KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

BACKGROUND AND PURPOSE

Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca²⁺ concentration ([Ca²⁺]_i) by opening of K channels and release of H₂S.

EXPERIMENTAL APPROACH

Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca²⁺]_i, and the expression of voltage-gated K channels K_V7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K_Ca1.1) was examined. Voltage clamp assessed the role of K_V7 channels in hypoxia.

KEY RESULTS

Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca²⁺]_i in PGF_2α (10 μM)-contracted arteries whereas no fall in [Ca²⁺]_i was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K_Ca1.1 inhibited hypoxia-induced dilatation in PGF_2α-contracted arteries; this inhibition was more marked in the presence of the K_v7 channel blockers, XE991 and linopirdine, while a K_V7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H₂S- and adenosine-induced vasodilatation. PCR revealed the expression of K_V7.1, K_V7.4, K_V7.5 and K_Ca1.1 channels, and K_Ca1.1, K_V7.4 and K_V7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions.

CONCLUSION

The K_V7.4 and K_V7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K_V7 channels in this vasodilatation. Activation of these K_V7 channels may be induced by H₂S and adenosine.     

Furosemide interactions with brain GABAA receptors

1. The loop diuretic furosemide is known to antagonize the function of γ-aminobutyric acid type A (GABA_A) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABA_A receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits.
2. Autoradiography with [³⁵S]-t-butylbicyclophosphorothionate ([³⁵S]-TBPS) as a ligand indicated that furosemide (0.1–1 mM) reversed the 5 μM GABA-induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mM decreased [³⁵S]-TBPS binding only in a limited number of brain regions, but facilitation of the GABA-inhibition of the binding was much more widespread.
3. In well-washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [³⁵S]-TBPS binding over basal level in the absence of added GABA. The GABA_A antagonist, SR 95531, and the convulsant, Ro 5-4864, blocked this furosemide-induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [³H]-SR 95531 and [³H]-Ro 5-4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA-antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5-4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed.
4. In recombinant GABA_A receptors, furosemide antagonism of GABA-inhibition of [³⁵S]-TBPS binding depended only on the presence of α6 and β2/3 subunits, irrespective of the presence or absence of γ2 or δ subunits.
5. In α6β3γ2 receptors, clozapine reversed the enhancement of [³⁵S]-TBPS binding by furosemide in the absence of GABA. However, it failed to affect the GABA-antagonism of furosemide, suggesting that the enhancement of basal binding and the GABA antagonism might represent two different allosteric actions of furosemide.
6. In conclusion, the present results indicate that furosemide is a subtype-selective GABA_A antagonist with a mode of action not shared by several other antagonists, which makes furosemide a unique compound for development of potential GABA_A receptor subtype-specific and -selective ligands.

     

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.     

Structurally diverse amphiphiles exhibit biphasic modulation of GABAA receptors: similarities and differences with neurosteroid actions

Background and purpose:

Some neurosteroids, notably 3α-hydroxysteroids, positively modulate GABA_A receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions.

Experimental approach:

Xenopus laevis oocytes were challenged with Triton X-100, octyl-β-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations.

Key results:

These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 µM) promoted potentiation and a high concentration (20 µM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (α1V256S) and positive (α1Q241L or α1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in α1V256S-mutated receptors, but potentiation was unaffected. In α1Q241L- or α1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact.

Conclusions and implications:

Structurally diverse amphiphiles have antagonist actions at GABA_A receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA_A receptors does not have a clear pharmacophore requirement.     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Phosphorylation influences neurosteroid modulation of synaptic GABAA receptors in rat CA1 and dentate gyrus neurones

The neurosteroid 5beta-pregnan-3alpha-ol-20-one (5beta3alpha) is a potent, endogenous, positive allosteric modulator of the GABA(A) receptor. Relatively low concentrations of 5beta3alpha (10-100 nM), thought to occur physiologically, caused a concentration-dependent slowing of the decay of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from hippocampal CA1 pyramidal neurones. However, much greater concentrations of this neurosteroid (> or =300 nM) were required to similarly influence dentate granule cell mIPSCs. By contrast, the allosteric modulators pentobarbitone and flunitrazepam were equi-effective in prolonging mIPSCs in both neuronal types. Hence, the neurosteroid selectively differentiates between the synaptic GABA(A) receptors of these hippocampal neurones. Inhibition of either protein kinase A, or C, greatly reduced the sensitivity of CA1 synaptic GABA(A) receptors to 5beta3alpha, but not pentobarbitone, whereas stimulation of PKC had no effect on steroid sensitivity. However, in dentate gyrus granule cells, activation of PKC made mIPSCs sensitive to a previously ineffective concentration of 5beta3alpha. Collectively, these results suggest that the GABA-modulatory effects of physiological levels of the neurosteroid will not be uniformly experienced throughout the central nervous system, or even within the same brain region such as the hippocampus, but will be neurone-specific and will be dependent on the phosphorylation status of the GABA(A) receptor, or associated proteins.     

