A pharmacological characterization of GABA, THIP and DS2 at binary α4ß3 and ß3δ receptors: GABA activates ß3δ receptors via the ß3(+)δ(-) interface.

There is growing evidence that GABA (γ-aminobutyric acid) can activate GABAA receptors (GABAARs) in the absence of an α subunit. In this study, we compared the pharmacology of homomeric and binary α4, ß3 or δ subunits with ternary α4ß3δ to identify subunit interfaces that contribute to the pharmacology of GABA, THIP, and DS2, and the antagonists, Zn(2+), gabazine and bicuculline. ß3δ receptors form functional GABA-gated channels when expressed in Xenopus oocytes with a pharmacology that differs to homomeric ß3, binary α4ß3 and ternary α4ß3δ receptors. GABA had similar potency at α4ß3 and ß3δ receptors (25µM and 26µM, respectively) but differed at α4ß3δ receptors where GABA exhibited a biphasic concentration-response (EC50 (1)=12.6nM; EC50 (2)=6.3µM). THIP activated ß3δ receptors (EC50=456µM) but was a more potent activator of α4ß3 (EC50=27µM) and α4ß3δ receptors (EC50 (1)=27.5nM; EC50 (2)=29.5µΜ), indicating that the α4 subunit significantly contribute to its potency. The δ-preferring modulator, DS2 had marginal or no effect at ß3δ and α4ß3 receptors, indicating a role for both the α4 and δ subunits for its potency. Gabazine inhibited GABA-elicited currents at ß3δ receptors whereas bicuculline activated these receptors. Mutational analysis verified that GABA binds to the ß3(+)δ(-) interface formed by the ß3 and δ subunits. In conclusion, evaluating agents against binary GABAARs such as ß3δ and α4ß3 receptors enables identification of interfaces that may contribute to the pharmacology of the more complex ternary α4ß3δ receptors.

GABAA α5 subunit-containing receptors do not contribute to reversal of inflammatory-induced spinal sensitization as indicated by the unique selectivity profile of the GABAA receptor allosteric modulator NS16085.

GABAA receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain. Restoration of diminished spinal GABAA-α2 and -α3 subunit-containing receptor function is a principal contributor to this analgesia, albeit involvement of GABAA-α5-receptors has not been excluded. Thus, we compared NS11394 and TPA023 (PAMs with selectivity/efficacy at GABAA-α2/α3/α5 receptors) with TP003 (a reportedly GABAA-α3 selective PAM) against spinal sensitization. However, in-house electrophysiology studies designed to confirm the selectivity of TPA023 and TP003 for human GABAA receptors did not corroborate published data, with TP003 displaying considerable GABAA-α5 receptor efficacy. Therefore, we identified a novel PAM, NS16085, which possesses negligible efficacy at GABAA-α5 receptors, but with GABAA-α2/α3 efficacy equivalent to NS11394. At the GABAA-α1 receptor the compound gives low level of negative modulation further separating it from the other compounds. Rat pups with carrageenan-induced hindpaw inflammatory hyperalgesia were used to make ex vivo spinal dorsal root-evoked ventral root recordings. Some spontaneous activity and large numbers of spikes to repetitive stimulation of dorsal roots at C-fibre intensity, indicative of wind-up and sensitization were observed. Equimolar concentrations of NS11394, TP003 and NS16085 all attenuated wind-up to a similar degree; TPA023 was clearly less effective. In adult rats, NS16085 (3-30 mg/kg, p.o.) dose-dependently reduced formalin-induced hindpaw flinching with efficacy comparable to NS11394. Thus, potentiation of GABAA-α2 and-α3 receptors is sufficient to depress spinal sensitization and mediate analgesia after inflammatory injury. Positive modulation at GABAA-α5-receptors is apparently dispensable for this process, an important consideration given the role of this receptor subtype in cognitive function.

