-mediated cardiotoxicity remains unclear. SK2 knockout mice (SK) and wild-type mice (WT) were divided into four groups, with 12 mice per group. We determined the difference in bupivacaine cardiotoxicity between SK2 knockout and WT mice by measuring the time to the first arrhythmia (T) and the time to asystole (T). Secondary indicators of cardiotoxicity were the time from the beginning of bupivacaine infusion to 20% prolongation of the QT interval (T) and the time to 20% widening of the QRS complex (T). T and T were significantly longer in the SK-bupi group than in the WT-bupi group (both < 0.05). T and T were longer in the SK-bupi group than in the WT-bupi group (all < 0.05). The time to 25%, 50%, and 75% reduction in HR in the SK-bupi group was significantly longer than in the WT-bupi group (all < 0.05). Knocking out the SK2 channel can reduce bupivacaine-induced cardiotoxicity in the mouse.
10.1177/09603271211010912
Small-conductance calcium-activated potassium type 2 channels (SK2, KCa2.2) in human brain.
Willis Michael,Trieb Maria,Leitner Irmgard,Wietzorrek Georg,Marksteiner Josef,Knaus Hans-Günther
Brain structure & function
SK2 (KCa2.2) channels are voltage-independent Ca-activated K channels that regulate neuronal excitability in brain regions important for memory formation. In this study, we investigated the distribution and expression of SK2 channels in human brain by Western blot analysis and immunohistochemistry. Immunoblot analysis of human brain indicated expression of four distinct SK2 channel isoforms: the standard, the long and two short isoforms. Immunohistochemistry in paraffin-embedded post-mortem brain sections was performed in the hippocampal formation, amygdala and neocortex. In hippocampus, SK2-like immunoreactivity could be detected in strata oriens and radiatum of area CA1-CA2 and in the molecular layer. In the amygdala, SK2-like immunoreactivity was highest in the basolateral nuclei, while in neocortex, staining was mainly found enriched in layer V. Activation of SK2 channels is thought to regulate neuronal excitability in brain by contributing to the medium afterhyperpolarization. However, SK2 channels are blocked by apamin with a sensitivity that suggests heteromeric channels. The herein first shown expression of SK2 human isoform b in brain could explain the variability of electrophysiological findings observed with SK2 channels.
10.1007/s00429-016-1258-1
Neonatal exposure to sevoflurane caused cognitive deficits by dysregulating SK2 channels and GluA2-lacking AMPA receptors in juvenile rat hippocampus.
Yu Xiangdi,Zhang Fangxiang,Shi Jinshan
Neuropharmacology
Anesthetics exposure to neonates leads to impairment of hippocampal synaptic plasticity and cognitive functions later in life. This phenomenon complies with the concept of metaplasticity: a priming stimulation can affect induction of synaptic plasticity mins or days later. We aimed to understand whether small conductance Ca-activated potassium channel type2 (SK2) and subunit composition of AMPA receptors are altered and contribute to sevoflurane-induced metaplasticity. To fulfill this goal, we exposed neonatal rats (postnatal day 7) to 2% sevoflurane for 2 h (sevoflurane rats) and examined synaptic plasticity in the hippocampus and cognitive function in juvenile rats (postnatal day 30-35). We observed that the juvenile sevoflurane rats showed elevation in the threshold for LTP induction, facilitation of LTD induction, and cognitive dysfunctions. Meanwhile, these rats also exhibited increased surface expression of SK2 and enhanced synaptic recruitment of GluA2-lacking AMPA receptors, which possess stronger inward rectification. Blocking SK2 eliminated inward rectification of AMPA receptors in juvenile sevoflurane rats. Interestingly, blocking either SK2 channels or GluA2-lacking AMPA receptors normalized LTP, LTD, and spatial memory in juvenile sevoflurane rats. Our data indicate that neonatal sevoflurane anesthesia have negative impact on cognitive function extended to juvenile rats probably through increasing surface expression of SK2 and synaptic recruitment of GluA2-lacking AMPA receptors. This study provides a new sight for sevoflurane induced metaplasticity.
10.1016/j.neuropharm.2018.08.014
Modulation of small conductance calcium-activated potassium (SK) channels: a new challenge in medicinal chemistry.
