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-1β.
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.
•Potassium channels might be regarded as a new group of therapeutic targets for IBD.•Immunosuppressive peptide from human endogenous retroviral envelope protein, ENV59 inhibits KCa3.1 potassium channel.•Inhibition of KCa3.1 with ENV59-GP3 peptide or KCa3.1-blocker NS6180 protects against DSS-induced acute colitis.•Virus derived channel blockers might be effective for ameliorating of autoimmune diseases.
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Extrasynaptically located γ-aminobutyric acid (GABA) receptors type A are often characterized by the presence of a δ subunit in the receptor complex. δ-Containing receptors respond to ...low ambient concentrations of GABA, or respond to spillover of GABA from the synapse, and give rise to tonic inhibitory currents. In certain brain regions, e.g. thalamocortical neurons, tonic inhibition is estimated to represent the majority of total GABA-mediated inhibition, which has raised substantial interest in extrasynaptic receptors as potential drug targets. Thalamocortical neurons typically express α4β2/3δ receptors, however, these have proven difficult to study in recombinant in vitro expression systems due to the inherently low current levels elicited in response to GABA.
In this study, we sought to characterize a range of agonists and positive allosteric modulators at α4β2δ and α4β2γ2 receptors. All tested agonists (GABA, THIP, muscimol, and taurine) displayed between 8 and 22 fold increase in potency at the α4β2δ receptor. In contrast, modulatory potencies of steroids (allopregnanolone, THDOC and alfaxalone), anesthetics (etomidate, pentobarbital) and Delta-Selective agents 1 and 2 (DS1 and DS2) were similar at α4β2δ and α4β2γ2 receptors. When evaluating modulatory efficacies, the neurosteroids and anesthetics displayed highest efficacy at α4β2γ2 receptors whereas DS1 and in particular DS2 had highest efficacy at α4β2δ receptors. Overall, several key messages emerged: (i) none of the tested compounds displayed significant selectivity and a great need for identifying new δ-selective compounds remains; (ii) α4β2δ and α4β2γ2 receptors have such divergent intrinsic activation properties that valid comparisons of modulator efficacies are at best challenging.
Small conductance Ca2+ ‐activated K+ (SK) channels play a prominent role in modulating the spontaneous activity of dopamine (DA) neurons as well as their response to synaptically‐released glutamate. ...SK channel gating is dependent on Ca2+ binding to constitutively bound calmodulin, which itself is subject to endogenous and exogenous modulation. In the present study, patch‐clamp recording techniques were used to examine the relationship between the apparent Ca2+ affinity of cloned SK3 channels expressed in cultured human embryonic kidney 293 cells and the excitability of DA neurons in slices from rat substantia nigra using the positive and negative SK channel modulators, 6,7‐dichloro‐1H‐indole‐2,3‐dione‐3‐oxime and R‐N‐(benzimidazol‐2‐yl)‐1,2,3,4‐tetrohydro‐1‐naphtylamine. Increasing the apparent Ca2+ affinity of SK channels decreased the responsiveness of DA neurons to depolarizing current pulses, enhanced spike frequency adaptation and slowed spontaneous firing, effects attributable to an increase in the amplitude and duration of an apamin‐sensitive afterhyperpolarization. In contrast, decreasing the apparent Ca2+ affinity of SK channels enhanced DA neuronal excitability and changed the firing pattern from a pacemaker to an irregular or bursting discharge. Both the reduction in apparent Ca2+ affinity and the bursting associated with negative SK channel modulation were gradually surmounted by co‐application of the positive SK channel modulator. These results underscore the importance of SK channels in ‘tuning’ the excitability of DA neurons and demonstrate that gating modulation, in a manner analogous to physiological regulation of SK channels in vivo, represents a means of altering the response of DA neurons to membrane depolarization.
Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca(2+)-dependence of the small-conductance Ca(2+)-activated K(+) ...channels K(Ca)2.1-2.3 to higher Ca(2+) concentrations. Similar to the positive K(Ca) channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca(2+) sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K(Ca)2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K(Ca)3.1 channel to K(Ca)2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K(Ca)2.3. In contrast, when we transferred the K(Ca)2.3 pore to K(Ca)3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K(Ca)2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K(Ca)3.1 (Thr250 and Val275) made K(Ca)2.3 insensitive to NS8593, whereas introduction of serine and alanine into K(Ca)3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K(Ca)3.1 to clotrimazole and 1-(2-chlorophenyl)diphenylmethyl-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K(Ca)2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.
SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of ...neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.
The KCa3.1 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 KCa3.1. The channel expression was demonstrated as a significant increase in the voltage‐independent current by NS309, a KCa3.1/KCa2 activator, followed by full inhibition upon co‐application with NS6180, a highly selective KCa3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa3.1, and the difference in current between full activation and inhibition (ΔKCa3.1) was estimated at 292 ± 48 pA at −40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa3.1 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 KCa3.1 current nor the fraction of KCa3.1 expressing cells. In contrast, the anti‐inflammatory cytokine IL‐4 slightly increased the KCa3.1 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 KCa1.1 modulators NS1619 and Paxilline and an inward‐rectifying current with the characteristics of a Kir channel. The high functional expression of KCa3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065–2078
Main Points
K+ currents in primary human microglia are mainly due to Ca2+‐activated K+ channels.
Electrophysiology and pharmacology revealed functional KCa3.1 and KCa1.1 in a majority of microglia.
IL‐4 or LPS treatment did not change the KCa3.1 current density.
