Distinct neurodevelopmental and epileptic phenotypes associated with gain- and loss-of-function GABRB2 variants.

BACKGROUND: Variants in GABRB2, encoding the ß2 subunit of the γ-aminobutyric acid type A (GABAA) receptor, can result in a diverse range of conditions, ranging from febrile seizures to severe developmental and epileptic encephalopathies. However, the mechanisms underlying the risk of developing milder vs more severe forms of disorder remain unclear. In this study, we conducted a comprehensive genotype-phenotype correlation analysis in a cohort of individuals with GABRB2 variants. METHODS: Genetic and electroclinical data of 42 individuals harbouring 26 different GABRB2 variants were collected and accompanied by electrophysiological analysis of the effects of the variants on receptor function. FINDINGS: Electrophysiological assessments of α1ß2γ2 receptors revealed that 25/26 variants caused dysfunction to core receptor properties such as GABA sensitivity. Of these, 17 resulted in gain-of-function (GOF) while eight yielded loss-of-function traits (LOF). Genotype-phenotype correlation analysis revealed that individuals harbouring GOF variants suffered from severe developmental delay/intellectual disability (DD/ID, 74%), movement disorders such as dystonia or dyskinesia (59%), microcephaly (50%) and high risk of early mortality (26%). Conversely, LOF variants were associated with milder disease manifestations. Individuals with these variants typically exhibited fever-triggered seizures (92%), milder degrees of DD/ID (85%), and maintained ambulatory function (85%). Notably, severe movement disorders or microcephaly were not reported in individuals with loss-of-function variants. INTERPRETATION: The data reveals that genetic variants in GABRB2 can lead to both gain and loss-of-function, and this divergence is correlated with distinct disease manifestations. Utilising this information, we constructed a diagnostic flowchart that aids in predicting the pathogenicity of recently identified variants by considering clinical phenotypes. FUNDING: This work was funded by the Australian National Health & Medical Research Council, the Novo Nordisk Foundation and The Lundbeck Foundation.

The shortcoming of using glibenclamide in exploratory clinical headache provocation studies.

OBJECTIVE: Preclinical and clinical studies implicate the vascular ATP-sensitive potassium (KATP) channel in the signaling cascades underlying headache and migraine. However, attempts to demonstrate that the KATP channel inhibitor glibenclamide would attenuate triggered headache in healthy volunteers have proven unsuccessful. It is questionable, however, whether target engagement was achieved in these clinical studies. METHODS: Literature data for human glibenclamide pharmacokinetics, plasma protein binding and functional IC50 values were used to predict the KATP receptor occupancy (RO) levels obtained after glibenclamide dosing in the published exploratory clinical headache provocation studies. RO vs. time profiles of glibenclamide were simulated for the pancreatic KATP channel subtype Kir6.2/SUR1 and the vascular subtype Kir6.1/SUR2B. RESULTS: At the clinical dose of 10 mg of glibenclamide used in the headache provocation studies, predicted maximal occupancy levels of up to 90% and up to 26% were found for Kir6.2/SUR1 and Kir6.1/SUR2B, respectively. CONCLUSIONS: The findings of the present study indicate that effective Kir6.1/SUR2B target engagement was not achieved in the clinical headache provocation studies using glibenclamide. Therefore, development of novel selective Kir6.1/SUR2B inhibitors, with good bioavailability and low plasma protein binding, is required to reveal the potential of KATP channel inhibition in the treatment of migraine.

Pharmacological Profiling of KATP Channel Modulators: An Outlook for New Treatment Opportunities for Migraine.

Migraine is a highly disabling pain disorder with huge socioeconomic and personal costs. It is genetically heterogenous leading to variability in response to current treatments and frequent lack of response. Thus, new treatment strategies are needed. A combination of preclinical and clinical data indicate that ATP-sensitive potassium (KATP) channel inhibitors could be novel and highly effective drugs in the treatment of migraine. The subtype Kir6.1/SUR2B is of particular interest and inhibitors specific for this cranio-vascular KATP channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize KATP channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different KATP subtypes is difficult to assess. Here, we characterize available KATP channel activators and inhibitors in fluorescence-based thallium-flux assays using HEK293 cells stably expressing human Kir6.1/SUR2B, Kir6.2/SUR1, and Kir6.2/SUR2A KATP channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the KATP channel subtype Kir6.1/SUR2B, whereas BMS-191095, NN414, and VU0071306 demonstrated preferred activation of the Kir6.2/SUR1 subtype. In the group of KATP channel blockers, only Rosiglitazone and PNU-37783A showed selective inhibition of the Kir6.1/SUR2B subtype. PNU-37783A was stopped in clinical development and Rosiglitazone has a low potency for the vascular KATP channel subtype. Therefore, development of novel selective KATP channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.