Liégeois J-F,Mercier F,Graulich A,Graulich-Lorge F,Scuvée-Moreau J,Seutin V
Current medicinal chemistry
Small conductance calcium-activated potassium (SK) channels are found in many types of neurons as well as in some other cell types. These channels are selective for K(+) and open when intracellular Ca(2+) rises to omega 500 nM. In neurons, this occurs during and after an action potential. Activation of SK channels hyperpolarizes the membrane, thus reducing cell excitability for several tens or hundreds of milliseconds. This phenomenon is called a afterhyperpolarization (AHP). Three subtypes of SK channels (SK1, SK2, SK3) have been cloned and exhibit a differential localization in the brain. SK channels may play a role in physiological and pathological conditions. They may be involved in the control of memory and cognition. Moreover, they are heavily expressed in the basal ganglia (in particular in the substantia nigra, pars compacta) and in the limbic system, suggesting that they may modulate motricity and emotional behaviour. Based on these facts, SK channel subtypes may be a suitable target for developing novel therapeutic agents, but more work is needed to validate these targets. Hence, there is a great need for selective ligands. Moreover, although the risk of peripheral side-effects for SK channel modulators appears to be low, some questions remain to be investigated. Currently, different molecules are known as SK channel modulators. Apamin is a very potent peptidic agent; it produces a strong blockade of these targets which is only very slowly reversible and it has limited selectivity. Dequalinium was found to be an effective blocker. Different chemical modulations on the dequalinium structure led to the discovery of highly potent bis-quinolinium derivatives such as UCL 1684. Other bis-(2-amino-benzimidazole) derivatives are in development. On the other hand, quaternary salts of bicuculline were reported to be effective in inhibiting AHPs. More recent developments on structurally-related molecules revealed that methyl-laudanosine is a new interesting tool for exploring SK channel pharmacology. Finally, a family of compounds has been shown to facilitate SK channel opening. Such compounds may be useful in treating disorders involving neuronal hyperexcitability.
Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons.
Dolga A M,de Andrade A,Meissner L,Knaus H-G,Höllerhage M,Christophersen P,Zischka H,Plesnila N,Höglinger G U,Culmsee C
Cell death & disease
Small-conductance Ca(2+)-activated K(+) channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson's disease, where dopaminergic cell loss is associated with progression of the disease.
10.1038/cddis.2013.530
Down-regulation of the small conductance calcium-activated potassium channels in diabetic mouse atria.
Yi Fu,Ling Tian-You,Lu Tong,Wang Xiao-Li,Li Jingchao,Claycomb William C,Shen Win-Kuang,Lee Hon-Chi
The Journal of biological chemistry
The small conductance Ca(2+)-activated K(+) (SK) channels have recently been found to be expressed in the heart, and genome-wide association studies have shown that they are implicated in atrial fibrillation. Diabetes mellitus is an independent risk factor of atrial fibrillation, but the ionic mechanism underlying this relationship remains unclear. We hypothesized that SK channel function is abnormal in diabetes mellitus, leading to altered cardiac electrophysiology. We found that in streptozotocin-induced diabetic mice, the expression of SK2 and SK3 isoforms was down-regulated by 85 and 92%, respectively, whereas that of SK1 was not changed. SK currents from isolated diabetic mouse atrial myocytes were significantly reduced compared with controls. The resting potentials of isolated atrial preparations were similar between control and diabetic mice, but action potential durations were significantly prolonged in the diabetic atria. Exposure to apamin significantly prolonged action potential durations in control but not in diabetic atria. Production of reactive oxygen species was significantly increased in diabetic atria and in high glucose-cultured HL-1 cells, whereas exposure of HL-1 cells in normal glucose culture to H2O2 reduced the expression of SK2 and SK3. Tyrosine nitration in SK2 and SK3 was significantly increased by high glucose culture, leading to accelerated channel turnover. Treatment with Tiron prevented these changes. Our results suggest that increased oxidative stress in diabetes results in SK channel-associated electrical remodeling in diabetic atria and may promote arrhythmogenesis.
10.1074/jbc.M114.607952
Activity-Dependent Plasticity of Spike Pauses in Cerebellar Purkinje Cells.
Grasselli Giorgio,He Qionger,Wan Vivian,Adelman John P,Ohtsuki Gen,Hansel Christian
Cell reports
The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2(-/-) mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity-by altering the Purkinje cell output-may be crucial to cerebellar information storage and learning.
10.1016/j.celrep.2016.02.054
Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I(AHP) and modulates the firing properties of hippocampal pyramidal neurons.
Pedarzani Paola,McCutcheon Jaime E,Rogge Gregor,Jensen Bo Skaaning,Christophersen Palle,Hougaard Charlotte,Strøbaek Dorte,Stocker Martin
The Journal of biological chemistry
SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.
10.1074/jbc.M509610200
The antidepressant fluoxetine blocks the human small conductance calcium-activated potassium channels SK1, SK2 and SK3.
Terstappen Georg C,Pellacani Annalisa,Aldegheri Laura,Graziani Francesca,Carignani Corrado,Pula Giordano,Virginio Caterina
Neuroscience letters
The effects of fluoxetine (Prozac) on the activity of human small-conductance calcium-activated potassium (SK) channels were investigated utilizing a functional fluorescence assay with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)). Fluoxetine blocked SK channels stably expressed in HEK 293 cells in a concentration-dependent manner displaying half-maximal inhibitory concentrations (IC(50)) of 9 microM for hSK1, 7 microM for hSK2 and 20 microM for hSK3. The block of hSK3 channels was confirmed by whole cell patch-clamp recordings of the recombinant cells and human TE 671 cells. Fluoxetine also inhibited [(125)I]apamin binding in a concentration-dependent manner displaying IC(50) values of 63 microM for hSK1, 148 microM for hSK2 and 295 microM for hSK3. These results provide new information concerning the mechanism of therapeutic and/or side effects of one of the most widely used antidepressant drugs.
