Opening Of SK Channels Research Articles

Metabolic escape to glycolysis through SK channel activation inhibits ferroptosis and increases the life span of C. elegans in conditions of heat stress

Metabolic flexibility is a characteristic of essentially all cells in order to cope with environmental alterations. One example of adaptation is represented by a mitochondrial switch from oxidative phosphorylation (OXPHOS) to glycolysis. Attenuation of mitochondrial respiration and cell metabolic shifts have been proposed as therapeutic strategies against oxidative stress and neurodegeneration. Activation of small‐conductance Ca2+‐activated K+(SK) channels contribute to neuroprotection in conditions of oxidative stress by attenuating mitochondrial calcium ([Ca2+]m), mitochondrial reactive oxygen species (ROS) generation and mitochondrial respiration in neuronal cells. To date, potential effects of SK channels have never been studied in relation to metabolic reprogramming nor the subsequent effects on survival in whole organisms in conditions of oxidative stress. Therefore, we aim to understand the mechanism by which SK channels confer neuroprotection during ferroptosis, a form of cell death associated with oxidative stress, and whether metabolic reprogramming can be part of neuroprotection.Our data show that CyPPA, a positive modulator of SK channels, prevented cell death in conditions of ferroptosis and preserved mitochondrial function. High‐resolution respiratory revealed that opening of SK channels alone resulted in a mild decrease of mitochondrial complex I activity. These effects were accompanied by a slight increase in mitochondrial ROS production and depolarization of the mitochondrial membrane, suggesting preconditioning mechanisms to be also involved in the neuroprotection. Experiments where mitochondrial ROS were scavenged showed reduced capacity of CyPPA to mediate neuroprotection. Interestingly, extracellular flux analysis by Seahorse flux analysis technology revealed a CyPPA‐mediated initial increase in glycolysis followed by a slight reduction of OXPHOS activity. Induction of glycolysis was crucial for the neuroprotective effects of the SK channel opener, since substances that inhibit glycolysis were able to restore the cell sensitivity to ferroptosis even in the presence of CyPPA. Importantly, we found CyPPA to increase survival of C. elegans challenged with mitochondrial toxins in conditions of heat stress. These findings shed light on new molecular mechanisms mediated by SK channels that could explain the increased neuroprotective capacity in conditions of ferroptosis and neurodegeneration.Support or Funding InformationA.M.D. is the recipient of a Rosalind Franklin Fellowship co‐funded by the European Union and the University of Groningen.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of excitability in tonic firing substantia gelatinosa neurons of the spinal cord by small-conductance Ca2+-activated K+ channels

The excitability of substantia gelatinosa (SG) neurons in the spinal dorsal horn determines the processing of nociceptive information from the periphery to the central nervous system. Small conductance Ca2+-activated K+ (SK) channels on neurons supply strong negative feedback control on neuronal excitability by affecting afterhyperpolarization (AHP). However, the role of SK channels in regulating tonic-firing SG neuron excitability remains elusive. In the present study, whole-cell recordings were conducted in SG neurons from acute spinal cord slices of adult rats. The SK channel opener 1-ethyl-2-benzimidazolinone (1-EBIO) attenuated spike discharges and increased AHP amplitudes; this effect was mimicked by a high Ca2+ external solution. Systemic administration of 1-EBIO attenuated the thermal-induced nociception behavior. Conversely, the inhibition of SK channels with apamin, a specific SK channel inhibitor, increased neuronal excitability and decreased the AHP amplitudes; this effect was mimicked by a Ca2+-free external solution. Apamin increased excitatory synaptic transmission by increasing the amplitudes of evoked excitatory postsynaptic potentials (eEPSPs). This facilitation depended on N-methyl-d-aspartate (NMDA) receptors, extracellular Mg2+ and intracellular Ca2+. Voltage-gated Ca2+ channels (VGCCs) were also involved in the apamin-induced effects. Strikingly, 1-EBIO action on decreasing excitability persisted in the presence of apamin, indicating that 1-EBIO manipulates SK channels via a pathway rather than via apamin-sensitive SK channels. The data reveal a previously uncharacterized mechanism for manipulating SG neuronal excitability by Ca2+ conductances via both apamin-sensitive and apamin-insensitive pathways. Because SG neurons in the dorsal horn are involved in regulating nociception, manipulating neuronal excitability via SK channels indicates a potential therapeutic target.

Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels

The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25–0.5%) ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

American Clinical Neurophysiology Society's standardized critical care EEG terminology: Interrater reliability and 2012 version

American Clinical Neurophysiology Society's standardized critical care EEG terminology: Interrater reliability and 2012 version

Anticonvulsant actions of Ska-31 in pilocarpine treated chronic epileptic rats

Purpose: Pilocarpine-induced status epilepticus or limbic seizure model is use to test activity of new molecules with anticonvulsant potential. In the present study we focused on anticonvulsant effect of SKa-31 (sK-channel opener) in pilocarpine-induced epileptiform activities.

Impaired vasodilation of human obese visceral resistance arteries is improved by apocynin through hydrogen peroxide and activation of SK and IK channels

Previous studies indicate that in human obesity, visceral adipose tissue (VAT) is associated with endothelial dysfunction compared to subcutaneous adipose tissue (SAT). We hypothesized that inhibition of NADPH oxidase will reverse impaired Flow‐induced dilation (FID) in resistance arteries of VAT. Arterioles were dissected from VAT and SAT biopsies (BMI: 48±2, n=15) and cannulated for measurements of luminal diameters. Maximum dilations to papaverine (10−4 M) were determined. Production of hydrogen peroxide (H2O2) was measured by fluorescence microscopy. FID was reduced in VAT to 38±4% (p<0.01, at 60 cm H2O pressure gradient) compared to SAT (92±4%). Apocynin restored FID (73±5%) in VAT and had no effect in SAT. PEG‐catalase abolished the restored FID in the presence of apocynin. Incubation with apamin and tram‐34, blockers of small (SK)‐ and intermediate (IK)‐ conductance, Ca2+‐activated potassium channels, respectively, decreased this restored FID. Improved FID induced by the SK channel opener CyPPA was blocked by apamin. Flow‐ induced H2O2 generation was increased (1.7 fold) in the presence of apocynin compared to baseline and PEG‐catalase attenuated this increase. Taken together these data suggest that H2O2 and the activation of SK and IK channels contribute to the restored effect of apocynin on flow induced dilation in visceral fat during obesity. NIH (R01HL095701).

Unstructured to structured transition of an intrinsically disordered protein peptide in coupling Ca 2+ -sensing and SK channel activation

Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1-S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca(2+)-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca(2+)-bound calmodulin (CaM). Upon binding of Ca(2+) to CaM, a 2 × 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca(2+) concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca(2+) sensing by CaM and mechanical opening of SK channels.

Role of small conductance calcium‐activated potassium channels (SK2) expressed in hypothalamic PVN neurons in regulating sympathetic nerve activity (SNA) in rats

Recently, we reported that SK channels regulate membrane properties of RVLM projecting PVN neurons and that inhibition of SK channels increase in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating SNA and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blockade with apamin (0, 0.125, 1.25, 3.75, 12.5, 25 pmol; 50 nl) increased splanchnic SNA (SSNA), renal SNA (RSNA), and MAP in a dose‐dependent manner. Maximum increases in SNA and MAP elicited by apamin (12.5 pmol) in SSNA, RSNA and MAP were 330 ± 40% (n=8), 271 ± 40% (n=6), and 29 ± 4 mmHg (n=7), respectively. Pre‐treatment with DCEBIO (2.5 nmol), a SK channel opener, microinjected into the PVN significantly (p<0.05–0.001, n=5) diminished the sympathoexcitatory response evoked by apamin (102 ± 24% for SSNA, 78 ± 40% for RSNA and 16 ± 3 mmHg for MAP). DCEBIO alone was without effect on baseline SNA and MAP. Western‐blot examination of the PVN showed abundant protein expression of SK2 channels. Further immunohistochemistry studies showed that SK2 channels were primarily expressed in dorsal and lateral parvocellular divisions of the PVN. The majority of SK2 channel expression was localized in PVN‐RVLM neurons identified by retrograde labeling. We conclude that SK currents in the PVN inhibit ongoing SNA. Inhibition of SK2 channels may underlie the mechanism of increased SNA induced by apamin. Support: AHA 10SDG2640130 (QHC).

