Structural Titration of Slo2.2, a Na+-Dependent K+ Channel.

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel.

The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.

Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

Slo2/KNa Channels in Drosophila Protect against Spontaneous and Induced Seizure-like Behavior Associated with an Increased Persistent Na+ Current.

Na+ sensitivity is a unique feature of Na+-activated K+ (KNa) channels, making them naturally suited to counter a sudden influx in Na+ ions. As such, it has long been suggested that KNa channels may serve a protective function against excessive excitation associated with neuronal injury and disease. This hypothesis, however, has remained largely untested. Here, we examine KNa channels encoded by the Drosophila Slo2 (dSlo2) gene in males and females. We show that dSlo2/KNa channels are selectively expressed in cholinergic neurons in the adult brain, as well as in glutamatergic motor neurons, where dampening excitation may function to inhibit global hyperactivity and seizure-like behavior. Indeed, we show that effects of feeding Drosophila a cholinergic agonist are exacerbated by the loss of dSlo2/KNa channels. Similar to mammalian Slo2/KNa channels, we show that dSlo2/KNa channels encode a TTX-sensitive K+ conductance, indicating that dSlo2/KNa channels can be activated by Na+ carried by voltage-dependent Na+ channels. We then tested the role of dSlo2/KNa channels in established genetic seizure models in which the voltage-dependent persistent Na+ current (INap) is elevated. We show that the absence of dSlo2/KNa channels increased susceptibility to mechanically induced seizure-like behavior. Similar results were observed in WT flies treated with veratridine, an enhancer of INap Finally, we show that loss of dSlo2/KNa channels in both genetic and pharmacologically primed seizure models resulted in the appearance of spontaneous seizures. Together, our results support a model in which dSlo2/KNa channels, activated by neuronal overexcitation, contribute to a protective threshold to suppress the induction of seizure-like activity.SIGNIFICANCE STATEMENT Slo2/KNa channels are unique in that they constitute a repolarizing K+ pore that is activated by the depolarizing Na+ ion, making them naturally suited to function as a protective "brake" against overexcitation and Na+ overload. Here, we test this hypothesis in vivo by examining how a null mutation of the Drosophila Slo2 (dSlo2)/KNa gene affects seizure-like behavior in genetic and pharmacological models of epilepsy. We show that indeed the loss of dSlo2/KNa channels results in increased incidence and severity of induced seizure behavior, as well as the appearance of spontaneous seizure activity. Our results advance our understanding of neuronal excitability and protective mechanisms that preserve normal physiology and the suppression of seizure susceptibility.

Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

Structure-activity relationship studies in a new series of 2-amino-N-phenylacetamide inhibitors of Slack potassium channels.

In this Letter we describe structure-activity relationship (SAR) studies conducted in five distinct regions of a new 2-amino-N-phenylacetamides series of Slack potassium channel inhibitors exemplified by recently disclosed high-throughput screening (HTS) hit VU0606170 (4). New analogs were screened in a thallium (Tl+) flux assay in HEK-293 cells stably expressing wild-type human (WT) Slack. Selected analogs were screened in Tl+ flux versus A934T Slack and other Slo family members Slick and Maxi-K and evaluated in whole-cell electrophysiology (EP) assays using an automated patch clamp system. Results revealed the series to have flat SAR with significant structural modifications resulting in a loss of Slack activity. More minor changes led to compounds with Slack activity and Slo family selectivity similar to the HTS hit.

Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids.

Recent identification of several different types of RNA editing factors in plant organelles suggests complex RNA editosomes within which each factor has a different task. However, the precise protein interactions between the different editing factors are still poorly understood. In this paper, we show that the E+-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine deaminase-like DYW domain, interacts in vivo with the DYW-type PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones. These results may reflect a protein scaffold or complex stabilization role of NUWA between E+-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP overexpressing lines show broad editing defects in both organelles, with predominant specificity for sites edited by E+-type PPR proteins. The latter suggests a coordinated regulation of organellar multiple site editing through DYW2, which probably provides the deaminase activity to E+ editosomes.

