Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a ... more Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a “target class” approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous ...
American journal of physiology. Lung cellular and molecular physiology, Jan 23, 2018
Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial... more Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial hypertension (PAH), however the pathogenesis of HIV-related PAH remains unclear. Since K+ channel dysfunction is a common marker in most forms of PAH, our aim was to analyse if the expression of HIV proteins is associated with impairment of K+ channel function in the pulmonary vascular bed. HIV transgenic mice (Tg26) expressing seven of the nine HIV viral proteins and wild type (Wt) mice were used. Hemodynamic assessment was performed by echocardiography and catheterization. Vascular reactivity was studied in endothelium-intact pulmonary arteries (PA). K+ currents were recorded in freshly isolated PA smooth muscle cells (PASMC) using the patch-clamp technique. Gene expression was assessed using RT-PCR. PASMC from Tg26 mice had reduced K+ currents and were more depolarized that those from Wt. While Kv1.5 currents were preserved, pH-sensitive non-inactivating background currents (IKN) wer...
TREK two pore domain potassium channels play a critical role in regulating the excitability of so... more TREK two pore domain potassium channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study we describe a new, selective opener of TREK channels, GI-530159 (4,4'-(Hexafluoroisopropylidene)bis(p-phenyleneoxy)dianiline). The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single channel patch clamp recordings from recombinant TREK channels. TREK1, TREK2 and TRAAK expression was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. For recombinant human TREK1 channels GI-530...
Schistosomiasis (bilharzia) is a neglected parasitic disease caused by trematode flatworms of the... more Schistosomiasis (bilharzia) is a neglected parasitic disease caused by trematode flatworms of the genus Schistosoma which affects over 240 million people worldwide. It is characterized by the formation of inflammatory granulomas around deposited parasite eggs. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of schistosomiasis. The aim of this paper is to systematically evaluate the number and distribution of inflammatory cells in S. mansoni-infected mice at different doses and time points. Immunohistochemistry was performed on lung and liver tissue sections from Schistosoma-infected mice and uninfected healthy controls. Positively stained cells in whole-lung/liver tissue sections, surrounding the eggs, and in the different compartments of the tissues, were counted. We found a significant increase in the number of mast cells (toluidine blue(+)), CD3(+) cells, CD14(+) cells, CD68(+) cells, and CD15(+) cells in Schistosoma-infecte...
Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling... more Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.
Current Opinion in Investigational Drugs, Jul 1, 2007
Two-pore domain potassium (K2P) channels are expressed in cells throughout the body and give rise... more Two-pore domain potassium (K2P) channels are expressed in cells throughout the body and give rise to leak potassium currents which control the excitability of these cells. Although not inhibited by classical potassium channel-blocking drugs, such as tetraethylammonium and 4-aminopyridine, K2P channels are regulated by a diverse array of pharmacological mediators. There are six main families of K2P channels and among these certain members of the TREK family (ie, TREK-1 and TREK-2) are activated by general anesthetic agents such as halothane, xenon and nitrous oxide. In addition, all members of the TREK familyare activated by neuroprotective agents, such as riluzole, polyunsaturated fatty acids and lysophospholipids, suggesting that these channels play an important role in neuroprotection. TREK channels are also inhibited by chlorpromazine, local anesthetics and the antidepressant fluoxetine. Furthermore, all members of the TASK family are inhibited by cannabinoids and local anesthetics, and TASK-3 is selectively inhibited by ruthenium red. Thus, the diversity and physiological importance of K2P channels suggest that the development of selective compounds to target these proteins has therapeutic potential for CNS disorders such as stroke, depression and epilepsy.
Transmitter-activated channels in freshly isolated neurones from sympathetic ganglia of young rat... more Transmitter-activated channels in freshly isolated neurones from sympathetic ganglia of young rats have been examined using the patch-clamp technique. Acetylcholine (ACh), gamma-aminobutyric acid (GABA) and (in about one third of cells) glycine produced an inward current and an increase in current noise when perfused onto voltage-clamped neurones. The ACh noise was fitted with a single Lorentzian spectrum with a time constant of 13.1 ms at -70 mV. Outside-out membrane patches allowed high-resolution measurements of single ACh- and GABA-activated channels. The ACh-channels had a conductance of 30 pS.