10.1016/s0304-3940(03)00574-3
Small-Conductance Calcium-Activated Potassium Current in Normal Rabbit Cardiac Purkinje Cells.
Reher Thomas A,Wang Zhuo,Hsueh Chia-Hsiang,Chang Po-Cheng,Pan Zhenwei,Kumar Mohineesh,Patel Jheel,Tan Jian,Shen Changyu,Chen Zhenhui,Fishbein Michael C,Rubart Michael,Boyden Penelope,Chen Peng-Sheng
Journal of the American Heart Association
BACKGROUND:Purkinje cells (PCs) are important in cardiac arrhythmogenesis. Whether small-conductance calcium-activated potassium (SK) channels are present in PCs remains unclear. We tested the hypotheses that subtype 2 SK (SK2) channel proteins and apamin-sensitive SK currents are abundantly present in PCs. METHODS AND RESULTS:We studied 25 normal rabbit ventricles, including 13 patch-clamp studies, 4 for Western blotting, and 8 for immunohistochemical staining. Transmembrane action potentials were recorded in current-clamp mode using the perforated-patch technique. For PCs, the apamin (100 nmol/L) significantly prolonged action potential duration measured to 80% repolarization by an average of 10.4 ms (95% CI, 0.11-20.72) (n=9, =0.047). Voltage-clamp study showed that apamin-sensitive SK current density was significantly larger in PCs compared with ventricular myocytes at potentials ≥0 mV. Western blotting of SK2 expression showed that the SK2 protein expression in the midmyocardium was 58% (=0.028) and the epicardium was 50% (=0.018) of that in the pseudotendons. Immunostaining of SK2 protein showed that PCs stained stronger than ventricular myocytes. Confocal microscope study showed SK2 protein was distributed to the periphery of the PCs. CONCLUSIONS:SK2 proteins are more abundantly present in the PCs than in the ventricular myocytes of normal rabbit ventricles. Apamin-sensitive SK current is important in ventricular repolarization of normal PCs.
10.1161/JAHA.117.005471
Differential distribution of three Ca(2+)-activated K(+) channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system.
Stocker M,Pedarzani P
Molecular and cellular neurosciences
Ca(2+)-activated, voltage-independent K(+) channels are present in most neurons and mediate the afterhyperpolarizations (AHPs) following action potentials. They present distinct physiological and pharmacological properties and play an important role in controlling neuronal firing frequency and spike frequency adaptation. We used in situ hybridization to characterize the distribution patterns of the three cloned SK channel subunits (SK1-3), the prime candidates likely to underlie Ca(2+)-dependent AHPs in the central nervous system. We found high levels of expression in regions presenting prominent AHP currents, such as, for example, neocortex and CA1-3 layers of the hippocampus (SK1 and SK2), reticularis thalami (SK1 and SK2), supraoptic nucleus (SK3), and inferior olivary nucleus (SK2 and SK3). Our results reveal the functional role of SK channels with defined subunit compositions in some neurons and open the way to the identification of the molecular determinants of AHP currents in many brain regions.
10.1006/mcne.2000.0842
Developmental mapping of small-conductance calcium-activated potassium channel expression in the rat nervous system.
Gymnopoulos Marco,Cingolani Lorenzo A,Pedarzani Paola,Stocker Martin
The Journal of comparative neurology
Early electrical activity and calcium influx regulate crucial aspects of neuronal development. Small-conductance calcium-activated potassium (SK) channels regulate action potential firing and shape calcium influx through feedback regulation in mature neurons. These functions, observed in the adult nervous system, make them ideal candidates to regulate activity- and calcium-dependent processes in neurodevelopment. However, to date little is known about the onset of expression and regions expressing SK channel subunits in the embryonic and postnatal development of the central nervous system (CNS). To allow studies on the contribution of SK channels to different phases of development of single neurons and networks, we have performed a detailed in situ hybridization mapping study, providing comprehensive distribution profiles of all three SK subunits (SK1, SK2, and SK3) in the rat CNS during embryonic and postnatal development. SK channel transcripts are expressed at early stages of prenatal CNS development. The three SK channel subunits display different developmental expression gradients in distinct CNS regions, with time points of expression and up- or downregulation that can be associated with a range of diverse developmental events. Their early expression in embryonic development suggests an involvement of SK channels in the regulation of developmental processes. Additionally, this study shows how the postnatal ontogenetic patterns lead to the adult expression map for each SK channel subunit and how their coexpression in the same regions or neurons varies throughout development.
10.1002/cne.23466
Increasing small conductance Ca2+-activated potassium channel activity reverses ischemia-induced impairment of long-term potentiation.
Orfila J E,Shimizu K,Garske A K,Deng G,Maylie J,Traystman R J,Quillinan N,Adelman J P,Herson P S
The European journal of neuroscience
Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.
10.1111/ejn.12683
Comparative immunohistochemical distribution of three small-conductance Ca2+-activated potassium channel subunits, SK1, SK2, and SK3 in mouse brain.
