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Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene ( CFTR ). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e. , function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 ( 4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid ),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC 50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

The synthesis and characterization of a series of selective, orally bioavailable 1-(chroman-4-yl)urea TRPV1 antagonists is described. Whereas first-generation antagonists that inhibit all modes of TRPV1 activation can elicit hyperthermia, the compounds disclosed herein do not elevate core body temperature in preclinical models and only partially block acid activation of TRPV1. Advancing the SAR of this series led to the eventual identification of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442, 52), an analogue that possesses excellent pharmacological selectivity, has a favorable pharmacokinetic profile, and demonstrates good efficacy against osteoarthritis pain in rodents.

The KCNQ2/3 channel has emerged as a drug target for a number of neurological disorders including pain and epilepsy. Known KCNQ2/3 openers have effects on two distinct biophysical properties of the channel: (1) a hyperpolarizing shift in the voltage dependence of channel activation (V(1/2)), and (2) an increase in channel open probability or peak whole-cell current. The current high-throughput screening assays for KCNQ2/3 openers measure changes of channel activity at sub-peak conductances and the output measure is a combination of effects on V(1/2) shift and peak current. Here, we describe a medium-throughput electrophysiological assay for screening KCNQ2/3 openers using the QPatch HT platform. We employed a double-pulse protocol that measures the shift in V(1/2) and the change in current amplitude at peak conductance voltage. Retigabine along with novel KCNQ2/3 openers were evaluated in this assay. Three classes of KCNQ2/3 openers were identified based on the hyperpolarizing shift in V(1/2) and the change in peak current. All three classes of compounds caused a hyperpolarizing shift in V(1/2), but they were differentiated by their respective effects on peak current amplitude (increase, decrease, or only modestly affecting peak current amplitude). KCNQ2/3 blockers were also identified with this assay. These compounds blocked currents without affecting voltage-dependent activation. In summary, we have developed a medium-throughput assay that can reliably detect changes in the biophysical properties of the KCNQ2/3 channel, V(1/2), and peak current amplitude, and therefore may serve as a reliable assay to evaluate KCNQ2/3 openers and blockers.

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca(2+) influx through the activated channel followed by Ca(2+)-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain.

Objective: The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays., Methods: The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology., Results: N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable., Conclusions: The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.

Elevated temperature and decreased extracellular pH are hallmarks of inflammatory pain states. Dorsal root ganglia (DRG) neurons are integral in transferring painful stimuli from the periphery to central sites. This study investigated the effect of elevated temperatures on the response of DRG neurons to acute application of acidic solutions. At room temperature (22 degrees C), in response to pH 5.5, there were a variety of kinetic responses consistent with differential expression of TRPV1 and ASIC channels. Increasing the temperature resulted in a significant increase in the peak and total current mediated by TRPV1 in response to an acidic solution. In contrast, the amplitude of a fast activating, rapidly inactivating ASIC1-like current was not affected by increasing the temperature but did result in an increased rate of desensitization that reduced the total current level. This effect on the rate of desensitization was temperature-dependent and could be reversed by returning to 22 degrees C. Likewise, cells exhibiting slowly inactivating ASIC2-like responses also had temperature-dependent increase in the rate of desensitization. The ASIC2-like responses and the TRPV1 responses tended to decrease in amplitude with repetitive application of pH 5.5 even at 22 degrees C. The rate of desensitization of ASIC-like currents activated by less acidic solutions (pH 6.8) was also increased in a temperature-dependent manner. Finally, acidic pH reduced threshold to trigger action potentials, however, the pattern of action potential firing was shaped by the distribution of ASIC and TRPV1 channels. These results indicate that the ambient temperature at which acidosis occurs has a profound effect on the contribution of ASIC and TRPV1 channels, therefore, altering the neuronal excitability., (Copyright 2010 Elsevier B.V. All rights reserved.)

TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.