Effect of the SK/IK channel modulator 4,5‐dichloro‐1,3‐diethyl‐1,3‐dihydro‐benzoimidazol‐2‐one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle

• To investigate the importance of small (SK)- and intermediate (IK)-conductance Ca2(+) -activated K(+) channels on bladder function, by studying the effects of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a new modulator of SK/IK channels, on contractions induced by electrical field stimulation (EFS) and carbachol in rat, pig and human detrusor. • Detrusor biopsies were obtained from rats, pigs and male patients undergoing cystectomy because of bladder cancer. • Force was recorded using myographs. • Intracellular free Ca(2+) was measured in myocytes using microfluorimetry. • In rat bladder rings subjected to EFS, cumulative addition of NS4591 (0.1-30 µM) decreased force by 82 ± 2.9% (n = 6).This effect was reduced by 64 ± 5.2% in the presence of 0.3 µM apamin, a specific inhibitor of SK channels. Apamin increased the force evoked by EFS significantly: force was increased by 14.2 ± 3.4% (n = 5) and 10.1 ± 2.6% (n = 7) in pig and human detrusor strips, respectively (P = 0.04 and P = 0.02). • The cumulative addition of NS4591 (0.3-30 µM) significantly reduced the amplitude of carbachol-induced rhythmic oscillations by 62.0 ± 12.0% (n = 12) and the minimum force between oscillations by 30 ± 5% (n = 9) in pig detrusor strips (P < 0.005). In the presence of 10 µM NS4591, carbachol (1 µM) induced rhythmic contractions with an amplitude and normalized mean power frequency (nmeanPF) of 8.4 ± 5.1% and 0.11 ± 0.06 mN root mean square (rms) Hz (n = 12), respectively, vs. 21 ± 3.4% and 0.17 ± 0.04 mN rms Hz in control strips (n = 13). Apamin induced 6- and 11-fold increases in amplitude and nmeanPF vs. 1.3- and 2-fold increases in control strips. • In human detrusor strips (n = 15), the cumulative addition of NS4591 (1-30 µM) significantly reduced the amplitude by 69 ± 11%, the nmeanPF by 78 ± 6% and the minimum force between carbachol-induced oscillations by 59 ± 5% (P < 0.008). The addition of apamin (0.3 µM) before application of 1 µM carbachol abolished the effects of NS4591 on amplitude and partially abolished its effect on nmeanPF by 41 ± 7%, vs. a 78 ± 6% reduction in the absence of apamin (n = 8). • In spontaneously active detrusor preparations, NS4591 reduced or abolished contractions. • Furthermore, NS4591 (10 µM) decreased the carbachol-induced increase in the fura-2 ratio by 43 ± 3% compared with control (n = 12) (P < 0.03). • The SK/IK channel modulator NS4591 inhibits EFS- and carbachol-induced contractions in rat, pig and human detrusor muscle. • NS4591 may have therapeutic potential for treatment of detrusor overactivity.

SK (KCa2) Channels Do Not Control Somatic Excitability in CA1 Pyramidal Neurons But Can Be Activated by Dendritic Excitatory Synapses and Regulate Their Impact

Calcium-activated K(+) channels of the K(Ca)2 type (SK channels) are prominently expressed in the mammalian brain, including hippocampus. These channels are thought to underlie neuronal excitability control and have been implicated in plasticity, memory, and neural disease. Contrary to previous reports, we found that somatic spike-evoked medium afterhyperpolarizations (mAHPs) and corresponding excitability control were not caused by SK channels but mainly by Kv7/KCNQ/M channels in CA1 hippocampal pyramidal neurons. Thus apparently, these SK channels are hardly activated by somatic Na(+) spikes. To further test this conclusion, we used sharp electrode, whole cell, and perforated-patch recordings from rat CA1 pyramidal neurons. We found that SK channel blockers consistently failed to suppress mAHPs under a range of experimental conditions: mAHPs following single spikes or spike trains, at -60 or -80 mV, at 20-30 degrees C, in low or elevated extracellular [K(+)], or spike trains triggered by synaptic stimulation after blocking N-methyl-d-aspartic acid receptors (NMDARs). Nevertheless, we found that SK channels in these cells were readily activated by artificially enhanced Ca(2+) spikes, and an SK channel opener (1-ethyl-2-benzimidazolinone) enhanced somatic AHPs following Na(+) spikes, thus reducing excitability. In contrast to CA1 pyramidal cells, bursting pyramidal cells in the subiculum showed a Na(+) spike-evoked mAHP that was reduced by apamin, indicating cell-type-dependent differences in mAHP mechanisms. Testing for other SK channel functions in CA1, we found that field excitatory postsynaptic potentials mediated by NMDARs were enhanced by apamin, supporting the idea that dendritic SK channels are activated by NMDAR-dependent calcium influx. We conclude that SK channels in rat CA1 pyramidal cells can be activated by NMDAR-mediated synaptic input and cause feedback regulation of synaptic efficacy but are normally not appreciably activated by somatic Na(+) spikes in this cell type.