Sailer Claudia A,Kaufmann Walter A,Marksteiner Josef,Knaus Hans-Günther
Molecular and cellular neurosciences
To investigate the distribution of all three SK channel subunits in the mouse central nervous system, we performed immunohistochemistry using sequence-specific antibodies directed against SK1, SK2, and SK3 proteins. Expression of SK1 and SK2 proteins revealed a partly overlapping distribution pattern restricted to a limited number of brain areas (e.g., neocortex, hippocampal formation). In contrast, SK3 immunoreactivity was rather complementary and predominantly detected in phylogenetically older brain regions like basal ganglia, thalamus, and various brain stem nuclei (e.g., locus coeruleus, tegmental nuclei). At the cellular level, SK1- and SK2-like immunoreactivity was primarily localized to somatic and dendritic structures, whereas the majority of SK3-like immunoreactivity was associated with varicose fibers.
10.1016/j.mcn.2004.03.002
SK Channel Modulates Synaptic Plasticity by Tuning CaMKIIα/β Dynamics.
Frontiers in synaptic neuroscience
N-Methyl-D-Aspartate Receptor 1 (NMDAR)-linked Ca current represents a significant percentage of post-synaptic transient that modulates synaptic strength and is pertinent to dendritic spine plasticity. In the hippocampus, Ca transient produced by glutamatergic ionotropic neurotransmission facilitates Ca-Calmodulin-dependent kinase 2 (CaMKII) Thr286 phosphorylation and promote long-term potentiation (LTP) expression. At CA1 post-synaptic densities, Ca transients equally activate small conductance (SK2) channel which regulates excitability by suppressing Ca movement. Here, we demonstrate that upstream attenuation of GluN1 function in the hippocampus led to a decrease in Thr286 CaMKIIα phosphorylation, and increased SK2 expression. Consistent with the loss of GluN1 function, potentiation of SK channel in wild type hippocampus reduced CaMKIIα expression and abrogate synaptic localization of T286 pCaMKIIα. Our results demonstrate that positive modulation of SK channel at hippocampal synapses likely refine GluN1-linked plasticity by tuning dendritic localization of CaMKIIα.
10.3389/fnsyn.2019.00018
Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase.
Ren Yajun,Barnwell Lyndon F,Alexander Jon C,Lubin Farah D,Adelman John P,Pfaffinger Paul J,Schrader Laura A,Anderson Anne E
The Journal of biological chemistry
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 microm) 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.
10.1074/jbc.M513125200
Transcriptional regulation of intronic calcium-activated potassium channel SK2 promoters by nuclear factor-kappa B and glucocorticoids.
Kye Min-Jeong,Spiess Joachim,Blank Thomas
Molecular and cellular biochemistry
Small-conductance Ca(2+)-activated K(+) channels (SK) of the SK2 subtype are widely expressed in the central nervous system where they contribute to the control of neuronal excitability. Two SK2 isoforms, SK2-S and SK2-L, the latter representing an N-terminally extended protein of SK2-S, are expressed in similar patterns in the brain. However, our understanding of mechanisms by which the expression of SK2 is regulated is limited. We identified one functional glucocorticoid response element (GRE) at position -2248 bp and two functional nuclear factor-kappB (NF-kappaB) response elements at positions -1652 and -1586 bp in the SK2-S promoter. An increase in SK2-S promoter activity was observed in PC12 cells transiently transfected with a wild-type SK2-S promoter-luciferase reporter gene construct and treated with aldosterone or dexamethasone. The mineralocorticoid receptor (MR) antagonist spironolactone or the glucocorticoid receptor (GR) antagonist mifepristone fully inhibited aldosterone or dexamethasone activation of the SK2-S promoter, respectively. SK2-S promoter activity was also induced by the cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide). Antisense oligonucleotides directed to NF-kappaB p65 or p50 suppressed SK2-S transcription induced by C2-ceramide. Deletion studies showed that only the -1586 bp NF-kappaB binding site was necessary for maximum C2-ceramide response. Finally, we showed that activation of GRs but not of MRs repressed the NF-kappaB-mediated induction of SK2-S transcription. These findings suggest a possible transcriptional cross talk between GRs and NF-kappaB in the intronic promoter regulation of SK2-S channel gene transcription.
10.1007/s11010-006-9320-6
Identification and characterization of a novel, shorter isoform of the small conductance Ca2+ -activated K+ channel SK2.
Murthy Saravana R K,Teodorescu Georgeta,Nijholt Ingrid M,Dolga Amalia M,Grissmer Stephan,Spiess Joachim,Blank Thomas
Journal of neurochemistry
Throughout the CNS, small conductance Ca(2+)-activated potassium (SK) channels modulate firing frequency and neuronal excitability. We have identified a novel, shorter isoform of standard SK2 (SK2-std) in mouse brain which we named SK2-sh. SK2-sh is alternatively spliced at exon 3 and therefore lacks 140 amino acids, which include transmembrane domains S3, S4 and S5, compared with SK2-std. Western blot analysis of mouse hippocampal tissue revealed a 47 kDa protein product as predicted for SK2-sh along with a 64 kDa band representing the standard SK2 isoform. Electrophysiological recordings from transiently expressed SK2-sh revealed no functional channel activity or interaction with SK2-std. With the help of real-time PCR, we found significantly higher expression levels of SK2-sh mRNA in cortical tissue from AD cases when compared with age-matched controls. A similar increase in SK2-sh expression was induced in cortical neurons from mice by cytokine exposure. Substantial clinical evidence suggests that excess cytokines are centrally involved in the pathogenesis of Alzheimer's disease. Thus, SK2-sh as a downstream target of cytokines, provide a promising target for additional investigation regarding potential therapeutic intervention.
