Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1.

TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.

The biophysical and molecular basis of TRPV1 proton gating.

The capsaicin receptor TRPV1, a member of the transient receptor potential family of non-selective cation channels is a polymodal nociceptor. Noxious thermal stimuli, protons, and the alkaloid irritant capsaicin open the channel. The mechanisms of heat and capsaicin activation have been linked to voltage-dependent gating in TRPV1. However, until now it was unclear whether proton activation or potentiation or both are linked to a similar voltage-dependent mechanism and which molecular determinants underlie the proton gating. Using the whole-cell patch-clamp technique, we show that protons activate and potentiate TRPV1 by shifting the voltage dependence of the activation curves towards more physiological membrane potentials. We further identified a key residue within the pore region of TRPV1, F660, to be critical for voltage-dependent proton activation and potentiation. We conclude that proton activation and potentiation of TRPV1 are both voltage dependent and that amino acid 660 is essential for proton-mediated gating of TRPV1.

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Influence of the N terminus on the biophysical properties and pharmacology of TREK1 potassium channels.

TWIK-related K(+) 1 (TREK1) potassium channels are members of the two-pore domain potassium channel family and contribute to background potassium conductances in many cell types, where their activity can be regulated by a variety of physiologic and pharmacologic mediators. Fenamates such as FFA (flufenamic acid; 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid), MFA [mefenamic acid; 2-(2,3-dimethylphenyl)aminobenzoic acid], NFA [niflumic acid; 2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid], and diclofenac [2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid] and the related experimental drug BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] enhance the activity of TREK1 currents, and we show that BL-1249 is the most potent of these compounds. Alternative translation initiation produces a shorter, N terminus truncated form of TREK1 with a much reduced open probability and a proposed increased permeability to sodium compared with the longer form. We show that both forms of TREK1 can be activated by fenamates and that a number of mutations that affect TREK1 channel gating occlude the action of fenamates but only in the longer form of TREK1. Furthermore, fenamates produce a marked enhancement of current through the shorter, truncated form of TREK1 and reveal a K(+)-selective channel, like the long form. These results provide insight into the mechanism of TREK1 channel activation by fenamates, and, given the role of TREK1 channels in pain, they suggest a novel analgesic mechanism for these compounds.

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum.

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation.

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.

BACKGROUND AND PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl- channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP-628006 with a distinct chemical structure. EXPERIMENTAL APPROACH: Using electrophysiological assays with CFTR-expressing heterologous cells and CF patient-derived human bronchial epithelial (hBE) cells, we compared the effects of CP-628006 with the marketed CFTR potentiator ivacaftor. KEY RESULTS: CP-628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del-CFTR were larger than those with the gating variant G551D-CFTR. In excised inside-out membrane patches, CP-628006 potentiated wild-type, F508del-CFTR, and G551D-CFTR by increasing the frequency and duration of channel openings. CP-628006 increased the affinity and efficacy of F508del-CFTR gating by ATP. In these respects, CP-628006 behaved like ivacaftor. CP-628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP-628006 conferred ATP-dependent gating on G551D-CFTR, whereas the action of ivacaftor was ATP-independent. For G551D-CFTR, but not F508del-CFTR, the action of CP-628006 plus ivacaftor was greater than ivacaftor alone. CP-628006 delayed, but did not prevent, the deactivation of F508del-CFTR at the plasma membrane, whereas ivacaftor accentuated F508del-CFTR deactivation. CONCLUSIONS AND IMPLICATIONS: CP-628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.

Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification.

Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn(333) and Asp(349) lie respectively in external and internal vestibules. Substitutions at Asn(333), Thr(336), and Ser(340) were particularly likely to cause spontaneously active channels. At Thr(336), Thr(339), and Ser(340), the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 A crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn(333) to Thr(339) and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.

Role of the domain encompassing Arg304-Ile328 in rat P2X2 receptor conformation revealed by alterations in complex glycosylation at Asn298.