Retroviral glycoprotein-mediated immune suppression via the potassium channel KCa3.1 - A new strategy for amelioration of inflammatory bowel diseases.

Peptides derived from retroviral envelope proteins have been shown to possess a wide range of immunosuppressive and anti-inflammatory activities. We have previously reported identification of such a peptide derived from the envelope protein coded by a human endogenous retrovirus (HERV). In this study, we identify that in vitro the peptide inhibits the KCa3.1 potassium channel, a potential target for therapy of immune diseases. We describe in vitro ENV59-GP3 effects with respect to potency of inhibition on KCa3.1 channels and calcium influx. Furthermore, we asses in vivo the effect of blocking KCa3.1 with ENV59-GP3 peptide or KCa3.1-blocker NS6180 on protection against DSS-induced acute colitis. ENV59-GP3 peptide treatment showed reduction of the disease score in the DSS-induced acute colitis mice model, which was comparable to effects of the KCa3.1 channel blocker NS6180. Analysis of cytokine production from DSS-mice model treated animals revealed equipotent inhibitory effects of the ENV59-GP3 and NS6180 compounds on the production of IL-6, TNF-α, IL-1ß. These findings altogether suggest that ENV59-GP3 functions as a KCa3.1 channel inhibitor and underline the implications of using virus derived channel blockers for treatment of autoimmune diseases. Additionally, they open the possibilities whether KCa3.1 inhibition is efficacious in patients with inflammatory bowel diseases.

Four phase 1 trials to evaluate the safety and pharmacokinetic profile of single and repeated dosing of SCO-101 in adult male and female volunteers.

SCO-101 (Endovion) was discontinued 20 years ago as a new drug under development against sickle cell anaemia. Data from the phase 1 studies remained unpublished. New data indicate that SCO-101 might be efficacious as add-on therapy in cancer. Thus, we report the results from the four phase 1 trials performed between 2001 and 2002. Adult volunteers received SCO-101 or placebo in four independent trials. Adverse events were recorded, and SCO-101 was determined for pharmacokinetic analysis. Ninety-two volunteers completed the trials. The most remarkable adverse effect was a transient and dose-dependent increase in unconjugated bilirubin. Plasma SCO-101 elimination was approximately log linear, with apparent oral clearances of between 315 and 2103 mL/h for single doses, and between 121 and 2433 mL/h at steady state following oral administration. There was a marked decrease in clearance with increasing dose, and for repeated dose versus single dose. Tmax was greater, and Cmax and AUC∞ were lower in the fed state compared to the fasted state. Exposure was equivalent in males and females and for African Americans and Caucasians. In conclusion, SCO-101 appears to be a safe drug with a predictable PK profile. Its efficacy as add-on to standard anticancer drugs has yet to be defined.

Characterization of AN317, a novel selective agonist of α6ß2-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6ß2-containing (α6ß2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

Characterization of AN6001, a positive allosteric modulator of α6ß2-containing nicotinic acetylcholine receptors.

α6ß2-Containing nicotinic acetylcholine receptors (α6ß2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4ß2. Here, we report the discovery and characterization of AN6001, a novel selective α6ß2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3ß2ß3V9'S nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3ß2ß3V9'S and a concatenated ß3-α6-ß2-α6-ß2 receptor in Xenopus oocytes, displaying EC50 values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4ß2, α3ß4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca2+ imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP+-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6ß2* nAChRs and that positive modulation of native α6ß2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6ß2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson's disease.

Pharmacology of Small- and Intermediate-Conductance Calcium-Activated Potassium Channels.

The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K+ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.

Antibodies and venom peptides: new modalities for ion channels.