10.1111/j.1471-4159.2008.05557.x
Developmental profile of SK2 channel expression and function in CA1 neurons.
Ballesteros-Merino Carmen,Lin Mike,Wu Wendy W,Ferrandiz-Huertas Clotilde,Cabañero María J,Watanabe Masahiko,Fukazawa Yugo,Shigemoto Ryuichi,Maylie James,Adelman John P,Luján Rafael
Hippocampus
We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development.
10.1002/hipo.20986
SK2 Channels Associate With mGlu Receptors and Ca2.1 Channels in Purkinje Cells.
Luján Rafael,Aguado Carolina,Ciruela Francisco,Arus Xavier Morató,Martín-Belmonte Alejandro,Alfaro-Ruiz Rocío,Martínez-Gómez Jesús,de la Ossa Luis,Watanabe Masahiko,Adelman John P,Shigemoto Ryuichi,Fukazawa Yugo
Frontiers in cellular neuroscience
The small-conductance, Ca-activated K (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca: Ca2.1 channels and mGlu receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for Ca2.1 channels and mGlu receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to Ca2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca, and suggest that the ultrastructural association of SK2 with Ca2.1 and mGlu provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca transients in PCs.
10.3389/fncel.2018.00311
Metformin Regulates the Expression of SK2 and SK3 in the Atria of Rats With Type 2 Diabetes Mellitus Through the NOX4/p38MAPK Signaling Pathway.
Liu Changhe,Hua Na,Fu Xi,Pan Yilong,Li Bin,Li Xiaodong
Journal of cardiovascular pharmacology
We previously found that metformin regulates the ion current conducted by the small conductance calcium-activated potassium channels (SK channels) in the atria of rats with type 2 diabetes mellitus (T2DM) as well as the mRNA and protein expression of the SK2 and SK3 subtypes of SK channels. In this study, we hypothesized that the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway was involved in the metformin-mediated regulation of SK2 and SK3 expression in the atria of rats with T2DM. We randomly divided Wistar rats into the control group, the untreated T2DM group, the metformin-treated group, the group receiving subcutaneous injections of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor diphenyleneiodonium (DPI), and the group receiving tail vein injections of the p38MAPK agonist anisomycin. Real-time polymerase chain reaction, Western blot, and immunohistochemistry were applied to examine the expression levels of SK2, SK3, NOX4, and phospho-p38MAPK (p-p38MAPK) mRNAs and proteins in the atrial tissue of relevant groups. We observed that the expression levels of NOX4 mRNA and protein and p-p38MAPK protein were significantly elevated in the atria of rats with T2DM compared with the control group. In addition, SK2 protein expression was reduced, whereas SK3 protein expression was increased. The 8-week treatment with metformin markedly reduced the expression levels of NOX4 mRNA and protein and p-p38MAPK protein, upregulated the SK2 expression, and downregulated the SK3 expression. Tail vein injection with anisomycin significantly increased the p-p38MAPK expression while further inhibiting the expression of SK2 and enhancing the expression of SK3. Subcutaneous injection with DPI considerably inhibited the expression of NOX4, further enhanced the expression of SK2 and suppressed the expression of SK3. In addition, subcutaneous injection with DPI significantly suppressed the phosphorylation of p38MAPK. In conclusion, the NOX4/p38MAPK signaling pathway mediates the downregulation of SK2 and the upregulation of SK3 in the atria of rats with T2DM. Long-term metformin treatment upregulates SK2 protein expression and downregulates SK3 protein expression by inhibiting the NOX4/p38MAPK signaling pathway.
10.1097/FJC.0000000000000615
Metformin regulates atrial SK2 and SK3 expression through inhibiting the PKC/ERK signaling pathway in type 2 diabetic rats.