HRPU-2, a Homolog of Mammalian hnRNP U, Regulates Synaptic Transmission by Controlling the Expression of SLO-2 Potassium Channel in Caenorhabditis elegans.

Slo2 channels are large-conductance potassium channels abundantly expressed in the nervous system. However, it is unclear how their expression level in neurons is regulated. Here we report that HRPU-2, an RNA-binding protein homologous to mammalian heterogeneous nuclear ribonucleoprotein U (hnRNP U), plays an important role in regulating the expression of SLO-2 (a homolog of mammalian Slo2) in Caenorhabditis elegans Loss-of-function (lf) mutants of hrpu-2 were isolated in a genetic screen for suppressors of a sluggish phenotype caused by a hyperactive SLO-2. In hrpu-2(lf) mutants, SLO-2-mediated delayed outward currents in neurons are greatly decreased, and neuromuscular synaptic transmission is enhanced. These mutant phenotypes can be rescued by expressing wild-type HRPU-2 in neurons. HRPU-2 binds to slo-2 mRNA, and hrpu-2(lf) mutants show decreased SLO-2 protein expression. In contrast, hrpu-2(lf) does not alter the expression of either the BK channel SLO-1 or the Shaker type potassium channel SHK-1. hrpu-2(lf) mutants are indistinguishable from wild type in gross motor neuron morphology and locomotion behavior. Together, these observations suggest that HRPU-2 plays important roles in SLO-2 function by regulating SLO-2 protein expression, and that SLO-2 is likely among a restricted set of proteins regulated by HRPU-2. Mutations of human Slo2 channel and hnRNP U are strongly linked to epileptic disorders and intellectual disability. The findings of this study suggest a potential link between these two molecules in human patients.SIGNIFICANCE STATEMENT Heterogeneous nuclear ribonucleoprotein U (hnRNP U) belongs to a family of RNA-binding proteins that play important roles in controlling gene expression. Recent studies have established a strong link between mutations of hnRNP U and human epilepsies and intellectual disability. However, it is unclear how mutations of hnRNP U may cause such disorders. This study shows that mutations of HRPU-2, a worm homolog of mammalian hnRNP U, result in dysfunction of a Slo2 potassium channel, which is critical to neuronal function. Because mutations of Slo2 channels are also strongly associated with epileptic encephalopathies and intellectual disability in humans, the findings of this study point to a potential mechanism underlying neurological disorders caused by hnRNP U mutations.

Oxytocin can regulate myometrial smooth muscle excitability by inhibiting the Na+ -activated K+ channel, Slo2.1.

KEY POINTS: At the end of pregnancy, the uterus transitions from a quiescent state to a highly contractile state. This transition requires that the uterine (myometrial) smooth muscle cells increase their excitability, although how this occurs is not fully understood. We identified SLO2.1, a potassium channel previously unknown in uterine smooth muscle, as a potential significant contributor to the electrical excitability of myometrial smooth muscle cells. We found that activity of the SLO2.1 channel is negatively regulated by oxytocin via Gαq-protein-coupled receptor activation of protein kinase C. This results in depolarization of the uterine smooth muscle cells and calcium entry, which may contribute to uterine contraction. These findings provide novel insights into a previously unknown mechanism by which oxytocin may act to modulate myometrial smooth muscle cell excitability. Our findings also reveal a new potential pharmacological target for modulating uterine excitability. ABSTRACT: During pregnancy, the uterus transitions from a quiescent state to a more excitable contractile state. This is considered to be at least partly a result of changes in the myometrial smooth muscle cell (MSMC) resting membrane potential. However, the ion channels controlling the myometrial resting membrane potential and the mechanism of transition to a more excitable state have not been fully clarified. In the present study, we show that the sodium-activated, high-conductance, potassium leak channel, SLO2.1, is expressed and active at the resting membrane potential in MSMCs. Additionally, we report that SLO2.1 is inhibited by oxytocin binding to the oxytocin receptor. Inhibition of SLO2.1 leads to membrane depolarization and activation of voltage-dependent calcium channels, resulting in calcium influx. The results of the present study reveal that oxytocin may modulate MSMC electrical activity by inhibiting SLO2.1 potassium channels.