Ion channels play fundamental roles in both excitable and non-excitable tissues and therefore constitute attractive drug targets for myriad neurological, cardiovascular and metabolic diseases as well as for cancer and immunomodulation. However, achieving selectivity for specific ion channel subtypes with small-molecule drugs has been challenging, and there currently is a growing trend to target ion channels with biologics. One approach is to improve the pharmacokinetics of existing or novel venom-derived peptides. In parallel, after initial studies with polyclonal antibodies demonstrated the technical feasibility of inhibiting channel function with antibodies, multiple preclinical programmes are now using the full spectrum of available technologies to generate conventional monoclonal and engineered antibodies or nanobodies against extracellular loops of ion channels. After a summary of the current state of ion channel drug discovery, this Review discusses recent developments using the purinergic receptor channel P2X purinoceptor 7 (P2X7), the voltage-gated potassium channel KV1.3 and the voltage-gated sodium channel NaV1.7 as examples of targeting ion channels with biologics.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Potassium channel expression and function in microglia: Plasticity and possible species variations.

Potassium channels play important roles in microglia functions and thus constitute potential targets for the treatment of neurodegenerative diseases like Alzheimer, Parkinson and stroke. However, uncertainty still prevails as to which potassium channels are expressed and at what levels in different species, how the expression pattern changes upon activation with M1 or M2 polarizing stimuli compared with more complex exposure paradigms, and - most importantly - how these findings relate to the in vivo situation. In this mini-review we discuss the functional potassium channel expression pattern in cultured neonatal mouse microglia in the light of data obtained previously from animal disease models and immunohistochemical studies and compare it with a recent study of adult human microglia isolated from epilepsy patients. Overall, microglial potassium channel expression is very plastic and possibly shows species differences and therefore should be studied carefully in each disease setting and respective animal models.

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

The KCa 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of KCa 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a KCa 3.1/KCa 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the KCa 3.1 current nor the fraction of KCa 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the KCa 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the KCa 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a Kir channel. The high functional expression of KCa 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078.

Pharmacological gating modulation of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2.x and KCa3.1).

This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2 and KCa3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite KCa channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative KCa2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective KCa modulators entering into drug development for these indications.

NS19504: a novel BK channel activator with relaxing effect on bladder smooth muscle spontaneous phasic contractions.

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

Kv7 channels as targets for anti-epileptic and psychiatric drug-development.

The Kv7 channels, a family of voltage-dependent K(+) channels (Kv7.1-Kv7.5), have gained much attention in drug discovery especially because four members are genetically linked to diseases. For disorders of the CNS focus was originally on epilepsy and pain, but it is becoming increasingly evident that Kv7 channels can also be valid targets for psychiatric disorders, such as anxiety and mania. The common denominator is probably neuronal hyperexcitability in different brain areas, which can be successfully attenuated by pharmacological increment of Kv7 channel activity. This perspective attempts to review the current status and challenges for CNS drug discovery based on Kv7 channels as targets for neurological and psychiatric indications with special focus on selectivity and mode-of-actions.

Characterization of a novel high-potency positive modulator of K(v)7 channels.

K(v)7 channel activators decrease neuronal excitability and might potentially treat neuronal hyperexcitability disorders like epilepsy and mania. Here we introduce NS15370 ((2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3-pyridyl]acetamide)hydrochloride, an in vitro high-potency chemical analogue of retigabine, without effects on GABA(A) receptors. NS15370 activates recombinant homo- and heteromeric K(v)7.2-K(v)7.5 channels in HEK293 cells at sub-micromolar concentrations (EC50~100 nM, as quantified by a fluorescence based Tl⁺-influx assay). In voltage clamp experiments NS15370 exhibits a complex, concentration-dependent mode-of-action: At low concentrations it accelerates voltage-dependent activation rates, slows deactivations, and increases steady-state current amplitudes. Quantified by the peak-tail current method, the V½ value of the steady-state activation curve is shifted towards hyperpolarized potentials at concentrations ~100 times lower than retigabine. However, in contrast to retigabine, NS15370 also introduces a distinct time-dependent current decrease, which eventually, at higher concentrations, causes suppression of the current at depolarized potentials, and an apparent "cross-over" of the voltage-activation curve. In brain slices, NS15370 hyperpolarizes and increases spike frequency adaptation of hippocampal CA1 neurons and the compound reduces the autonomous firing of dopaminergic neurons in the substantia-nigra pars compacta. NS15370 is effective in rodent models of hyperexcitability: (i) it yields full protection against mouse 6 Hz seizures and rat amygdala kindling discharges, two models of partial epilepsia; (ii) it reduces (+)-MK-801 hydrogen maleate (MK-801)-induced hyperactivity as well as chlordiazepoxide (CDP)+d-amphetamine (AMP)-induced hyperactivity, models sensitive to classic anti-psychotic and anti-manic treatments, respectively. Our findings with NS15370 consolidate neuronal K(v)7 channels as targets for anti-epileptic and psychiatric drug development.