SK Ca2+-activated K+ channel ligands alter the firing pattern of dopamine-containing neurons in vivo

Apamin-sensitive, SK channels play an important role in generating the rhythmic firing patterns exhibited by midbrain dopamine neurons in vitro. However, their contribution to the firing properties of these cells in intact animals has yet to be determined. In the present series of experiments, extracellular single unit recording techniques were used to assess the central effects of prototypical SK channel ligands on the firing pattern of dopamine neurons in the substantia nigra of the chloral hydrate anesthetized rat. I.v. administration of the SK channel blocker apamin (0.4 mg/kg), increased bursting activity in approximately 50% of the dopamine neurons tested without altering average firing rate. The majority of these cells responded slowly to the effects of apamin, gradually transitioning from an irregular single spike to a phasic discharge composed of the same relative proportion of long (≥three spike) and short (two spike) bursts as “natural” bursting activity recorded in drug naive animals. Local administration of apamin increased bursting activity in all cells tested. Systemic administration of the SK channel opener, 1-ethyl-2-benzimidazolinone (5–25 mg/kg) also had no effect on average firing rate but suppressed bursting activity and increased the precision of firing. The effects of 1-ethyl-2-benzimidazolinon on firing pattern were abolished when recording electrodes contained apamin (125 μM). These results suggest that SK channels actively contribute to the spontaneous firing patterns exhibited by dopamine neurons in vivo and provide additional support for the proposition that this channel could serve as a useful target for modifying their activity.

Modulation of calcium-dependent chloride secretion by basolateral SK4-like channels in a human bronchial cell line.

The human bronchial cell line16HBE14o- was used as a model of airway epithelial cells to study the Ca(2+)-dependent Cl(-) secretion and the identity of K(Ca) channels involved in the generation of a favorable driving force for Cl(-) exit. After ionomycin application, a calcium-activated short-circuit current ( I(sc)) developed, presenting a transient peak followed by a plateau phase. Both phases were inhibited to different degrees by NFA, glybenclamide and NPPB but DIDS was only effective on the peak phase. (86)Rb effluxes through both apical and basolateral membranes were stimulated by calcium, blocked by charybdotoxin, clotrimazole and TPA. 1-EBIO, a SK-channel opener, stimulated (86)Rb effluxes. Block of basolateral K(Ca) channels resulted in I(sc) inhibition but, while reduced, I(sc) was still observed if mucosal Cl(-) was lowered. Among SK family members, only SK4 and SK1 mRNAs were detected by RT-PCR. KCNQ1 mRNAs were also identified, but involvement of K(cAMP) channels in Cl(-) secretion was unlikely, since cAMP application had no effect on (86)Rb effluxes. Moreover, chromanol 293B or clofilium, specific inhibitors of KCNQ1 channels, had no effect on cAMP-dependent I(sc). In conclusion, two distinct components of Cl(-) secretion were identified by a pharmacological approach after a Cai2+ rise. K(Ca) channels presenting the pharmacology of SK4 channels are present on both apical and basolateral membranes, but it is the basolateral SK4-like channels that play a major role in calcium-dependent chloride secretion in 16HBE14o- cells.

Partial apamin sensitivity of human small conductance Ca2+-activated K+ channels stably expressed in Chinese hamster ovary cells.