Liu Chang-He,Hua Na,Fu Xi,Pan Yi-Long,Li Bin,Li Xiao-Dong
BMC cardiovascular disorders
BACKGROUND:Our previous study showed that metformin regulates the mRNA and protein levels of type 2 small conductance calcium-activated potassium channel (SK2) and type 3 small conductance calcium-activated potassium channels (SK3) in atrial tissue as well as the ion current of atrial myocytes in rats with type 2 diabetes mellitus (T2DM), but the underlying signaling mechanism is unknown. This study aimed to investigate whether metformin regulates atrial SK2 and SK3 protein expression in T2DM rats though the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signaling pathway. METHODS:A T2DM rat model was established using a high-fat and high-sugar diet combined with a low-dose intraperitoneal injection of streptozotocin (STZ). The rats were randomly divided into the following five groups: the control group, the untreated T2DM group, the metformin-treated only group, the phorbol 12-myristate 13-acetate (PMA; a PKC agonist administered by intraperitoneal injection) treatment group, and the recombinant human epidermal growth factor (rh-EGF; an ERK agonist administered by tail vein injection) treatment group. The activity of PKC in atrial tissues was assayed by a PKC kinase activity assay kit. The protein expression of SK2, SK3, and phosphorylated ERK (pERK) were determined by western blotting and immunohistochemistry. RESULTS:Compared with the Control group, atrial PKC activity and pERK and SK3 protein expression were increased, while SK2 protein expression was decreased in atrial tissues of T2DM rats. Eight weeks of metformin treatment inhibited the PKC activity and pERK and SK3 expression, and elevated SK2 expression compared with the T2DM group. Compared with the metformin-treated only group, the injection of rh-EGF increased pERK and SK3 expression, and decreased SK2 expression; the injection of PMA increased PKC activity and SK3 expression, and decreased SK2 expression. In addition, the injection with PMA significantly elevated the expression of pERK. CONCLUSIONS:The PKC/ERK signaling pathway is involved in the downregulation of SK2 expression and the upregulation of SK3 expression in the atrium of T2DM rats. Long-term metformin treatment prevents the SK2 downregulation and the SK3 upregulation through inhibiting the PKC/ERK signaling pathway.
10.1186/s12872-018-0950-x
Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity.
Hammond Rebecca S,Bond Chris T,Strassmaier Timothy,Ngo-Anh Thu Jennifer,Adelman John P,Maylie James,Stackman Robert W
The Journal of neuroscience : the official journal of the Society for Neuroscience
Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.
10.1523/JNEUROSCI.4106-05.2006
Alpha-actinin2 cytoskeletal protein is required for the functional membrane localization of a Ca2+-activated K+ channel (SK2 channel).
Lu Ling,Timofeyev Valeriy,Li Ning,Rafizadeh Sassan,Singapuri Anil,Harris Todd R,Chiamvimonvat Nipavan
Proceedings of the National Academy of Sciences of the United States of America
The importance of proper ion channel trafficking is underpinned by a number of channel-linked genetic diseases whose defect is associated with failure to reach the cell surface. Conceptually, it is reasonable to suggest that the function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane, which is determined jointly by the secretory and endocytic pathways. Yet the precise mechanisms of the entire ion channel trafficking pathway remain unknown. Here, we directly demonstrate that proper membrane localization of a small-conductance Ca(2+)-activated K(+) channel (SK2 or K(Ca)2.2) is dependent on its interacting protein, alpha-actinin2, a major F-actin crosslinking protein. SK2 channel localization on the cell-surface membrane is dynamically regulated, and one of the critical steps includes the process of cytoskeletal anchoring of SK2 channel by its interacting protein, alpha-actinin2, as well as endocytic recycling via early endosome back to the cell membrane. Consequently, alteration of these components of SK2 channel recycling results in profound changes in channel surface expression. The importance of our findings may transcend the area of K(+) channels, given that similar cytoskeletal interaction and anchoring may be critical for the membrane localization of other ion channels in neurons and other excitable cells.
10.1073/pnas.0908207106
Apamin-Sensitive K+ Current Upregulation in Volume-Overload Heart Failure is Associated with the Decreased Interaction of CK2 with SK2.
Yang Dandan,Wang Tingzhong,Ni Yajuan,Song Bingxue,Ning Feifei,Hu Peijing,Luo Ling,Wang Ya,Ma Aiqun
The Journal of membrane biology
Recent studies have shown that the sensitivity of apamin-sensitive K(+) current (I KAS, mediated by apamin-sensitive small conductance calcium-activated potassium channels subunits) to intracellular Ca(2+) is increased in heart failure (HF), leading to I KAS upregulation, action potential duration shortening, early after depolarization, and recurrent spontaneous ventricular fibrillation. We hypothesized that casein kinase 2 (CK2) interacted with small conductance calcium-activated potassium channels (SK) is decreased in HF, and protein phosphatase 2A (PP2A) is increased on the opposite, upregulating the sensitivity of I KAS to intracellular Ca(2+) in HF. Rat model of volume-overload HF was established by an abdominal arteriovenous fistula procedure. The expression of SK channels, PP2A and CK2 was detected by Western blot analysis. Interaction and colocalization of CK2 with SK channel were detected by co-immunoprecipitation analysis and double immunofluorescence staining. In HF rat left ventricle, SK3 was increased by 100 % (P < 0.05), and SK2 was not significantly changed. PP2A protein was increased by 94.7 % in HF rats (P < 0.05), whereas the level of CK2 was almost unchanged. We found that CK2 colocalized with SK2 and SK3 in rat left ventricle. With anti-CK2α antibody, SK2 and SK3 were immunoprecipitated, the level of precipitated SK2 decreased by half, whereas precipitated SK3 was almost unchanged. In conclusion, the increased expression of total PP2A and decreased interaction of CK2 with SK2 may underlie enhanced sensitivity of I KAS to intracellular Ca(2+) in volume-overload HF rat.