Unravelling the mechanism of action of NS9283, a positive allosteric modulator of (α4)3(ß2)2 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Strong implications in major neurological diseases make the neuronal α4ß2 nicotinic ACh receptor (nAChR) a highly interesting drug target. In this study, we present a detailed electrophysiological characterization of NS9283, a potent positive allosteric modulator acting selectively at 3α:2ß stoichiometry of α2* and α4* nAChRs. EXPERIMENTAL APPROACH: The whole-cell patch-clamp technique equipped with an ultra-fast drug application system was used to perform electrophysiological characterization of NS9283 modulatory actions on human α4ß2 nAChRs stably expressed in HEK293 cells (HEK293-hα4ß2). KEY RESULTS: NS9283 was demonstrated to increase the potency of ACh-evoked currents in HEK293-hα4ß2 cells by left-shifting the concentration-response curve ~60-fold. Interestingly, this modulation did not significantly alter maximal efficacy levels of ACh. Further, NS9283 did not affect the rate of desensitization of ACh-evoked currents, was incapable of reactivating desensitized receptors and only moderately slowed recovery from desensitization. However, NS9283 strongly decreased the rate of deactivation kinetics and also modestly decreased the rate of activation. This resulted in a left-shift of the ACh window current of (α4)3(ß2)2 nAChRs in the presence of NS9283. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that NS9283 increases responsiveness of human (α4)3(ß2)2 nAChR to ACh with no change in maximum efficacy. We propose that this potentiation is due to a significant slowing of deactivation kinetics. In summary, the mechanism of action of NS9283 bears high resemblance to that of benzodiazepines at the GABAA receptor and to our knowledge, NS9283 constitutes the first nAChR compound of this class.

Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Positive allosteric modulation of α4ß2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively. EXPERIMENTAL APPROACH: Effects of NS9283 were evaluated in vitro using fluorescence-based Ca(2+) imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats. KEY RESULTS: NS9283 was shown to increase agonist-evoked response amplitude of (α4)(3) (ß2)(2) nACh receptors in electrophysiology paradigms. (α2)(3) (ß2)(2) , (α2)(3) (ß4)(2) and (α4)(3) (ß4)(2) were modulated to comparable extents, but no effects were detected at α3-containing or any 2α : 3ß stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHßE-sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP-disrupted pre-pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five-choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors. CONCLUSIONS AND IMPLICATIONS: These results provide compelling evidence that positive allosteric modulators acting at the (α4)(3) (ß2)(2) nACh receptors can augment activity across a broad range of cognitive domains, and that α4ß2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment.

Comparison of the novel subtype-selective GABAA receptor-positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] with diazepam, zolpidem, bretazenil, and gaboxadol in rat models of inflammatory and neuropathic pain.

Spinal administration of GABA(A) receptor modulators, such as the benzodiazepine drug diazepam, partially alleviates neuropathic hypersensitivity that manifests as spontaneous pain, allodynia, and hyperalgesia. However, benzodiazepines are hindered by sedative impairments and other side effect issues occurring mainly as a consequence of binding to GABA(A) receptors containing the alpha(1) subunit. Here, we report on the novel subtype-selective GABA(A) receptor-positive modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], which possesses a functional efficacy selectivity profile of alpha(5) > alpha(3) > alpha(2) > alpha(1) at GABA(A) alpha subunit-containing receptors. Oral administration of NS11394 (1-30 mg/kg) to rats attenuated spontaneous nociceptive behaviors in response to hindpaw injection of formalin and capsaicin, effects that were blocked by the benzodiazepine site antagonist flumazenil. Ongoing inflammatory nociception, observed as hindpaw weight-bearing deficits after Freund's adjuvant injection, was also completely reversed by NS11394. Likewise, hindpaw mechanical allodynia was fully reversed by NS11394 in two rat models of peripheral neuropathic pain. Importantly, NS11394-mediated antinociception occurred at doses 20 to 40-fold lower than those inducing minor sedative or ataxic impairments. In contrast, putative antinociception associated with administration of either diazepam, zolpidem, or gaboxadol only occurred at doses producing intolerable side effects, whereas bretazenil was completely inactive despite minor influences on motoric function. In electrophysiological studies, NS11394 selectively attenuated spinal nociceptive reflexes and C-fiber-mediated wind-up in vitro pointing to involvement of a spinal site of action. The robust therapeutic window seen with NS11394 in animals suggests that compounds with this in vitro selectivity profile could have potential benefit in clinical treatment of pain in humans.

NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], a unique subtype-selective GABAA receptor positive allosteric modulator: in vitro actions, pharmacokinetic properties and in vivo anxiolytic efficacy.