ABSTRACT TREK1, two pore domain potassium (K2P) channels are responsible for background currents ... more ABSTRACT TREK1, two pore domain potassium (K2P) channels are responsible for background currents that regulate neuronal excitability. Deletion of TREK1 has previously been shown to result in a depression-resistant phenotype in mice (Heurteaux et al. 2006). In this study we investigated the effect of the tricyclic antidepressant, amitriptyline, on TREK1 channels. Additionally, amitriptyline is used for relief of neuropathic pain and TREK-1 is also involved in pain perception (Alloui et al. 2006). tsA-201 cells were transiently transfected with wild type and mutated K2P channels. The whole cell patch clamp technique was used to obtain current recordings. Homology models for TREK1 were constructed using Modeller. Amitriptyline was docked to these models using the MOE program. Amitriptyline (50 μM) significantly inhibited WT TREK1 channel current by 77.4 ± 3.7 % (n=9). While the inhibitory effect of amitriptyline was reversible, on wash, it was often followed by an over-recovery of current to above the baseline level. We have previously investigated the effect of the antidepressant, citalopram, on TREK1, and found that it inhibited the channel. Subsequently we identified a number of amino acids in both the pore region and transmembrane domains M2 and M4 of TREK1 that were important for its effect (Al-Moubarak et al. 2013). Interestingly, mutations of several amino acids, T142A, L289A and T251A, which reduced the effectiveness of citalopram had no effect on the observed inhibition by amitriptyline. Other mutations, L169A and I167A had no effect on inhibition by either compound. Mutation L174A (in the M2 transmembrane domain of TREK1), however, significantly reduced inhibition by both citalopram (Al-Moubarak et al 2013) and amitriptyline (50 μM, 49.0 ± 3.1 %, n=6, p < 0.05). As previously found for citalopram, the E306A gain of function mutation completely prevented inhibition by amitriptyline. Docking of amitriptyline into the TREK1 homology model, guided by the obtained electrophysiology results, predicts a different binding profile for amitriptyline compared to citalopram. Thus we have shown that amitriptyline is a potent inhibitor of TREK1 potassium channels. This action may contribute to the therapeutic usefulness of this compound in both the treatment of depression and pain.
Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a ... more Two-pore domain potassium (K2P) channels carry background (or leak) potassium current and play a key role in regulating resting membrane potential and cellular excitability. Accumulating evidence points to a role for K2Ps in human pathophysiologies, most notably in pain and migraine, making them attractive targets for therapeutic intervention. However, there remains a lack of selective pharmacological tools. The aim of this work was to apply a “target class” approach to investigate the K2P superfamily and identify novel activators across all the described subclasses of K2P channels. Target class drug discovery allows for the leveraging of accumulated knowledge and maximizing synergies across a family of targets and serves as an additional approach to standard target-based screening. A common assay platform using baculovirus (BacMam) to transiently express K2P channels in mammalian cells and a thallium flux assay to determine channel activity was developed, allowing the simultaneous ...
American journal of physiology. Lung cellular and molecular physiology, Jan 23, 2018
Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial... more Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial hypertension (PAH), however the pathogenesis of HIV-related PAH remains unclear. Since K+ channel dysfunction is a common marker in most forms of PAH, our aim was to analyse if the expression of HIV proteins is associated with impairment of K+ channel function in the pulmonary vascular bed. HIV transgenic mice (Tg26) expressing seven of the nine HIV viral proteins and wild type (Wt) mice were used. Hemodynamic assessment was performed by echocardiography and catheterization. Vascular reactivity was studied in endothelium-intact pulmonary arteries (PA). K+ currents were recorded in freshly isolated PA smooth muscle cells (PASMC) using the patch-clamp technique. Gene expression was assessed using RT-PCR. PASMC from Tg26 mice had reduced K+ currents and were more depolarized that those from Wt. While Kv1.5 currents were preserved, pH-sensitive non-inactivating background currents (IKN) wer...
TREK two pore domain potassium channels play a critical role in regulating the excitability of so... more TREK two pore domain potassium channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study we describe a new, selective opener of TREK channels, GI-530159 (4,4'-(Hexafluoroisopropylidene)bis(p-phenyleneoxy)dianiline). The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single channel patch clamp recordings from recombinant TREK channels. TREK1, TREK2 and TRAAK expression was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. For recombinant human TREK1 channels GI-530...
Schistosomiasis (bilharzia) is a neglected parasitic disease caused by trematode flatworms of the... more Schistosomiasis (bilharzia) is a neglected parasitic disease caused by trematode flatworms of the genus Schistosoma which affects over 240 million people worldwide. It is characterized by the formation of inflammatory granulomas around deposited parasite eggs. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of schistosomiasis. The aim of this paper is to systematically evaluate the number and distribution of inflammatory cells in S. mansoni-infected mice at different doses and time points. Immunohistochemistry was performed on lung and liver tissue sections from Schistosoma-infected mice and uninfected healthy controls. Positively stained cells in whole-lung/liver tissue sections, surrounding the eggs, and in the different compartments of the tissues, were counted. We found a significant increase in the number of mast cells (toluidine blue(+)), CD3(+) cells, CD14(+) cells, CD68(+) cells, and CD15(+) cells in Schistosoma-infecte...
Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling... more Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.
Current Opinion in Investigational Drugs, Jul 1, 2007
Two-pore domain potassium (K2P) channels are expressed in cells throughout the body and give rise... more Two-pore domain potassium (K2P) channels are expressed in cells throughout the body and give rise to leak potassium currents which control the excitability of these cells. Although not inhibited by classical potassium channel-blocking drugs, such as tetraethylammonium and 4-aminopyridine, K2P channels are regulated by a diverse array of pharmacological mediators. There are six main families of K2P channels and among these certain members of the TREK family (ie, TREK-1 and TREK-2) are activated by general anesthetic agents such as halothane, xenon and nitrous oxide. In addition, all members of the TREK familyare activated by neuroprotective agents, such as riluzole, polyunsaturated fatty acids and lysophospholipids, suggesting that these channels play an important role in neuroprotection. TREK channels are also inhibited by chlorpromazine, local anesthetics and the antidepressant fluoxetine. Furthermore, all members of the TASK family are inhibited by cannabinoids and local anesthetics, and TASK-3 is selectively inhibited by ruthenium red. Thus, the diversity and physiological importance of K2P channels suggest that the development of selective compounds to target these proteins has therapeutic potential for CNS disorders such as stroke, depression and epilepsy.
Transmitter-activated channels in freshly isolated neurones from sympathetic ganglia of young rat... more Transmitter-activated channels in freshly isolated neurones from sympathetic ganglia of young rats have been examined using the patch-clamp technique. Acetylcholine (ACh), gamma-aminobutyric acid (GABA) and (in about one third of cells) glycine produced an inward current and an increase in current noise when perfused onto voltage-clamped neurones. The ACh noise was fitted with a single Lorentzian spectrum with a time constant of 13.1 ms at -70 mV. Outside-out membrane patches allowed high-resolution measurements of single ACh- and GABA-activated channels. The ACh-channels had a conductance of 30 pS.
ABSTRACT TREK1, two pore domain potassium (K2P) channels are responsible for background currents ... more ABSTRACT TREK1, two pore domain potassium (K2P) channels are responsible for background currents that regulate neuronal excitability. Deletion of TREK1 has previously been shown to result in a depression-resistant phenotype in mice (Heurteaux et al. 2006). In this study we investigated the effect of the tricyclic antidepressant, amitriptyline, on TREK1 channels. Additionally, amitriptyline is used for relief of neuropathic pain and TREK-1 is also involved in pain perception (Alloui et al. 2006). tsA-201 cells were transiently transfected with wild type and mutated K2P channels. The whole cell patch clamp technique was used to obtain current recordings. Homology models for TREK1 were constructed using Modeller. Amitriptyline was docked to these models using the MOE program. Amitriptyline (50 μM) significantly inhibited WT TREK1 channel current by 77.4 ± 3.7 % (n=9). While the inhibitory effect of amitriptyline was reversible, on wash, it was often followed by an over-recovery of current to above the baseline level. We have previously investigated the effect of the antidepressant, citalopram, on TREK1, and found that it inhibited the channel. Subsequently we identified a number of amino acids in both the pore region and transmembrane domains M2 and M4 of TREK1 that were important for its effect (Al-Moubarak et al. 2013). Interestingly, mutations of several amino acids, T142A, L289A and T251A, which reduced the effectiveness of citalopram had no effect on the observed inhibition by amitriptyline. Other mutations, L169A and I167A had no effect on inhibition by either compound. Mutation L174A (in the M2 transmembrane domain of TREK1), however, significantly reduced inhibition by both citalopram (Al-Moubarak et al 2013) and amitriptyline (50 μM, 49.0 ± 3.1 %, n=6, p < 0.05). As previously found for citalopram, the E306A gain of function mutation completely prevented inhibition by amitriptyline. Docking of amitriptyline into the TREK1 homology model, guided by the obtained electrophysiology results, predicts a different binding profile for amitriptyline compared to citalopram. Thus we have shown that amitriptyline is a potent inhibitor of TREK1 potassium channels. This action may contribute to the therapeutic usefulness of this compound in both the treatment of depression and pain.
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