10.1007/s00232-015-9839-0
SK2 channels are neuroprotective for ischemia-induced neuronal cell death.
Allen Duane,Nakayama Shin,Kuroiwa Masayuki,Nakano Takaaki,Palmateer Julie,Kosaka Yasuharu,Ballesteros Carmen,Watanabe Masahiko,Bond Chris T,Luján Rafael,Maylie James,Adelman John P,Herson Paco S
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
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.
10.1038/jcbfm.2011.90
SK2 and SK3 expression differentially affect firing frequency and precision in dopamine neurons.
Deignan J,Luján R,Bond C,Riegel A,Watanabe M,Williams J T,Maylie J,Adelman J P
Neuroscience
The firing properties of dopamine (DA) neurons in the substantia nigra (SN) pars compacta are strongly influenced by the activity of apamin-sensitive small conductance Ca(2+)-activated K(+) (SK) channels. Of the three SK channel genes expressed in central neurons, only SK3 expression has been identified in DA neurons. The present findings show that SK2 was also expressed in DA neurons. Immuno-electron microscopy (iEM) showed that SK2 was primarily expressed in the distal dendrites, while SK3 was heavily expressed in the soma and, to a lesser extent, throughout the dendritic arbor. Electrophysiological recordings of the effects of the SK channel blocker apamin on DA neurons from wild type and SK(-/-) mice show that SK2-containing channels contributed to the precision of action potential (AP) timing, while SK3-containing channels influenced AP frequency. The expression of SK2 in DA neurons may endow distinct signaling and subcellular localization to SK2-containing channels.
10.1016/j.neuroscience.2012.04.053
A V-to-F substitution in SK2 channels causes Ca hypersensitivity and improves locomotion in a C. elegans ALS model.
Nam Young-Woo,Baskoylu Saba N,Gazgalis Dimitris,Orfali Razan,Cui Meng,Hart Anne C,Zhang Miao
Scientific reports
Small-conductance Ca-activated K (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP-interacting residue K405, and subsequently enhanced Ca sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca with increased activity in vivo.
10.1038/s41598-018-28783-2
Functional interaction with filamin A and intracellular Ca2+ enhance the surface membrane expression of a small-conductance Ca2+-activated K+ (SK2) channel.
Rafizadeh Sassan,Zhang Zheng,Woltz Ryan L,Kim Hyo Jeong,Myers Richard E,Lu Ling,Tuteja Dipika,Singapuri Anil,Bigdeli Amir Ali Ziaei,Harchache Sana Ben,Knowlton Anne A,Yarov-Yarovoy Vladimir,Yamoah Ebenezer N,Chiamvimonvat Nipavan
Proceedings of the National Academy of Sciences of the United States of America
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, α-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.
10.1073/pnas.1323541111
SK2 channel regulation of neuronal excitability, synaptic transmission, and brain rhythmic activity in health and diseases.
Biochimica et biophysica acta. Molecular cell research
Small conductance calcium-activated potassium channels (SKs) are solely activated by intracellular Ca and their activation leads to potassium efflux, thereby repolarizing/hyperpolarizing membrane potential. Thus, these channels play a critical role in synaptic transmission, and consequently in information transmission along the neuronal circuits expressing them. SKs are widely but not homogeneously distributed in the central nervous system (CNS). Activation of SKs requires submicromolar cytoplasmic Ca concentrations, which are reached following either Ca release from intracellular Ca stores or influx through Ca permeable membrane channels. Both Ca sensitivity and synaptic levels of SKs are regulated by protein kinases and phosphatases, and degradation pathways. SKs in turn control the activity of multiple Ca channels. They are therefore critically involved in coordinating diverse Ca signaling pathways and controlling Ca signal amplitude and duration. This review highlights recent advances in our understanding of the regulation of SK2 channels and of their roles in normal brain functions, including synaptic plasticity, learning and memory, and rhythmic activities. It will also discuss how alterations in their expression and regulation might contribute to various brain disorders such as Angelman Syndrome, Alzheimer's disease and Parkinson's disease.
10.1016/j.bbamcr.2020.118834
SK2 channels regulate mitochondrial respiration and mitochondrial Ca uptake.
Honrath Birgit,Matschke Lina,Meyer Tammo,Magerhans Lena,Perocchi Fabiana,Ganjam Goutham K,Zischka Hans,Krasel Cornelius,Gerding Albert,Bakker Barbara M,Bünemann Moritz,Strack Stefan,Decher Niels,Culmsee Carsten,Dolga Amalia M
Cell death and differentiation
Mitochondrial calcium ([Ca]) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca] uptake upon SK channel activation as detected by time lapse mitochondrial Ca measurements with the Ca-binding mitochondria-targeted aequorin and FRET-based [Ca] probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca] uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.
10.1038/cdd.2017.2
Coupled activity-dependent trafficking of synaptic SK2 channels and AMPA receptors.