Sodium-activated potassium channels moderate excitability in vascular smooth muscle.

KEY POINTS: We report that a sodium-activated potassium current, IKNa , has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells. IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice. The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild-type with raised blood pressure when challenged with vasoconstrictive agents. IKNa is negatively regulated by angiotensin II acting through Gαq protein-coupled receptors. In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+ -dependent action potentials. ABSTRACT: Although several potassium currents have been reported to play a role in arterial smooth muscle (ASM), we find that one of the largest contributors to membrane conductance in both conduit and resistance ASMs has been inadvertently overlooked. In the present study, we show that IKNa , a sodium-activated potassium current, contributes a major portion of macroscopic outward current in a critical physiological voltage range that determines intrinsic cell excitability; IKNa is the largest contributor to ASM cell resting conductance. A genetic knockout (KO) mouse strain lacking KNa channels (KCNT1 and KCNT2) shows only a modest hypertensive phenotype. However, acute administration of vasoconstrictive agents such as angiotensin II (Ang II) and phenylephrine results in an abnormally large increase in blood pressure in the KO animals. In wild-type animals Ang II acting through Gαq protein-coupled receptors down-regulates IKNa , which increases the excitability of the ASMs. The complete genetic removal of IKNa in KO mice makes the mutant animal more vulnerable to vasoconstrictive agents, thus producing a paroxysmal-hypertensive phenotype. This may result from the lowering of cell resting K+ conductance allowing the cells to depolarize more readily to a variety of excitable stimuli. Thus, the sodium-activated potassium current may serve to moderate blood pressure in instances of heightened stress. IKNa may represent a new therapeutic target for hypertension and stroke.

Cardiac metabolic effects of KNa1.2 channel deletion and evidence for its mitochondrial localization.

Controversy surrounds the molecular identity of mitochondrial K+ channels that are important for protection against cardiac ischemia-reperfusion injury. Although KNa1.2 (sodium-activated potassium channel encoded by Kcn2) is necessary for cardioprotection by volatile anesthetics, electrophysiological evidence for a channel of this type in mitochondria is lacking. The endogenous physiological role of a potential mito-KNa1.2 channel is also unclear. In this study, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild-type (WT) mice yielded 6 channels matching the known ion sensitivity, ion selectivity, pharmacology, and conductance properties of KNa1.2 (slope conductance, 138 ± 1 pS). However, similar experiments on 40 preparations from Kcnt2-/- mice yielded no such channels. The KNa opener bithionol uncoupled respiration in WT but not Kcnt2-/- cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2-/- cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2-/- mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial KNa1.2 channel, and a role for cardiac KNa1.2 in regulating metabolism under conditions of high energetic demand.-Smith, C. O., Wang, Y. T., Nadtochiy, S. M., Miller, J. H., Jonas, E. A., Dirksen, R. T., Nehrke, K., Brookes, P. S. Cardiac metabolic effects of KNa1.2 channel deletion and evidence for its mitochondrial localization.

Morphophysiological and molecular evidence supporting the augmentative role of Piriformospora indica in mitigation of salinity in Cucumis melo L.

Salinity is one of the major limiting factors in plant growth and productivity. Cucumis melo L. is a widely cultivated plant, but its productivity is significantly influenced by the level of salinity in soil. Symbiotic colonization of plants with Piriformospora indica has shown a promotion in plants growth and tolerance against biotic stress. In this study, physiological markers such as ion analysis, antioxidant determination, proline content, electrolyte leakage and chlorophyll measurement were assessed in melon cultivar under two concentrations (100 and 200 mM) of NaCl with and without P. indica inoculation. Results showed that the endophytic inoculation consistently upregulated the level of antioxidants, enhanced plants to antagonize salinity stress. The expression level of an RNA editing factor (SLO2) which is known to participate in mitochondria electron transport chain was analyzed, and its full mRNA sequence was obtained by rapid amplification of cDNA ends (RACE). Under salinity stress, the expression level of SLO2 was increased, enhancing the plant's capability to adapt to the stress. However, P. indica inoculation further elevated the expression level of SLO2. These findings suggested that the symbiotic association of fungi could help the plants to tolerate the salinity stress.

SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels.

Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 µmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions.

Hydrophobic interactions between the S5 segment and the pore helix stabilizes the closed state of Slo2.1 potassium channels.

Under normal physiological conditions, Slo2.1K(+) channels are in a closed state unless activated by an elevation in [Na(+)]i. Fenamates such as niflumic acid also activate Slo2.1. Previous studies suggest that activation of Slo2.1 channels is mediated by a conformational change in the selectivity filter, and not a widening of the aperture formed by the S6 segment bundle crossing as occurs in voltage-gated K(+) channels. It is unclear how binding of Na(+) or fenamates is allosterically linked to opening of the presumed selectivity filter activation gate in Slo2.1. Here we examined the role of the S5 transmembrane segment in the activation of Slo2.1. Channels were heterologously expressed in Xenopus laevis oocytes and whole cell currents measured with the voltage-clamp technique. Ala substitution of five residues located on a single face of the S5 α-helical segment induced constitutive channel activity. Leu-209, predicted to face towards Phe-240 in the pore helix was investigated by further mutagenesis. Mutation of Leu-209 to Glu or Gln induced maximal channel activation as did the combined mutation to Ala of all three hydrophobic S5 residues predicted to be adjacent to Phe-240. Together these results suggest that hydrophobic interactions between residues in S5 and the C-terminal end of the pore helix stabilize Slo2.1 channels in a closed state.

Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels.

Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na(+)]i (e.g. during ischemia). An intracellular Na(+) coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na(+) coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na(+) sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 M NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]i of 70 mM. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na(+). In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mM niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]i from 3 to 100 mM. Thus, relative to fenamates, intracellular Na(+) is a poor activator of Slo2.1.

SLO2.1/NALCN Functional Complex Activity in Mouse Myometrial Smooth Muscle Cells During Pregnancy.

At the end of pregnancy, the uterus transitions from a quiescent to a highly contractile state. This is partly due to depolarization of the resting membrane potential in uterine (myometrial) smooth muscle cells (MSMCs). Experiments with human MSMCs showed that the membrane potential is regulated by a functional complex between the sodium (Na+)-activated potassium (K+) channel SLO2.1 and the Na+ Leak Channel Non-Selective (NALCN). In human MSMCs, Na+ entering through NALCN activates SLO2.1, leading to K+ efflux, membrane hyperpolarization (cells become more negative inside), and reduced contractility. Decreased SLO2.1/NALCN activity results in reduced K+ efflux, leading to membrane depolarization, Ca2+ influx via voltage-dependent calcium channels, and increased MSMC contractility. However, all of these experiments were performed with MSMCs isolated from women at term, so the role of the SLO2.1/NALCN complex early in pregnancy was speculative. To address this question here, we examined the role of the SLO2.1/NALCN complex in regulating mouse MSMC membrane potential across pregnancy. We report that Slo2.1 and Nalcn expression change along pregnancy, being more highly expressed in MSMCs from non-pregnant and early pregnant mice than in those from late-pregnant mice. Functional studies revealed that SLO2.1 channels mediate a significant portion of the K+ current in mouse MSMCs, particularly in cells from non-pregnant and early pregnant mice. Activation of SLO2.1 by Na+ influx through NALCN led to membrane hyperpolarization in MSMCs from early pregnancy but not in MSMCs from later pregnancy. Moreover, we found that the NALCN/SLO2.1 complex regulates intracellular Ca2+ responses more in MSMCs from non-pregnant and early pregnancy mice than in MSMCs from late pregnancy. Together, these findings reveal that the SLO2.1/NALCN functional complex is conserved between mouse and humans and functions throughout pregnancy. This work could open avenues for targeted pharmacological interventions in pregnancy-related complications.

Slack K+ channels confer protection against myocardial ischemia/reperfusion injury.

