Presence of Small-Conductance Calcium-Activated Potassium (SK) Channels in the Central and Peripheral Nervous Systems and Their Role in Health and Disease.

Potassium (K+) channels establish and maintain the resting potential of most living cells. Their activity is predominantly regulated by the membrane voltage or the K+ gradient across the cell membrane. However, many cells also express small-conductance calcium-activated potassium (SK) channels, which have the unique ability to translate changes in the level of the intracellular second messenger, Ca2+ to changes in the membrane K+ conductance and, therefore, the resting membrane potential. This article reviews the structure, presence, distribution, and function of SK channels, their pharmacological modulation, and their role in health and disease, emphasizing nociception and pain.

Small conductance calcium-activated potassium channels and nitric oxide/cGMP pathway mediate cardioprotective effects of Croton urucurana Baill. In hypertensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton urucurana Baill. (Euphorbiaceae), popularly known as 'sangue de dragão' is a Brazilian species widely used in traditional medicine for cardiovascular ailments. AIM: To investigate the cardiovascular effects of the C. urucurana extract in spontaneously hypertensive rats (SHRs). MATERIALS AND METHODS: Leaves from C. urucurana were collected and morphoanatomically characterized. The ethanol-soluble fraction (ESCU) was obtained and analyzed by LC-DAD-MS. Using female Wistar rats we investigated the acute toxicity of ESCU. Then, SHRs (six months old) received vehicle, hydrochlorothiazide (25 mg/kg), or ESCU (30, 100, 300 mg/kg) for 28 days. At the beginning and at the end of treatments, urine samples were obtained to assess renal function. At the end of the trial period, the blood pressure, mesenteric vascular beds (MVBs) reactivity, and electrocardiographic profile were evaluated. Serum angiotensin-converting enzyme activity, as well as urea, creatinine, sodium, potassium, nitrite, malondialdehyde, nitrotyrosine, and aldosterone levels were determined. Relative organ weights and histopathological analysis were performed. Finally, the cardiac function on a Langendorff system, as well as the molecular mechanisms involved in the vasodilator effects of ESCU in MVBs were also investigated. RESULTS: The compounds annotated from ESCU by LC-DAD-MS included mainly phenylpropanoid derivatives, alkaloids, O-glycosylated megastigmanes, glycosylated flavonoids, flavan-3-ols, and others, such as quercetin O-deoxyhexosyl-hexoside, magnoflorine, reticuline, and taspine. None of the animals showed any signs of toxicity. Male SHRs treated only with the vehicle showed important cardiovascular changes, including a reduction in renal function, increase in serum oxidative stress, and hemodynamic, electrocardiographic, and morphological changes typical of hypertensive disease. Moreover, parameters of cardiac function, including left ventricular developed pressure, peak rate of contraction, peak rate of relaxation, and the rate pressure product were significantly altered, showing a significant impairment of ventricular function. All ESCU-doses presented a significant cardioprotective effect in SHRs rats. The 28-day treatment normalized the hemodynamic, electrocardiographic, morphological, and renal impairments, as well as reversed the changes in ventricular function induced by hypertension. In MVBs with an intact endothelium, ESCU (0.1, 0.3, and 1 mg) dose-dependently induced vasodilation. Endothelium removal or the inhibition of nitric oxide synthase prevented the vasodilatory effect of ESCU. Perfusion with a physiological saline solution that contained KCl, tetraethylammonium, or apamin also abolished the vasodilatory effect of ESCU. CONCLUSION: Prolonged ESCU-treatment showed significant cardioprotective effects in SHRs. Moreover, the data showed the role of nitric oxide and calcium-activated small conductance potassium channels in the cardiovascular effects of ESCU.

Fibroblast growth factor 19 as a countermeasure to muscle and locomotion dysfunctions in experimental cerebral palsy.

BACKGROUND: Cerebral palsy (CP) associates cerebral function damages with strong locomotor defects and premature sarcopenia. We previously showed that fibroblast growth factor 19 (FGF19) exerts hypertrophic effects on skeletal muscle and improves muscle mass and strength in mouse models with muscle atrophy. Facing the lack of therapeutics to treat locomotor dysfunctions in CP, we investigated whether FGF19 treatment could have beneficial effects in an experimental rat model of CP. METHODS: Cerebral palsy was induced in male Wistar rat pups by perinatal anoxia immediately after birth and by sensorimotor restriction of hind paws maintained until Day 28. Daily subcutaneous injections with recombinant human FGF19 (0.1 mg/kg bw) were performed from Days 22 to 28. Locomotor activity and muscle strength were assessed before and after FGF19 treatment. At Day 29, motor coordination on rotarod and various musculoskeletal parameters (weight of tibia bone and of soleus and extensor digitorum longus (EDL) muscles; area of skeletal muscle fibres) were evaluated. In addition, expression of specific genes linked to human CP was measured in rat skeletal muscles. RESULTS: Compared to controls, CP rats had reduced locomotion activity (-37.8% of distance travelled, P < 0.05), motor coordination (-88.9% latency of falls on rotarod, P < 0.05) and muscle strength (-25.1%, P < 0.05). These defects were associated with reduction in soleus (-51.5%, P < 0.05) and EDL (-42.5%, P < 0.05) weight, smaller area of muscle fibres, and with lower tibia weight (-38%, P < 0.05). In muscles from rats submitted to CP, changes in the expression levels of several genes related to muscle development and neuromuscular junctions were similar to those found in wrist muscle of children with CP (increased mRNA levels of Igfbp5, Kcnn3, Gdf8, and MyH4 and decreased expression of Myog, Ucp2 and Lpl). Compared with vehicle-treated CP rats, FGF19 administration improved locomotor activity (+53.2%, P < 0.05) and muscle strength (+25.7%, P < 0.05), and increased tibia weight (+13.8%, P < 0.05) and soleus and EDL muscle weight (+28.6% and +27.3%, respectively, P < 0.05). In addition, it reduced a number of very small fibres in both muscles (P < 0.05). Finally, gene expression analyses revealed that FGF19 might counteract the immature state of skeletal muscles induced by CP. CONCLUSIONS: These results demonstrate that pharmacological intervention with recombinant FGF19 could restore musculoskeletal and locomotor dysfunction in an experimental CP model, suggesting that FGF19 may represent a potential therapeutic strategy to combat the locomotor disorders associated with CP.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.