The final 25 amino acids of the ectodomain of the P2X receptors, immediately prior to the second TM (transmembrane domain) (pre-TM2: Arg(304)-Ile(328) in rat P2X(2)), are highly conserved. Whole-cell patch clamp recordings showed that single cysteine substitutions in the N-terminal half of pre-TM2 (Arg(304)-Ile(314)) led to loss of function at Arg(304), Leu(306), Lys(308) and Ile(312). Cysteine substitutions within this region also resulted in a significant reduction in the apparent molecular mass of receptors, due to loss of complex glycosylation at the nearby acceptor site Asn(298), which was not seen for the C-terminal portion of pre-TM2 (Asp(315)-Ile(328)). The reduction in complex glycosylation was not due to reduced cell-surface presentation, demonstrating that glycosylation at Asn(298) was acting as a sensor of subtle changes in receptor conformation within the pre-TM2 region. When this N-glycan site was repositioned closer to the plasma membrane by mutagenesis (N298S together with G299N, T300N, T301N or T303N), glycosylation was restored at G299N and T300N, but was impaired for T301N and completely absent for T303N. These results suggest that the region in the vicinity of Asp(315) is at the plasma membrane interface and that the N-terminal portion of pre-TM2 (Arg(304)-Ile(314)) is important for the correct conformation of the receptor at the extracellular face of the membrane.

Publisher Correction: Molecular and functional variation in iPSC-derived sensory neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design and Identification of a Novel, Functionally Subtype Selective GABAA Positive Allosteric Modulator (PF-06372865).

The design, optimization, and evaluation of a series of novel imidazopyridazine-based subtype-selective positive allosteric modulators (PAMs) for the GABAA ligand-gated ion channel are described. From a set of initial hits multiple subseries were designed and evaluated based on binding affinity and functional activity. As designing in the desired level of functional selectivity proved difficult, a probability-based assessment was performed to focus the project's efforts on a single subseries that had the greatest odds of delivering the target profile. These efforts ultimately led to the identification of two precandidates from this subseries, which were advanced to preclinical safety studies and subsequently to the identification of the clinical candidate PF-06372865.

Thr339-to-serine substitution in rat P2X2 receptor second transmembrane domain causes constitutive opening and indicates a gating role for Lys308.

P2X2 receptors are ATP-gated ion channels widely expressed by neurons. Thr339 lies in the second of the two transmembrane domains of the rat P2X2 receptor protein, and is likely to be close to the narrowest part of the pore. Single-channel and whole-cell recording after expression in human embryonic kidney 293 cells showed that P2X2[T339S] receptors had pronounced spontaneous channel openings that were never seen in wild-type P2X2 receptors. P2X2[T339S] receptors were 10-fold more sensitive than wild type to exogenous ATP, and alphabeta meATP also increased channel opening. Two conserved ectodomain lysine residues (Lys69 and Lys308) are critical for function and have been proposed to contribute to the ATP binding site of P2X receptors. The spontaneous opening of P2X2[K69A/T339S] receptors was not different than that seen in P2X2[T339S], but for P2X2[K308A/T339S] the spontaneous activity was absent. Suramin, which is a noncompetitive antagonist at wild-type P2X2 receptors, had a pronounced agonist action at both P2X2[T339S] and P2X2[K69A/T339S] receptors but not at P2X2[K308A/T339S]. 2',3'-O-O-(2,4,6-Trinitrophenyl)-ATP (TNP-ATP), which is a competitive agonist at wild-type receptors, was also an agonist at P2X2[T339S] receptors, but not at either double mutant. The results indicate that the T339S mutation substantially destabilizes the closed channel and suggest an important role in channel gating. The correction of this gating defect, in the absence of any agonist, by the second mutation K308A shows that Lys308 is also involved in channel gating. A similar interpretation can account for the results with suramin and TNP-ATP.

GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability.