1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea (A-425619), a novel, potent, and selective transient receptor potential type V1 (TRPV1) antagonist, attenuates pain associated with inflammation and tissue injury in rats. The purpose of this study was to extend the in vitro characterization of A-425619 to native TRPV1 receptors and to compare the pharmacological properties of TRPV1 receptors in the dorsal root ganglion with trigeminal ganglion neurons. A robust increase in intracellular Ca(2+) was elicited by a variety of TRPV1 agonists with similar rank order of potency between both cultures: resiniferatoxin>tinyatoxin>capsaicin>N-arachidonoyl-dopamine (NADA). A-425619 blocked the 500 nM capsaicin response in both dorsal root ganglion with trigeminal ganglion cultures with IC(50) values of 78 nM and 115 nM, respectively, whereas capsazepine was significantly less potent (dorsal root ganglia: IC(50)=2.63 microM; trigeminal ganglia: IC(50)=6.31 microM). Furthermore, A-425619 was more potent in blocking the 3 microM NADA-evoked response in both dorsal root ganglia (IC(50)=36 nM) and trigeminal ganglia (IC(50)=37 nM) than capsazepine (dorsal root ganglia, IC(50)=741 nM; trigeminal ganglia, IC(50)=708 nM). Electrophysiology studies showed that 100 nM A-425619 completely inhibited TRPV1-mediated acid activated currents in dorsal root ganglia and trigeminal ganglia neurons. In addition, A-425619 blocked capsaicin- and NADA-evoked calcitonin gene-related peptide (CGRP) release in both cultures more effectively than capsazepine. These data show that A-425619 is a potent TRPV1 antagonist at the native TRPV1 receptors, and suggest that the pharmacological profile for TRPV1 receptors on dorsal root ganglia and trigeminal ganglia is very similar.

The transient receptor potential vanilloid (TRPV) 1 receptor, a nonselective cation channel expressed on peripheral sensory neurons and in the central nervous system, plays a key role in pain. TRPV1 receptor antagonism is a promising approach for pain management. In this report, we describe the pharmacological and functional characteristics of a structurally novel TRPV1 antagonist, (R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102), which has entered clinical trials. At the recombinant human TRPV1 receptor ABT-102 potently (IC(50) = 5-7 nM) inhibits agonist (capsaicin, N-arachidonyl dopamine, anandamide, and proton)-evoked increases in intracellular Ca(2+) levels. ABT-102 also potently (IC(50) = 1-16 nM) inhibits capsaicin-evoked currents in rat dorsal root ganglion (DRG) neurons and currents evoked through activation of recombinant rat TRPV1 currents by capsaicin, protons, or heat. ABT-102 is a competitive antagonist (pA(2) = 8.344) of capsaicin-evoked increased intracellular Ca(2+) and shows high selectivity for blocking TRPV1 receptors over other TRP receptors and a range of other receptors, ion channels, and transporters. In functional studies, ABT-102 blocks capsaicin-evoked calcitonin gene-related peptide release from rat DRG neurons. Intraplantar administration of ABT-102 blocks heat-evoked firing of wide dynamic range and nociceptive-specific neurons in the spinal cord dorsal horn of the rat. This effect is enhanced in a rat model of inflammatory pain induced by administration of complete Freund's adjuvant. Therefore, ABT-102 potently blocks multiple modes of TRPV1 receptor activation and effectively attenuates downstream consequences of receptor activity. ABT-102 is a novel and selective TRPV1 antagonist with pharmacological and functional properties that support its advancement into clinical studies.

The vanilloid receptor 1 (TRPV1) is activated by capsaicin, several endogenous lipids, acidic pH and elevated temperatures. Inflammatory mediators (BK, substance P) also modulate TRPV1 activity. In this study we investigated the effect of TRPV1 agonists and elevated temperatures on neuronal membrane excitability by electrophysiological techniques using freshly isolated rat dorsal root ganglion neurons (DRGs). Focal application of heated solutions demonstrated that the normal threshold (approximately 42 degrees C) of TRPV1 activation was reduced in the presence of capsaicin (1 microM) to approximately 30 degrees C. In current-clamp recordings, increasing the temperature of the solution resulted in larger membrane depolarizations and significantly altered the pattern and onset of the action potential train evoked by 1 microM capsaicin. These effects were blocked by the TRPV1 antagonist capsazepine (10 microM). In contrast to capsaicin, anandamide (10 microM) alone did not evoke action potentials, but it did alter the excitability of neurons to subsequent applications of heat (50 degrees C). Together these results provide evidence that a synergistic interaction of TRPV1 ligands and elevated temperature activates TRPV1 receptors and results in profound effects on membrane excitability.