The bee venom toxin apamin is an important drug tool for characterising small conductance Ca(2+)-activated K(+) channels (SK channels). In recombinant expression systems both rSK2 and rSK3 channels are potently blocked by apamin, whilst the sensitivity of SK1 channels is somewhat less clear. In the present study we have conducted a detailed analysis by patch clamp electrophysiology of the effects of apamin on human SK channels (SK1, SK2 and SK3) stably expressed in Chinese hamster ovary (CHO-K1) cells. CHO-K1 cell lines expressing either hSK1, 2 or 3 channels were first validated using specific antibodies and Western blotting. Specific protein bands of a size corresponding to the predicted channel tetramer (approximately 250-290 kDa) were detected. In each cell line, but not wild-type untransfected cells, large, time-independent inwardly rectifying Ca(2+)-dependent K(+) currents were observed under voltage-clamp. In CHO-hSK1, this current was markedly reduced by apamin (IC(50) value 8 nM), however, a significant fraction of the current remained unblocked (39+/-5%), even at saturating concentrations (1 microM apamin). The apamin-sensitive and -insensitive currents possess very similar biophysical and pharmacological properties. Each are Ca(2+)-dependent, inwardly rectify and have relative ionic permeabilities of K(+)>Cs(+)>Li(+)=Na(+). Both components were resistant to block by charybdotoxin and iberiotoxin, known IK and BK channel blockers, but were attenuated by the tricyclic antidepressant cyproheptadine (>95% block at 1 mM). The SK channel opener 1-EBIO could still produce channel activation in the presence of apamin. Importantly, hSK2 and hSK3 channels also exhibit partial apamin sensitivity in our experimental paradigm (IC(50) values of 0.14 nM and 1.1 nM, respectively, and maximal percentage inhibition values of 47+/-7% and 58+/-9%, respectively). Our data indicate that, at least in a recombinant expression system, all three SK channels can be partially apamin-sensitive. The explanation for this finding is presently unclear but may be due to regulatory subunits, phosphorylation or other types of post translational modification. Ascribing particular SK channels to physiological roles using apamin as a drug tool needs to be done cautiously in light of these findings.

Localization of the Activation Gate for Small Conductance Ca2+-activated K+Channels

Small conductance Ca2+-activated K+ (SK) channels open in response to increased cytosolic Ca2+ and contribute to the afterhyperpolarization in many excitable cell types. Opening of SK channels is initiated by Ca2+ binding to calmodulin that is bound to the C terminus of the channel. Based on structural information, a chemomechanical gating model has been proposed in which the chemical energy derived from Ca2+ binding is transduced into a mechanical force that restructures the protein to allow K+ ion conduction through the pore. However, the residues that comprise the physical gate of the SK channels have not been identified. In voltage-gated K+ (Kv) channels, access to the inner vestibule is controlled by a bundle crossing formed by the intracellular end of the sixth transmembrane domain (S6) of each of the four channel subunits. Probing SK channels with internally applied quaternary amines suggests that the inner vestibules of Kv and SK channels share structural similarity. Using substituted cysteine accessibility mutagenesis, the relatively large molecule [2-(trimethylammonium)] methanethiosulfonate accessed positions near the putative bundle crossing more rapidly in the open than the closed state but did not modify S6 positions closer to the selectivity filter. In contrast, the smaller compound, 2-(aminoethyl) methanethiosulfonate (MTSEA), modified a position predicted to lie in the lumen immediately intracellular to the selectivity filter equivalently in the open and closed states. The pore blocker tetrabutylammonium impeded MTSEA access to this position in both open and closed channels. The results suggest that the SK channel gate is not formed by the cytoplasmic end of S6 but resides deep in the channel pore in or near the selectivity filter.

Localization of the activation gate for small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels open in response to increased cytosolic Ca2+ and contribute to the afterhyperpolarization in many excitable cell types. Opening of SK channels is initiated by Ca2+ binding to calmodulin that is bound to the C terminus of the channel. Based on structural information, a chemomechanical gating model has been proposed in which the chemical energy derived from Ca2+ binding is transduced into a mechanical force that restructures the protein to allow K+ ion conduction through the pore. However, the residues that comprise the physical gate of the SK channels have not been identified. In voltage-gated K+ (Kv) channels, access to the inner vestibule is controlled by a bundle crossing formed by the intracellular end of the sixth transmembrane domain (S6) of each of the four channel subunits. Probing SK channels with internally applied quaternary amines suggests that the inner vestibules of Kv and SK channels share structural similarity. Using substituted cysteine accessibility mutagenesis, the relatively large molecule [2-(trimethylammonium)] methanethiosulfonate accessed positions near the putative bundle crossing more rapidly in the open than the closed state but did not modify S6 positions closer to the selectivity filter. In contrast, the smaller compound, 2-(aminoethyl) methanethiosulfonate (MTSEA), modified a position predicted to lie in the lumen immediately intracellular to the selectivity filter equivalently in the open and closed states. The pore blocker tetrabutylammonium impeded MTSEA access to this position in both open and closed channels. The results suggest that the SK channel gate is not formed by the cytoplasmic end of S6 but resides deep in the channel pore in or near the selectivity filter.