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension

Background and purpose:

Voltage-gated potassium (K_v) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K_v7 channels has been recently proposed. We investigated the effect of the K_v7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT⁺ mice).

Experimental approach:

Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg·kg⁻¹·day⁻¹). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K_v channel blocking drugs, including the K_v7 channel blocker linopirdine, were measured.

Key results:

In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT⁺ mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT⁺ mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K_v channel blockers did not.

Conclusions and implications:

Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT⁺ mice, apparently via K_v7 channel activation. These results provide the first direct evidence that drugs activating K_v7 channels may be of benefit in the treatment of PAH with different aetiologies.     

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer

1. γ-Aminobutyric acid (GABA), acting via the GABA_A receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer.
2. The optimal conditions for the measurement of GABA_A receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9–12×10⁵ cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25°C, with a stimulation time of 33 s.
3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC₅₀ of 2.0±0.2 μM (mean±s.e.mean, n=7 experiments) and maximal increase (E_max) over basal response of 15.4±1.2%.
4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 μV s⁻¹, GABA+flunitrazepam, 3.8 μV s⁻¹, mean increment over basal, n=7).
5. The GABA-stimulated increase in acidification rate was inhibited by the GABA_A antagonist, bicuculline (100 μM) (90% inhibition at 1 mM GABA).
6. The results of this study show that activation of GABA_A receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABA_A receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABA_A receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABA_A receptor in populations of neurones.

     

Modulation of presynaptic GABA(A) receptors by endogenous neurosteroids

BACKGROUND AND PURPOSE

Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABA_A receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown.

EXPERIMENTAL APPROACH

In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique.

KEY RESULTS

AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABA_A receptor blocker, tetrodotoxin or Cd²⁺, suggesting that AlloP acts on presynaptic GABA_A receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABA_A receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABA_A receptors to affect spontaneous glutamate release onto hilar neurons.

CONCLUSIONS AND IMPLICATIONS

The modulation of presynaptic GABA_A receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.     

Flavonoid modulation of GABA(A) receptors

There has been a resurgence of interest in synthetic and plant-derived flavonoids as modulators of γ-amino butyric acid-A (GABA(A) ) receptor function influencing inhibition mediated by the major inhibitory neurotransmitter GABA in the brain. Areas of interest include (i) flavonoids that show subtype selectivity in recombinant receptor studies in vitro consistent with their behavioural effects in vivo, (ii) flumazenil-insensitive modulation of GABA(A) receptor function by flavonoids, (iii) the ability of some flavonoids to act as second-order modulators of first-order modulation by benzodiazepines and (iv) the identification of the different sites of action of flavonoids on GABA(A) receptor complexes. An emerging area of interest is the activation of GABA(A) receptors by flavonoids in the absence of GABA. The relatively rigid shape of flavonoids means that they are useful scaffolds for the design of new therapeutic agents. Like steroids, flavonoids have wide-ranging effects on numerous biological targets. The challenge is to understand the structural determinants of flavonoid effects on particular targets and to develop agents specific for these targets.     

Neurosteroid Modulation of GABAA Receptor Function by Independent Action at Multiple Specific Binding Sites

Neurosteroids are endogenous modulators of GABA_A receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epi-allopregnanolone (3β-OH) are both prevalent in the mammalian brain and produce opposite effects on GABA_A receptor function, acting as positive and negative allosteric modulators, respectively. This Perspective provides a model to explain the actions of 3α-OH and 3β-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABA_A receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.     

Alcohol use disorders and current pharmacological therapies: the role of GABAA receptors

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABA_A receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABA_A receptors in the treatment of AUD and currently available and potentially novel pharmacological agents.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

Intrathecal injection of the neurosteroid, DHEAS, produces mechanical allodynia in mice: involvement of spinal sigma-1 and GABAA receptors

Background and purpose:

The neurosteroid, dehydroepiandrosterone sulphate (DHEAS) and its non-sulphated form, DHEA, are considered as crucial endogenous modulators of a number of important physiological events. Evidence suggests that DHEAS and DHEA modulate central nervous system-related functions by activating sigma-1 receptors and/or allosterically inhibiting γ-aminobutyric acic receptor type A (GABA_A) receptors. As both the sigma-1 receptor and the GABA_A receptor play important roles in spinal pain transmission, the present study was designed to examine whether intrathecally injected DHEAS or DHEA affect nociceptive signalling at the spinal cord level.

Experimental approach:

We first determined whether intrathecal (i.t.) DHEA or DHEAS injection was able to affect nociceptive thresholds to peripheral mechanical stimulation and subsequently examined whether this effect was mediated by sigma-1 or the GABA_A receptors.

Key results:

The i.t. DHEAS injection dose-dependently decreased the nociceptive threshold to mechanical stimulation, thus producing mechanical allodynia. Moreover, this DHEAS-induced mechanical allodynia was significantly reduced by administration of the sigma-1 receptor antagonist, BD-1047 or the GABA_A receptor agonist, muscimol. Conversely, i.t. DHEA had no effect on mechanical sensitivity. However, when i.t. DHEA was combined with the GABA_A receptor antagonist bicuculline, DHEA dose-dependently produced mechanical allodynia similar to that of DHEAS. This effect was blocked by BD-1047 and by muscimol.