Transient receptor potential (TRP)A1 channel has been implicated in various physiological processes, including thermosensation and pain. A recent study of TRPA1 knockout mice demonstrated deficits in sensing mechanical stimuli, suggesting a role for TRPA1 also in somatic mechanosensation. However, direct evidence of TRPA1 activation by mechanical forces has thus far been lacking. Here we show, using an intracellular calcium assay, that hypertonic solution (HTS) activates TRPA1 channels in human embryonic kidney 293 cells transiently expressing rat TRPA1. In contrast, hypotonic solution has no effect. Single-channel recordings reveal that HTS opens an ion channel that displays similar single-channel conductance to that evoked by the TRPA1 agonist allyl isothiocyanate (AITC) in both recombinant rat TRPA1 cell lines and rat dorsal root ganglia neurons. Ruthenium red reduces the open probability of the single-channel currents and blocks the whole-cell currents evoked by HTS. Camphor also blocks the whole-cell currents evoked by HTS. HTS-activated channel openings are only observed in patches that are also sensitive to AITC. Finally, like AITC, HTS depolarizes the membrane potential of dorsal root ganglia neurons leading to the generation of action potentials. Taken together, these findings indicate that TRPA1 mediates an osmotically-activated ion channel and support a role for TRPA1 in mechanosensation.

1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778317) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pIC50 = 8.31 +/- 0.13). The (S)-stereoisomer, 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A-778316), is 6.8-fold less potent (pIC50 = 7.47 +/- 0.07). A-778317 also potently blocks capsaicin and acid activation of native rat TRPV1 receptors in dorsal root ganglion neurons. A-778317 was tritiated ([3H]A-778317; 29.3 Ci/mmol) and used to study recombinant human TRPV1 (hTRPV1) receptors expressed in Chinese ovary cells (CHO) cells. [3H]A-778317 labeled a single class of binding sites in hTRPV1-expressing CHO cell membranes with high affinity (KD = 3.4 nM; Bmax = 4.0 pmol/mg protein). Specific binding of 2 nM [3H]A-778317 to hTRPV1-expressing CHO cell membranes was reversible. The rank-order potency of TRPV1 receptor antagonists to inhibit binding of 2 nM [3H]A-778317 correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A-778317 blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site and that [3H]A-778317 possesses favorable binding properties to facilitate further studies of hTRPV1 receptor pharmacology.

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.

Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N'-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.

TRPV1 (vanilloid receptor 1) receptors are activated by a variety of ligands such as capsaicin, as well as by acidic conditions and temperatures above 42 degrees C. These activators can enhance the potency of one another, shifting the activation curve for TRPV1 to the left. In this study, for example, we observed an approximately 10-fold shift in the capsaicin EC50 (640 nM to 45 nM) for rat TRPV1 receptors expressed in HEK-293 cells when the pH was lowered from 7.4 to 5.5. To investigate potential causes for this shift in capsaicin potency, the rates of current activation and deactivation of whole-cell currents were measured in individual cells exposed to treatments of pH 5.5, 1 microM capsaicin or in combination. Acidic pH was found to both increase the activation rate and decrease the deactivation rate of capsaicin-activated currents providing a possible mechanism for the enhanced potency of capsaicin under acidic conditions. Utilizing a paired-pulse protocol, acidic pH slowed the capsaicin deactivation rate and was readily reversible. Moreover, the effect could occur under modestly acidic conditions (pH 6.5) that did not directly activate TRPV1. When TRPV1 was maximally activated by capsaicin and acidic pH, the apparent affinity of the novel and selective capsaicin-site competitive TRPV1 antagonist, A-425619, was reduced approximately 35 fold. This shift was overcome by reducing the capsaicin concentration co-applied with acidic pH. Since inflammation is associated with tissue acidosis, these findings enhance understanding of TRPV1 receptor responses in inflammatory pain where tissue acidosis is prevalent.

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC50 = 5 nM). A-425619 showed similar potency (IC50 = 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC50 = 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol ester-sensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA2 = 2.5 nM) of capsaicin-evoked calcium flux. Moreover, A-425619 was 25- to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.