The Small Conductance Calcium-Activated Potassium Channel Inhibitors NS8593 and UCL1684 Prevent the Development of Atrial Fibrillation Through Atrial-Selective Inhibition of Sodium Channel Activity.

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase.

Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(ω)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 µM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.

Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia.

Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system.

Altered expression and function of small-conductance (SK) Ca(2+)-activated K+ channels in pilocarpine-treated epileptic rats.

Small conductance calcium (Ca(2+)) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold ( big up tri, open big up tri, openCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10days following status epilepticus (SE), but levels recovered at 1month and at more than 2months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100nM for 15min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1microM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE.

Constitutive expression of the alpha10 nicotinic acetylcholine receptor subunit fails to maintain cholinergic responses in inner hair cells after the onset of hearing.

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.

Developmental regulation of nicotinic synapses on cochlear inner hair cells.

In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both alpha9 and alpha10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that alpha9 mRNA is expressed in adult IHCs but that alpha10 mRNA disappears after the onset of hearing. In the present study, antibodies to alpha10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of alpha10, but not alpha9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.

Association and linkage studies between bipolar affective disorder and the polymorphic CAG/CTG repeat loci ERDA1, SEF2-1B, MAB21L and KCNN3.

Several reports have suggested the presence of anticipation in bipolar affective disorder (BPAD). In addition, independent studies using the RED (repeat expansion detection) have shown association between BPAD and longer CAG/CTG repeats. Therefore loci with large CAG/CTG repeats are plausible candidates in the inheritance of BPAD. The present study assesses the length of the repeats in four loci: the ERDA-1 locus which is known to account for most of the long CAG repeats detected by RED, the SEF2-1b locus which is placed in a region where positive linkage results have been reported and the loci MAB21L and KCNN3 as functional candidate genes. A Brazilian case-control sample with 115 unrelated BPAD patients and 196 healthy control subjects and 14 multiply affected bipolar families was investigated. With the case-control design the distribution of alleles between the two groups did not approach statistical significance. The extended transmission disequilibrium test (ETDT) performed in our families did not show evidence for linkage disequilibrium. Parametric and non-parametric linkage analysis also did not provide support for linkage between any of the four loci and BPAD. Our data do not support the hypothesis that variation at the polymorphic CAG/CTG repeat loci ERDA-1, SEF2-1b, MAB21L or KCNN3 influence susceptibility to BPAD in our sample.

Involvement of soluble guanylate cyclase and calcium-activated potassium channels in the long-lasting hyporesponsiveness to phenylephrine induced by nitric oxide in rat aorta.

Excessive nitric oxide (NO) production by inducible NO synthase has been implicated in the hyporesponsiveness to vasoconstrictors present in septic shock. Here we show that a brief incubation (30 min) of rat aorta rings with NO donors renders the vessels hyporesponsive to phenylephrine for several hours. Contraction of rings without endothelium by phenylephrine (0.1 nM to 100 microM) was decreased by 50-60% after incubation (30 min) with sodium nitroprusside (3-300 microM) or S-nitroso-acetyl-D,L-penicillamine (SNAP; 70-200 microM). This decrease was characterized by reductions in maximal response and rightwards shifts of phenylephrine concentration/response curves, present even 130 min after NO donor removal. Soluble guanylate cyclase inhibitors methylene blue ( 10 microM) and 1H-(1,2,4)-oxadiazol-(4,3-a)quinoxalin-1-one (ODQ, 1 microM) or the potassium channel blockers TEA (tetraethylammonium; 10 mM) and charybdotoxin (100 nM) inhibited the hyporesponsiveness to phenylephrine induced by the NO donors. In contrast, 4-aminopyridine (1 mM) and glibenclamide (10 microM) had no effect. Our results show that incubation with NO donors reproduces the hyporesponsiveness to phenylephrine and that NO alone accounts for most, if not all, the refractoriness to vasoconstrictors present in septic shock. In addition, soluble guanylate cyclase activation and opening of potassium channels, more specifically the calcium-activated subtype, play a predominant role in this NO-induced hyporesponsiveness to phenylephrine in the rat aorta.