Small conductance Ca2+-activated K+ channels (SK channels) participate in the control of neuronal excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic ...transmission. SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and neurological diseases such as depression, cognition impairment, and Parkinson’s disease. In the present study we describe the structure−activity relationship (SAR) of a class of 2-(N-substituted)-2-aminobenzimidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished by point mutations. These novel SK inhibitors shift the concentration−response curve for Ca2+ toward higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated currents with a K d value of 9 nM.
We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective K(Ca)2.1 channel activation by GW542573X (4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic ...acid tert-butyl ester). Now we show that Ser293 mediates both activation and inhibition of K(Ca)2.1: CM-TPMF (N-{7-1-(4-chloro-2-methylphenoxy)ethyl-1,2,4triazolo1,5-apyrimidin-2-yl}-N'-methoxy-formamidine) and B-TPMF (N-{7-1-(4-tert-butyl-phenoxy)ethyl-1,2,4triazolo1,5-apyrimidin-2-yl}-N'-methoxy-formamidine), two newly identified and structurally related 1,2,4triazolo1,5-apyrimidines, act either as activators or as inhibitors of the human K(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) value of 31 nM. In contrast, their (+)-enantiomers are 40 to 100 times less active. Both (-)-CM-TPMF and (-)-B-TPMF are subtype-selective, with 10- to 20-fold discrimination toward other K(Ca)2 channels and the K(Ca)3 channel. Coapplication experiments reveal competitive-like functional interactions between the effects of (-)-CM-TPMF and (-)-B-TPMF. Despite belonging to a different chemical class than GW542573X, the K(Ca)2.1 selectivity of (-)-CM-TPMF and (-)-B-TPMF depend critically on Ser293 as revealed by loss- and gain-of-function mutations. We conclude that compounds occupying the TPMF site may either positively or negatively influence the gating process depending on their substitution patterns. It is noteworthy that (-)-CM-TPMF is 10 times more potent on K(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect K(Ca)2.2, (-)-B-TPMF exhibits K(Ca)2.1 selectivity. These high-affinity compounds, which exert opposite effects on K(Ca)2.1 gating, may help define physiological or pathophysiological roles of this channel.
Three genes encode the small‐conductance Ca2+‐activated K+ channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using ...whole‐cell patch clamp recordings.
The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC50 values of 3.3 nM and 83 pM, respectively.
The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC50 value of 80 nM and rSK2 at 287 pM.
The potent small molecule blockers showed little differentiation between the channel subtypes. The bis‐quinolinium cyclophane UCL 1684 blocked hSK1 with an IC50 value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC50 values of 444 nM and 162 nM, respectively.
The nicotinic acetylcholine receptor antagonist d‐tubocurarine was found to block hSK1 and rSK2 with IC50 values of 27 μM and 17 μM when measured at +80 mV. The inhibition by d‐tubocurarine was voltage‐dependent with increasing affinities at more hyperpolarized potentials.
The GABAA receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage‐dependent manner with IC50 values of 15 and 25 μM when measured at +80 mV.
In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin‐insensitive afterhyperpolarization of neurones is mediated by hSK1.
British Journal of Pharmacology (2000) 129, 991–999; doi:10.1038/sj.bjp.0703120
Abstract only
The muscarinic receptor antagonist, Oxybutynin (Ox), and the β
3
‐receptor agonist, mirabegron (Mb), are used in the treatment of overactive bladder syndrome (OAB). However, the exact ...downstream mechanisms by which Ox and Mb opposes the enhanced contractility in OAB is unknown. The
KCNQ
genes encode five K
v
7 K
+
channel subunits (K
V
7.1–K
v
7.5). Of these, K
V
7.2–K
V
7.5 channels have been proposed to be involved in regulation of smooth muscle tone, i.e. the contractile state of the bladder. In this study, we investigated the involvement of K
V
7.2–K
V
7.5 channels in Ox‐ and Mb‐induced relaxation of the bladder. Furthermore, the direct effect of K
V
7.2–K
V
7.5 channel‐activation on bladder relaxation was determined. Patch clamp recordings from HEK cells expressing K
V
7.1–K
V
7.5 channels were used to determine the relative potency of five different activators; ICA‐27243, ML213, NS15370, Retigabine, and SciFluor, on the K
V
7.2–K
V
7.5 channels. Bladder strips from rats mounted in organ baths and contracted with electrical field stimulation were used to investigate the functional effects of inhibiting and activating K
V
7.2–K
V
7.5 channels. This study found that inhibiting K
V
7 channels with XE991 markedly reduced Ox‐ and Mb‐induced relaxation of bladder strips. Furthermore, bladder strip EC
50
values corresponded well with HEK cell EC
50
values for both K
V
7.2–3 and Kv7.4 channels, and arranged the five activators with the most potent first accordingly: NS15370>ML213=SciFluor>Retigabine>ICA‐27243. In conclusion, this study showed that K
V
7.2–K
V
7.5 channels are probably involved in the therapeutic relaxation of the bladder induced by Ox and Mb in OAB patients. In addition, direct pharmacological activation of K
V
7.2–K
V
7.5 channels induced a potent relaxation of the bladder, suggesting that K
V
7.2–K
V
7.5 channel activators may be a new target for treating OAB ‐ either as standalone treatment or as enhancers of the effect of currently available treatment options.
Support or Funding Information
This study was funded by the Innovation Fund Denmark.
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in
The FASEB Journal
.
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