(1) Rapid desensitization of ligand-gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast-channel activation difficult. Investigation of the antagonist pharmacology of ATP-sensitive homomeric P2X3 receptors is limited by agonist-evoked fast-desensitization kinetics. (2) In the present studies, chimeric receptors were created using the coding sequence for the N-terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast-desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C-terminus of the other receptor (designated P2X2-3 and P2X3-2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium-imaging methods. (3) Chimeric P2X2-3 and P2X3-2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild-type human P2X2a and P2X3 receptors, respectively. (4) The P2X2-3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild-type receptor being activated by low concentrations of both ATP and alpha,beta-meATP. In contrast, the P2X3-2 chimera had markedly reduced sensitivity to both agonists. (5) The P2X3 receptor antagonists TNP-ATP and A-317491 were shown to be potent, competitive antagonists of the P2X2-3 chimera (Ki=2.2 and 52.1 nm, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild-type homomeric P2X3 receptors.

Skeletal muscle hyperexcitability is characteristically associated with denervation. Expression of SK3, a small conductance Ca(2+)-activated K(+) channel (SK channel) in skeletal muscle is induced by denervation, and direct application of apamin, a peptide blocker of SK channels, dramatically reduces hyperexcitability. To investigate the role of SK3 channels in denervation- induced hyperexcitability, SK3 expression was manipulated using a transgenic mouse that harbors a tetracycline-regulated SK3 gene. Electromyographic (EMG) recordings from anterior tibial (AT) muscle showed that denervated muscle from transgenic or wild-type animals had equivalent hyperexcitability that was blocked by apamin. In contrast, denervated skeletal muscle from SK3tTA mice lacking SK3 channels showed little or no hyperexcitability, similar to results from wild-type innervated skeletal muscle. However, innervated skeletal muscle from SK3tTA mice containing SK3 channels did not show hyperexcitability. The results demonstrate that SK3 channels are necessary but not sufficient for denervation-induced skeletal muscle hyperexcitability., (Copyright 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 817-822, 2002)

1. Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small-conductance Ca2+-activated K+ channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole-cell patch clamp configuration. 2. Depolarization activated a K+-selective, apamin-sensitive and iberiotoxin-insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mM EGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+-activated SK channels expressed only in denervated muscle. 3. Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+ due to release from the sarcoplasmic reticulum (SR) and influx through voltage-gated Ca2+ channels. 4. The envelope of SK tail currents was diminished by 10 microM ryanodine for all pulse durations, whereas 2 mM cobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+ release from the SR during short pulses primarily activated SK channels. 5. In current clamp mode with the resting membrane potential set at -70 mV, denervation decreased the action potential threshold by approximately 8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold. 6. These results are consistent with a model in which SK channel activity in the T-tubules of denervated skeletal muscle causes a local increase in K+ concentration that results in hyperexcitability.

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at -80 mV, barium currents (I(Ba)s) were evoked by voltage commands to above -35 mV that peaked at +5 mV. When holding potentials were reduced to -20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I(Ba) was fit with a Boltzmann curve (V(1/2) = -43.3 mV; slope = -17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I(Ba)s were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 microM nifedipine (19 vs. 25%), 10 microM conotoxin GVIA (27 vs. 25%), 10 microM conotoxin MVIIC (15 vs. 16%), and 1.75 microM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 microM cadmium. In the presence of nifedipine, GVIA, and MVIIC, approximately 35% of current remained, which was reduced further by SNX-482 (7-14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).

mRNA encoding the recently cloned gamma-aminobuytyric acid(A) receptor (GABAR) pi subunit is expressed in the hippocampus and in several non-neuronal tissues including the uterus and ovaries. Whereas native GABARs are pentamers composed primarily of alphabetagamma, alphabetadelta, or alphabetaepsilon subunits, it has not been demonstrated clearly that the pi subunit incorporates into functional GABARs to form alphabetapi receptors and, if so, with what properties. We provide electrophysiological evidence that the pi subunit can coassemble with either alpha5beta3 or alpha5beta3gamma3 subunits to produce recombinant GABARs with distinct pharmacological and biophysical properties. Compared with alpha5beta3 receptors, GABARs produced by coexpression of alpha5beta3pi subunits had a lower GABA EC(50) value, were enhanced to a lesser extent by loreclezole, had different IC(50) values for pregnenolone sulfate and lanthanum, and were insensitive to benzodiazepines. Incorporation of both pi and gamma3 subunits into an alpha5beta3gamma3pi isoform was suggested by reduced enhancement by diazepam and a high zinc IC(50) value. Current-voltage relations for the alpha5beta3pi subunit combination outwardly rectified more than currents from alpha5beta3gamma3 but less than alpha5beta3 combination GABARs. Single-channel alpha5beta3 GABAR currents had a main conductance state of 15.2 picoSeimens (pS). Coexpression of the pi subunit with alpha5beta3 subtypes increased the conductance level to 23.8 pS, similar to the conductance level of alpha5beta3gamma3 GABARs (26.9 pS). We conclude that the pi subunit coassembles with alpha, beta, and gamma subunits to form functional alphabetapi or alphabetagammapi GABARs and, thus, could have a significant impact on the function of native GABARs expressed in the brain or non-neuronal tissue.