A high-throughput screening campaign for detection of ca(2+)-activated k(+) channel activators and inhibitors using a fluorometric imaging plate reader-based tl(+)-influx assay.

The intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) has been proposed to play many physiological roles, and modulators of KCa3.1 activity are potentially interesting as new drugs. In order to identify new chemical scaffolds, high-throughput screening (HTS) assays are needed. In the current study, we present an HTS assay that has been optimized for the detection of inhibitors as well as activators of KCa3.1 in a combined assay. We used HEK293 cells heterologously expressing KCa3.1 in a fluorescence-based Tl(+) influx assay, where the permeability of potassium channels to Tl(+) is taken advantage of. We found the combined activator-and-inhibitor assay to be robust and insensitive to dimethyl sulfoxide (up to 1%), and conducted an HTS campaign of 217,119 small molecules. In total, 224 confirmed activators and 312 confirmed inhibitors were found, which corresponded to a hit rate of 0.10% and 0.14%, respectively. The confirmed hits were further characterized in a fluorometric imaging plate reader-based concentration-response assay, and selected compounds were subjected to secondary testing in an assay for endogenous KCa3.1 activity using human erythrocytes (red blood cell assay). Although the estimated potencies were slightly higher in the RBC assay, there was an overall good correlation across all clusters. The campaign led to the identification of several chemical series of KCa3.1 activators and inhibitors, comprising already known pharmacophores and new chemical series. One of these were the benzothiazinones that constitute a new class of highly potent KCa3.1 inhibitors, exemplified by 4-{[3-(trifluoromethyl)phenyl]methyl}-2H-1,4-benzothiazin-3(4H)-one (NS6180).

Selective positive modulator of calcium-activated potassium channels exerts beneficial effects in a mouse model of spinocerebellar ataxia type 2.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by a polyglutamine expansion within the Ataxin-2 (Atxn2) protein. Purkinje cells (PC) of the cerebellum fire irregularly and eventually die in SCA2. We show here that the type 2 small conductance calcium-activated potassium channel (SK2) play a key role in control of normal PC activity. Using cerebellar slices from transgenic SCA2 mice we demonstrate that SK channel modulators restore regular pacemaker activity of SCA2 PCs. Furthermore, we also show that oral delivery of a more selective positive modulator of SK2/3 channels (NS13001) alleviates behavioral and neuropathological phenotypes of aging SCA2 transgenic mice. We conclude that SK2 channels constitute a therapeutic target for SCA2 treatment and that the developed selective SK2/3 modulator NS13001 holds promise as a potential therapeutic agent for treatment of SCA2 and possibly other cerebellar ataxias.

CyPPA, a Positive SK3/SK2 Modulator, Reduces Activity of Dopaminergic Neurons, Inhibits Dopamine Release, and Counteracts Hyperdopaminergic Behaviors Induced by Methylphenidate.

Dopamine (DA) containing midbrain neurons play critical roles in several psychiatric and neurological diseases, including schizophrenia and attention deficit hyperactivity disorder, and the substantia nigra pars compacta neurons selectively degenerate in Parkinson's disease. Pharmacological modulation of DA receptors and transporters are well established approaches for treatment of DA-related disorders. Direct modulation of the DA system by influencing the discharge pattern of these autonomously firing neurons has yet to be exploited as a potential therapeutic strategy. Small conductance Ca(2+)-activated K(+) channels (SK channels), in particular the SK3 subtype, are important in the physiology of DA neurons, and agents modifying SK channel activity could potentially affect DA signaling and DA-related behaviors. Here we show that cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), a subtype-selective positive modulator of SK channels (SK3 > SK2 > > > SK1, IK), decreased spontaneous firing rate, increased the duration of the apamin-sensitive afterhyperpolarization, and caused an activity-dependent inhibition of current-evoked action potentials in DA neurons from both mouse and rat midbrain slices. Using an immunocytochemically and pharmacologically validated DA release assay employing cultured DA neurons from rats, we show that CyPPA repressed DA release in a concentration-dependent manner with a maximal effect equal to the D2 receptor agonist quinpirole. In vivo studies revealed that systemic administration of CyPPA attenuated methylphenidate-induced hyperactivity and stereotypic behaviors in mice. Taken together, the data accentuate the important role played by SK3 channels in the physiology of DA neurons, and indicate that their facilitation by CyPPA profoundly influences physiological as well as pharmacologically induced hyperdopaminergic behavior.