BACKGROUND AND PURPOSE: TREK two-pore-domain potassium (K2P ) 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. EXPERIMENTAL APPROACH: 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. The expression of K2P 2.1 (TREK1), K2P 10.1 (TREK2) and K2P 4.1 (TRAAK) channels 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. KEY RESULTS: For recombinant human TREK1 channels, GI-530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current-clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1 µM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential. CONCLUSIONS AND IMPLICATIONS: This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid in the development of novel analgesic agents. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Molecular and functional variation in iPSC-derived sensory neurons.

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.

Pharmacology in translation: the preclinical and early clinical profile of the novel α2/3 functionally selective GABAA receptor positive allosteric modulator PF-06372865.

BACKGROUND AND PURPOSE: Benzodiazepines, non-selective positive allosteric modulators (PAMs) of GABAA receptors, have significant side effects that limit their clinical utility. As many of these side effects are mediated by the α1 subunit, there has been a concerted effort to develop α2/3 subtype-selective PAMs. EXPERIMENTAL APPROACH: In vitro screening assays were used to identify molecules with functional selectivity for receptors containing α2/3 subunits over those containing α1 subunits. In vivo receptor occupancy (RO) was conducted, prior to confirmation of in vivo α2/3 and α1 pharmacology through quantitative EEG (qEEG) beta frequency and zolpidem drug discrimination in rats respectively. PF-06372865 was then progressed to Phase 1 clinical trials. KEY RESULTS: PF-06372865 exhibited functional selectivity for those receptors containing α2/3/5 subunits, with significant positive allosteric modulation (90-140%) but negligible activity (≤20%) at GABAA receptors containing α1 subunits. PF-06372865 exhibited concentration-dependent occupancy of GABAA receptors in preclinical species. There was an occupancy-dependent increase in qEEG beta frequency and no generalization to a GABAA α1 cue in the drug-discrimination assay, clearly demonstrating the lack of modulation at the GABAA receptors containing an α1 subtype. In a Phase 1 single ascending dose study in healthy volunteers, evaluation of the pharmacodynamics of PF-06372865 demonstrated a robust increase in saccadic peak velocity (a marker of α2/3 pharmacology), increases in beta frequency qEEG and a slight saturating increase in body sway. CONCLUSIONS AND IMPLICATIONS: PF-06372865 has a unique clinical pharmacology profile and a highly predictive translational data package from preclinical species to the clinical setting.

Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia.

In common with other chronic pain conditions, there is an unmet clinical need in the treatment of inherited erythromelalgia (IEM). TheSCN9Agene encoding the sodium channel Nav1.7 expressed in the peripheral nervous system plays a critical role in IEM. A gain-of-function mutation in this sodium channel leads to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. Five patients with IEM were treated with a new potent and selective compound that blocked the Nav1.7 sodium channel resulting in a decrease in heat-induced pain in most of the patients. We derived induced pluripotent stem cell (iPSC) lines from four of five subjects and produced sensory neurons that emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the hyperexcitability of the iPSC-derived sensory neurons, we saw a trend toward a correlation for individual mutations. The in vitro IEM phenotype was sensitive to Nav1.7 blockers, including the clinical test agent. Given the importance of peripherally expressed sodium channels in many pain conditions, our approach may have broader utility for a wide range of pain and sensory conditions.

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel.

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.

K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac.

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.

Rescue of functional DeltaF508-CFTR channels by co-expression with truncated CFTR constructs in COS-1 cells.

The most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR), DeltaF508-CFTR, is misprocessed and subsequently degraded in the endoplasmic reticulum. Using the patch-clamp technique, we showed that co-expressions of DeltaF508-CFTR with the N-terminal CFTR truncates containing bi-arginine (RXR) retention/retrieval motifs result in a functional rescue of the DeltaF508-CFTR mutant channel in COS-1 cells. This DeltaF508-CFTR rescue process was strongly impaired when truncated CFTR constructs possessed either the DeltaF508 mutation or arginine-to-lysine mutations in RXRs. In conclusions, our data demonstrated that expression of truncated CFTR constructs could be a novel promising approach to improve maturation of DeltaF508-CFTR channels.