Conclusions and implications:

These findings indicate that i.t. injection of DHEAS produces mechanical allodynia and that the development of this mechanical allodynia is mediated by sigma-1 and GABA_A receptors. The findings of this study raise several interesting questions for further investigations into the mechanisms underlying neurosteroid modulation of spinal pain transmission.British Journal of Pharmacology (2009) 157, 666–673; doi:10.1111/j.1476-5381.2009.00197.x; published online 30 April 2009     

Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.     

Pharmacological modulation of GABAA receptor-mediated postsynaptic potentials in the CA1 region of the rat hippocampus

1. It is unclear whether GABA_A receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP_As and DPSP_As, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA_A receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA_A receptor ligands, on each response.
2. The GABA uptake inhibitor NNC 05-711 (10 μM) enhanced whereas bicuculline (10 μM) inhibited both IPSP_As and DPSP_As.
3. (−)-Baclofen (5 μM), [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAGO; 0.5 μM), and carbachol (10 μM) caused substantial depressions (up to 99%) of DPSP_As that were reversed by CGP 55845A (1 μM), naloxone (10 μM) and atropine (5 μM), respectively. In contrast, 2-chloroadenosine (CADO; 10 μM) only slightly depressed DPSP_As. Quantitatively, the effect of each agonist was similar to that reported for IPSP_As.
4. The neurosteroid ORG 21465 (1–10 μM), the anaesthetic propofol (50–500 μM), the barbiturate pentobarbitone (100–300 μM) and zinc (50 μM) all enhanced DPSP_As and IPSP_As.
5. The benzodiazepine (BZ) agonist flunitrazepam (10–50 μM) and inverse agonist DMCM (1 μM) caused a respective enhancement and inhibition of both IPSP_As and DPSP_As. The BZω₁ site agonist zolpidem (10–30 μM) produced similar effects to flunitrazepam.
6. The anticonvulsant loreclezole (1–100 μM) did not affect either response.
7. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP_As and DPSP_As by activating GABA_A receptors that are subject to similar allosteric modulation.

     

Redox modulation of GABAA receptors obscured by Zn2+ complexation

Redox reagents are thought to modulate gamma-Aminobutyric acid type A (GABA(A)) receptors by regulating the redox state of the N-terminal disulphide bridge. Examining the redox sensitivity of recombinant GABA(A) receptors in human embryonic kidney cells, using whole-cell patch clamp techniques, revealed that alpha1beta2(H267A) and alpha1beta2gamma2 receptors, which are both less sensitive to Zn(2+) and H(+) modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for alpha1beta2 receptors. This effect could result from disruption to the redox signal transduction pathway or be due to DTT chelating Zn(2+) from its H267 inhibitory binding site, consequently potentiating GABA-activated currents in alpha1beta2 but not alpha1beta2(H267A) or alpha1beta2gamma2 receptors. A Zn(2+) chelating agent, tricine, potentiated GABA currents for the alphabeta constructs and vertically displaced GABA dose-response curves, suggesting that these receptors are subject to some inhibition by basal Zn(2+). Tricine, did not affect the GABA currents of either alpha1beta2(H267A) or alpha1beta2gamma2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on alpha1beta2 receptors. Thus, at low concentrations of DTT, a substantial component of the potentiation probably occurs via Zn(2+) chelation from H267 in the ion channel. In contrast, at higher DTT concentrations, it is more likely to be acting as a redox agent, which modulates both alphabeta and alphabetagamma subunit receptors.     

Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels

BACKGROUND AND PURPOSE

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on K_v7 K⁺ channels and compared its effects with the well-established K_v7 channel opener retigabine.

EXPERIMENTAL APPROACH

A perforated whole-cell patch technique was used to record K_v7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons.

KEY RESULTS

Celecoxib enhanced K_v7.2–7.4, K_v7.2/7.3 and K_v7.3/7.5 currents but inhibited K_v7.1 and K_v7.1/KCNE1 currents and these effects were concentration dependent. The IC₅₀ value for inhibition of K_v7.1 channels was approximately 4 µM and the EC₅₀ values for activation of K_v7.2–7.4, K_v7.2/K_v7.3 and K_v7.3/K_v7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of K_v7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on K_v7currents. K_v7.2(A235T) and K_v7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition.

CONCLUSIONS AND IMPLICATIONS

These results suggest that K_v7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing K_v7 modulators and for studying the structure–function relationship of K_v7 channels.     

Protein kinase C-independent inhibition of arterial smooth muscle K(+) channels by a diacylglycerol analogue

BACKGROUND AND PURPOSE

Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K⁺ and Ca²⁺ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K⁺ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner.

EXPERIMENTAL APPROACH

Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K⁺ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.

KEY RESULTS

Mesenteric artery smooth muscle K_v currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and K_ATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.

CONCLUSIONS AND IMPLICATIONS

These results show that DiC8 has a direct effect on arterial smooth muscle K⁺ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K⁺ channels.