During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.

A new gamma-aminobutyric acid (GABA)A receptor (GABAR) subunit class, epsilon, has recently been cloned and shown to form functional channels when coexpressed with both alpha and beta subunits. We report that the combination of alpha1beta3epsilon subunit subtypes expressed in L929 cells produced functional chloride ion channels that were both spontaneously active and gated by the application of extracellular GABA. When cells were voltage-clamped at -75 mV in the whole-cell configuration, holding currents of 50 to 300 pA associated with increased noise were consistently recorded. The application of pentobarbital and loreclezole, which increase GABAR currents, increased the holding current, whereas the application of zinc and picrotoxin, which reduce GABAR currents, reduced the holding current in a concentration-dependent manner. Coexpression of alpha1beta3gamma2L, alpha1beta3delta, alpha1epsilon, beta3epsilon, alpha1beta3, or epsilon subtypes did not produce holding currents that were sensitive to picrotoxin (30 microM). Cells expressing alpha1 beta3epsilon subtypes had concentration-dependent GABAR currents that were potentiated by pentobarbital, loreclezole, and lanthanum and inhibited by zinc and furosemide. Spontaneous and GABAR single-channel currents from alpha1beta3epsilon receptors had single-channel conductances of approximately 24 pS. The biophysical properties and the effects of allosteric modulators were similar for spontaneous and evoked GABAR currents, suggesting that a single GABAR isoform was responsible for both currents. These data extend the pharmacological characterization of epsilon-containing GABARs and demonstrate that incorporation of the epsilon subunit permits spontaneous channel gating while preserving the structural information necessary for GABA sensitivity.

Human NT2 teratocarcinoma cells differentiate into neuron-like NT2-N cells when treated with retinoic acid. GABA evoked concentration-dependent whole-cell currents in NT2-N cells with an EC50 of 21.8 microM and a Hill slope of 1.2. GABAA receptor (GABAR) currents reversed at ECl- and did not display voltage-dependent rectification. GABAR single channels opened in bursts to a 23 pS main conductance level and a 19 pS subconductance level, with infrequent openings to a 27 pS conductance level. Kinetic properties of the main conductance level were similar to other native and recombinant GABAR channels. Diazepam and zolpidem enhanced GABAR currents with moderate affinity, whereas methyl-6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate inhibited GABAR currents. Loreclezole enhanced GABAR currents with high affinity, but furosemide antagonized GABAR currents with low affinity. The neurosteroids alphaxalone and pregnenolone sulfate appropriately modulated GABAR currents. Zinc blocked GABAR currents with low affinity, but lanthanum did not significantly alter NT2-N GABAR currents. Reverse transcription PCR (RT-PCR) performed on RNA from NT2-N cells clearly detected transcripts encoding human alpha2, alpha3, alpha5, beta3, gamma3, and pi subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an alpha2 and/or alpha3 subtype combined with the beta3 and gamma3 subtypes. The data do not rule out receptors containing combinations of alpha2 and/or alpha3 subtypes with the alpha5 subtype or receptors with both beta1 and beta3 subtypes. The presence or absence or the pi subunit in functionally expressed receptors could not be determined.