The novel positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] possesses a functional selectivity profile at GABA(A) receptors of alpha(5) > alpha(3) > alpha(2) > alpha(1) based on oocyte electrophysiology with human GABA(A) receptors. Compared with other subtype-selective ligands, NS11394 is unique in having superior efficacy at GABA(A)-alpha(3) receptors while maintaining low efficacy at GABA(A)-alpha(1) receptors. NS11394 has an excellent pharmacokinetic profile, which correlates with pharmacodynamic endpoints (CNS receptor occupancy), yielding a high level of confidence in deriving in vivo conclusions anchored to an in vitro selectivity profile and allowing for translation to higher species. Specifically, we show that NS11394 is potent and highly effective in rodent anxiety models. The anxiolytic efficacy of NS11394 is most probably mediated through its high efficacy at GABA(A)-alpha(3) receptors, although a contributory role of GABA(A)-alpha(2) receptors cannot be excluded. Compared with benzodiazepines, NS11394 has a significantly reduced side effect profile in rat (sedation, ataxia, and ethanol interaction) and mouse (sedation), even at full CNS receptor occupancy. We attribute this benign side effect profile to very low efficacy of NS11394 at GABA(A)-alpha(1) receptors and an overall partial agonist profile across receptor subtypes. However, NS11394 impairs memory in both rats and mice, which is possibly attributable to its efficacy at GABA(A)-alpha(5) receptors, albeit activity at this receptor might be relevant to its antinociceptive effects (J Pharmacol Exp Ther 327:doi;10.1124/jpet.108.144, 2008). In conclusion, NS11394 has a unique subtype-selective GABA(A) receptor profile and represents an excellent pharmacological tool to further our understanding on the relative contributions of GABA(A) receptor subtypes in various therapeutic areas.

Anxiolytic effects of Maxipost (BMS-204352) and retigabine via activation of neuronal Kv7 channels.

Neuronal Kv7 channels are recognized as potential drug targets for treating hyperexcitability disorders such as pain, epilepsy, and mania. Hyperactivity of the amygdala has been described in clinical and preclinical studies of anxiety, and therefore, neuronal Kv7 channels may be a relevant target for this indication. In patch-clamp electrophysiology on cell lines expressing Kv7 channel subtypes, Maxipost (BMS-204352) exerted positive modulation of all neuronal Kv7 channels, whereas its R-enantiomer was a negative modulator. By contrast, at the Kv7.1 and the large conductance Ca2+-activated potassium channels, the two enantiomers showed the same effect, namely, negative and positive modulation at the two channels, respectively. At GABA(A) receptors (alpha1beta2gamma2s and alpha2beta2gamma2s) expressed in Xenopus oocytes, BMS-204352 was a negative modulator, and the R-enantiomer was a positive modulator. The observation that the S- and R-forms exhibited opposing effects on neuronal Kv7 channel subtypes allowed us to assess the potential role of Kv7 channels in anxiety. In vivo, BMS-204352 (3-30 mg/kg) was anxiolytic in the mouse zero maze and marble burying models of anxiety, with the effect in the burying model antagonized by the R-enantiomer (3 mg/kg). Likewise, the positive Kv7 channel modulator retigabine was anxiolytic in both models, and its effect in the burying model was blocked by the Kv7 channel inhibitor 10,10-bis-pyridin-4-ylmethyl-10H-anthracen-9-one (XE-991) (1 mg/kg). Doses at which BMS-204352 and retigabine induce anxiolysis could be dissociated from effects on sedation or memory impairment. In conclusion, these in vitro and in vivo studies provide compelling evidence that neuronal Kv7 channels are a target for developing novel anxiolytics.

Identification of a novel voltage-gated Na+ channel rNa(v)1.5a in the rat hippocampal progenitor stem cell line HiB5.

A conditionally immortalised cell line, HiB5, derived from embryonic hippocampal precursor cells expressed a voltage-gated Na+ channel with electrophysiological characteristics shifted to more negative voltages and a lower sensitivity to tetrodotoxin [concentration required for half-maximal inhibition (IC50) 0.9 microM] compared with endogenous neuronal Na+ channels. The channel activation and steady-state inactivation occurred at very negative potentials with the threshold for activation at -55 mV and half-maximal inactivation at -78.7 mV. The channel was blocked by lamotrigine and sipatrigine voltage and state dependently, with potencies 5-20 times higher (IC50 12 and 1.8 microM at -80 mV respectively) than the corresponding block of endogenous Na+ channels from neurones and cloned rNa(v)1.2a (rBIIA) alpha-subunits. Both compounds slowed the channel's recovery from inactivation. Whereas lamotrigine and sipatrigine had similar effects on the fast inactivated state, the effect of sipatrigine on the slow inactivation state was more pronounced, rendering this compound overall the more effective. The molecular subtype mainly expressed by HiB5 cells was identified using RT-PCR and was a novel splice variant of rNa(v)1.5 (rNa(v)1.5a). It differs from the known rNa(v)1.5 version in that it lacks 53 amino acids in the intracellular loop between domains II and III. rNa(v)1.5a was also detected in mRNA derived from rat whole brain.