Lin Mike T,Luján Rafael,Watanabe Masahiko,Frerking Matthew,Maylie James,Adelman John P
The Journal of neuroscience : the official journal of the Society for Neuroscience
Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By approximately 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the approximately 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
10.1523/JNEUROSCI.1411-10.2010
The SK2-long isoform directs synaptic localization and function of SK2-containing channels.
Allen Duane,Bond Chris T,Luján Rafael,Ballesteros-Merino Carmen,Lin Mike T,Wang Kang,Klett Nathan,Watanabe Masahiko,Shigemoto Ryuichi,Stackman Robert W,Maylie James,Adelman John P
Nature neuroscience
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.
10.1038/nn.2832
Activation of SK2 channels preserves ER Ca²⁺ homeostasis and protects against ER stress-induced cell death.
Richter M,Vidovic N,Honrath B,Mahavadi P,Dodel R,Dolga A M,Culmsee C
Cell death and differentiation
Alteration of endoplasmic reticulum (ER) Ca(2+) homeostasis leads to excessive cytosolic Ca(2+) accumulation and delayed neuronal cell death in acute and chronic neurodegenerative disorders. While our recent studies established a protective role for SK channels against excessive intracellular Ca(2+) accumulation, their functional role in the ER has not been elucidated yet. We show here that SK2 channels are present in ER membranes of neuronal HT-22 cells, and that positive pharmacological modulation of SK2 channels with CyPPA protects against cell death induced by the ER stressors brefeldin A and tunicamycin. Calcium imaging of HT-22 neurons revealed that elevated cytosolic Ca(2+) levels and decreased ER Ca(2+) load during sustained ER stress could be largely prevented by SK2 channel activation. Interestingly, SK2 channel activation reduced the amount of the unfolded protein response transcription factor ATF4, but further enhanced the induction of CHOP. Using siRNA approaches we confirmed a detrimental role for ATF4 in ER stress, whereas CHOP regulation was dispensable for both, brefeldin A toxicity and CyPPA-mediated protection. Cell death induced by blocking Ca(2+) influx into the ER with the SERCA inhibitor thapsigargin was not prevented by CyPPA. Blocking the K(+) efflux via K(+)/H(+) exchangers with quinine inhibited CyPPA-mediated neuroprotection, suggesting an essential role of proton uptake and K(+) release in the SK channel-mediated neuroprotection. Our data demonstrate that ER SK2 channel activation preserves ER Ca(2+) uptake and retention which determines cell survival in conditions where sustained ER stress contributes to progressive neuronal death.
10.1038/cdd.2015.146
UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.
Sun Jiandong,Zhu Guoqi,Liu Yan,Standley Steve,Ji Angela,Tunuguntla Rashmi,Wang Yubin,Claus Chad,Luo Yun,Baudry Michel,Bi Xiaoning
Cell reports
Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function.
10.1016/j.celrep.2015.06.023
Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function.
Kim Gukhan,Luján Rafael,Schwenk Jochen,Kelley Melissa H,Aguado Carolina,Watanabe Masahiko,Fakler Bernd,Maylie James,Adelman John P
eLife
Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
10.7554/eLife.12637
SK2 channels in cerebellar Purkinje cells contribute to excitability modulation in motor-learning-specific memory traces.
PLoS biology
Neurons store information by changing synaptic input weights. In addition, they can adjust their membrane excitability to alter spike output. Here, we demonstrate a role of such "intrinsic plasticity" in behavioral learning in a mouse model that allows us to detect specific consequences of absent excitability modulation. Mice with a Purkinje-cell-specific knockout (KO) of the calcium-activated K+ channel SK2 (L7-SK2) show intact vestibulo-ocular reflex (VOR) gain adaptation but impaired eyeblink conditioning (EBC), which relies on the ability to establish associations between stimuli, with the eyelid closure itself depending on a transient suppression of spike firing. In these mice, the intrinsic plasticity of Purkinje cells is prevented without affecting long-term depression or potentiation at their parallel fiber (PF) input. In contrast to the typical spike pattern of EBC-supporting zebrin-negative Purkinje cells, L7-SK2 neurons show reduced background spiking but enhanced excitability. Thus, SK2 plasticity and excitability modulation are essential for specific forms of motor learning.
10.1371/journal.pbio.3000596
PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome.
Sun Jiandong,Liu Yan,Zhu Guoqi,Cato Caleb,Hao Xiaoning,Qian Li,Lin Weiju,Adhikari Rachana,Luo Yun,Baudry Michel,Bi Xiaoning
Scientific reports
The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.
10.1038/s41598-020-66790-4
The MORN domain of Junctophilin2 regulates functional interactions with small-conductance Ca -activated potassium channel subtype2 (SK2).
BioFactors (Oxford, England)
Small-conductance Ca -activated K channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca homeostasis. Junctophilin-2 (JP2) is a membrane-binding protein, which provides functional crosstalk by physically linking with the cell-surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C-terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2-regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co-expressing the JP2-MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C-terminus.
10.1002/biof.1608