Functional studies have indicated that, unlike most divalent cations, lanthanum increases both native and recombinant gamma-aminobutyric acid (GABA) receptor (GABAR) currents. In the present study, we have examined whether lanthanum shows subunit-dependent selectivity for modification of currents from different GABAR isoforms. The effects of lanthanum on three different GABAR isoforms, alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta, were determined by transient expression of combinations of alpha1, alpha6, beta3, gamma2L, and delta subunit cDNAs in L929 fibroblasts. Whole-cell recording was used to determine the concentration-response curves for lanthanum for the three different isoforms at submaximal concentrations of GABA. Lanthanum displayed strong potentiation of alpha1beta3gamma2L GABAR currents consistent with earlier reports of potentiation of GABAR currents by lanthanum in neurons and recombinant GABAR isoforms. However, in contrast to the potentiation of alpha1beta3gamma2L GABAR currents by lanthanum, alpha6beta3delta GABAR currents were strongly inhibited and alpha6beta3gamma2L GABAR currents were weakly inhibited by lanthanum. Interaction of lanthanum with GABAR isoforms was competitive, with lanthanum decreasing the EC50 value for GABA of alpha1beta3gamma2L GABARs without changing the maximum current and increasing the EC50 value for GABA of alpha6beta3delta and alpha6beta3gamma2L GABAR currents (greater shift in EC50 value in the alpha6beta3delta compared with the alpha6beta3gamma2L GABARs) without changing the maximum GABAR current. Neither potentiation nor inhibition of GABAR currents by lanthanum showed any voltage dependence. These results suggest that 1) changing the alpha-subunit subtype from alpha1 to alpha6 altered the effect of lanthanum from potentiation to inhibition, 2) changing the gamma2L subunit to the delta-subunit changed the level of maximal inhibition of alpha6 subtype-containing GABAR currents by lanthanum, and 3) the site for interaction with lanthanum probably was on the extracellular surface of GABARs.

Transient cotransfection of fibroblasts, with plasmids encoding individual GABAA receptor (GABAAR) subunits, has provided a model to characterize the pharmacological and kinetic properties of receptor subtype combinations. However, identifying transfected cells for electrophysiological recording is often difficult due to low transfection efficiencies. Selection of transfected cells has required cotransfection with a marker gene and fluorescence microscopic localization prior to recording. To circumvent these problems, two GABAAR subtypes combinations in transfected L929 cells were isolated with a novel biomagnetic separation system. Cell selection was accomplished by cotransfection with a plasmid (pHook-1) encoding a single-stranded cell surface antibody (sFv), which bound to ferromagnetic beads, coated with an antigen (phOx). Bead-covered cells were then magnetically separated from non-transfected cells. Bead-selected cells cotransfected with alpha 6, beta 3 and gamma 2L subtypes, expressed GABAAR currents in 95% (41/43) of cells recorded. Cells cotransfected with alpha 5, beta 3 and gamma 2L subtypes had an EC50 for GABA of 5.4 microM and a Hill slope of 1.4. Membrane patches from cells expressing the alpha 5 beta 3 gamma 2L isoform demonstrated single channel currents with a main conductance state of 23 pS. Magnetic bead immunoselection provides a purified population of transfected cells well suited for whole cell and single channel recording.

The cDNAs encoding alpha 5 and gamma 2L subunit subtypes of the gamma-aminobutyric acid (GABA) type A receptor (GABAR) were transfected into L929 cells together with cDNAs encoding either the beta 1, beta 2, or beta 3 subunit subtype. Properties of expressed recombinant alpha 5 beta X gamma 2L (where X = 1,2, or 3) GABARs were studied with the use of whole-cell, patch-clamp techniques. In cells voltage-clamped at -70 mV with equlvalent bath and pipette chloride concentrations, the application of GABA produced a concentration-dependent inward chloride current with all three alpha 5 beta X gamma 2L isoforms. Minimal or no responses were recorded from cells transfected with only two subunit cDNAs, demonstrating that all three subunits were required for functional receptor assembly in these cells. The GABA concentration producing a half-maximal current was similar for beta 2 and beta 3 subtype-containing receptors (6 microM) but higher for beta 1 subtype-containing receptors (26 microM). alpha 5 beta 3 gamma 2L receptors were zinc and diazepam sensitive but zolpidem insensitive. In response to low GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed outward rectification of the current-voltage relationship, whereas current-voltage responses of beta 2 subtype-containing receptors were relatively linear. Likewise, at high GABA concentrations, beta 1 and beta 3 subtype-containing receptors showed less desensitization at positive than at negative membrane potentials. Beta 2 subtype-containing receptors displayed faster desensitization at depolarized potentials. These voltage-dependent properties were characteristic of alpha 5 but not alpha 1 or alpha 6 subtype-containing receptors and were similar to responses recorded from hippocampal CA1 pyramidal neurons. Based on the pharmacological and biophysical similarities to hippocampal GABAR responses, the alpha 5 beta 3 gamma 2L isoform could represent a native GABAR subtype.