Pharmacological characterization of small-conductance Ca(2+)-activated K(+) channels stably expressed in HEK 293 cells.

Three genes encode the small-conductance Ca(2+)-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 IC(50) 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 IC(50) 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 IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM 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.

Neuroprotection by a novel compound, NS521.

NS521 (1-(1-butyl)-4-(2-oxo-1-benzimidazolinyl)piperidine) belongs to a group of novel benzimidazolones, which exhibit neurotrophic-like activities. In vitro, NS521 rescued neuronal PC12 cells from death induced by serum and nerve growth factor deprivation. The survival effect of NS521 appeared to reflect a delay of the apoptotic process, because the extent of DNA fragmentation was attenuated transiently by NS521. NS521 did not preserve the neurites of the rescued cells, which, otherwise, appeared to be healthy and were able to regenerate when serum and nerve growth factor were added back to the culture. In vivo, NS521 provided significant protection against the delayed loss of hippocampal CA1 neurons in a gerbil model of transient global ischemia. A neuroprotective effect of NS521 in the peripheral nervous system also was observed in rats after transection of the sciatic nerve, where daily treatment with NS521 was found to inhibit retrograde degeneration of the transected nerve. The neuroprotective effect of NS521 is unlikely to be mediated through neurotrophin receptors, such as TrkA, because NS521 did not induce phosphorylation of the 44- and 42-kDa isoforms of mitogen-activated protein kinases (ERK1/2) in PC12 cells.

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel.

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) >> Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 microM), and cetiedil (IC50 79 microM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 microM.

Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells.

We have generated HEK293 cell lines stably expressing high levels of either the human BK channel alpha-subunit alone or the BK channel alpha-subunit and beta-subunit together. For co-expression a plasmid with three expression cassettes was constructed. Patch-clamp recordings on inside-out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200-800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The alpha-subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the beta-subunit the steady-state activation curves were shifted by -20 to -30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 microM) shifted the steady-state activation curves for the alpha-subunit as well as for the alphabeta-subunits by -40 to -50 mV.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Modulation of the Ca(2+)-dependent K+ channel, hslo, by the substituted diphenylurea NS 1608, paxilline and internal Ca2+.

The high-conductance Ca(2+)-activated K channel (BK channel) is not only regulated by a number of physiological stimuli, but it is also sensitive to pharmacological modulation. We have stably expressed the alpha-subunit of the human BK channel, hslo, in HEK 293 cells and studied by patch-clamp technique how its gating is modulated by the channel activator NS 1608, by the selective channel blocker paxilline, as well as by changes in [Ca2+]i and Vm. The cells expressed 200-800 hslo channels per patch. The channel activity was determined by tail current analysis, and the activation curves were fitted to single Boltzmann functions, from which a gating charge for the hslo channel of 1.2 elementary charges was deduced. The hslo channel was very sensitive to changes in [Ca2+]i within the physiological range, whereas Ca(2+)-independent openings were seen at Ca2+ concentrations of 15 nM or below. NS 1608 shifted the hslo channel activation curve towards negative membrane potentials with an EC50 of 2.1 microM and a maximal shift of -74 mV. The channels activated by NS 1608 were sensitive to block by paxilline, but the two molecules apparently did not interact within the same site, since paxilline reduced the size of the tail current at all voltages, whereas NS 1608 shifted the activation curve along the voltage axis. Further, the effect of paxilline was Ca(2+)-sensitive, whereas NS 1608 elicited identical effects in the presence of either < 0.5 nM or 500 nM [Ca2+]i. NS 1608 hyperpolarized the cells by -50 to -70 mV, and paxilline depolarized them towards 0 mV. In addition to the effects on the steady state current NS 1608 also significantly influenced the non-stationary channel kinetics. In the presence of NS 1608 the time constants for deactivation of tail currents were more than tripled at all potentials. We have shown, that NS 1608 modulates steady-state BK currents and channel gating kinetics through a Ca(2+)-independent interaction with the alpha-subunit of the channel.