AIMS: Na+-activated Slack potassium (K+) channels are increasingly recognized as regulators of neuronal activity, yet little is known about their role in the cardiovascular system. Slack activity increases when intracellular Na+ concentration ([Na+]i) reaches pathophysiological levels. Elevated [Na+]i is a major determinant of the ischemia and reperfusion (I/R)-induced myocardial injury, thus we hypothesized that Slack plays a role under these conditions. METHODS: and results: K+ currents in cardiomyocytes (CMs) obtained from wildtype (WT) but not from global Slack knockout (KO) mice were sensitive to electrical inactivation of voltage-sensitive Na+-channels. Live-cell imaging demonstrated that K+ fluxes across the sarcolemma rely on Slack, while the depolarized resting membrane potential in Slack-deficient CMs led to excessive cytosolic Ca2+ accumulation and finally to hypoxia/reoxygenation-induced cell death. Cardiac damage in an in vivo model of I/R was exacerbated in global and CM-specific conditional Slack mutants and largely insensitive to mechanical conditioning maneuvers. Finally, the protection conferred by mitochondrial ATP-dependent K+ channels required functional Slack in CMs. CONCLUSIONS: Collectively, our study provides evidence for Slack's crucial involvement in the ion homeostasis of no or low O2-stressed CMs. Thereby, Slack activity opposes the I/R-induced fatal Ca2+-uptake to CMs supporting the cardioprotective signaling widely attributed to mitoKATP function.

Euchromatin histone-lysine N-methyltransferase 2 regulates the expression of potassium-sodium-activated channel subfamily T member 1 in primary sensory neurons and contributes to remifentanil-induced pain sensitivity.

Intraoperative remifentanil administration has been linked to increased postoperative pain sensitivity. Recent studies have identified the involvement of euchromatic histone-lysine N-methyltransferase 2 (Ehmt2/G9a) in neuropathic pain associated with the transcriptional silencing of many potassium ion channel genes. This study investigates whether G9a regulates the potassium sodium-activated channel subfamily T member 1 (Slo2.2) in remifentanil-induced post-incisional hyperalgesia (RIH) in rodents. We performed remifentanil infusion (1 µg·kg-1·min-1 for 60 min) followed by plantar incision to induce RIH in rodents. Our results showed that RIH was accompanied by increased G9a and H3K9me2 production and decreased Slo2.2 expression 48 h postoperatively. Deletion of G9a rescued Slo2.2 expression in DRG and reduced RIH intensity. Slo2.2 overexpression also reversed this hyperalgesia phenotype. G9a overexpression decreased Slo2.2-mediated leak current and increased excitability in the small-diameter DRG neurons and laminal II small-diameter neurons in the spinal dorsal horn, which was implicated in peripheral and central sensitization. These results suggest that G9a contributes to the development of RIH by epigenetically silencing Slo2.2 in DRG neurons, leading to decreased central sensitization in the spinal cord. The findings may have implications for the development of novel therapeutic targets for the treatment of postoperative pain.

Structural basis of human Slo2.2 channel gating and modulation.

The sodium-activated Slo2.2 channel is abundantly expressed in the brain, playing a critical role in regulating neuronal excitability. The Na+-binding site and the underlying mechanisms of Na+-dependent activation remain unclear. Here, we present cryoelectron microscopy (cryo-EM) structures of human Slo2.2 in closed, open, and inhibitor-bound form at resolutions of 2.6-3.2 Å, revealing gating mechanisms of Slo2.2 regulation by cations and a potent inhibitor. The cytoplasmic gating ring domain of the closed Slo2.2 harbors multiple K+ and Zn2+ sites, which stabilize the channel in the closed conformation. The open Slo2.2 structure reveals at least two Na+-sensitive sites where Na+ binding induces expansion and rotation of the gating ring that opens the inner gate. Furthermore, a potent inhibitor wedges into a pocket formed by pore helix and S6 helix and blocks the pore. Together, our results provide a comprehensive structural framework for the investigation of Slo2.2 channel gating, Na+ sensation, and inhibition.