The 13th Annual Aurora Biomed Ion Channel Retreat: Three Days of Research,Technology,and Networking

Cardiac Safety and Pharmacology

Arthur Brown (ChanTest/Charles River) presented the comprehensive in vitro proarrhythmia assay (CiPA), which is a new screen for cardiac safety. CiPA shifts focus from off-target drug effects on human ether-à-go-go-related gene (hERG) channels to multiple ion channel effects (MICE). Dr. Brown showed that examining MICE, combined with in silico modeling and confirmatory experiments on induced pluripotent stem cell (iPSC)-derived human cardiomyocytes, allows compounds to be ranked by cardiac risk profile and a mechanism-based “proarrhythmia score.” Under the new CiPA guidelines, cardiac risk will be examined earlier in the drug development process and safety profiles will take into account interactions with a wider variety of cardiac channels.

Shouming Du (Hamamatsu) discussed a new 96-channel electrode array system that can be mounted on existing Functional Drug Screening System series FDSS/μCell image-based plate readers. This electrode array system sends electric field stimulations and monitors fluorescence in all 96 wells simultaneously. Dr. Du showed that the electric field stimulations allow for synchronized beating of human iPSC-derived cardiomyocytes across a 96-well plate. This pacing kit can lead to improved monitoring of intracellular events that occur during cardiomyocyte beating, such as oscillating Ca²⁺ levels.

Structure, Function, and Engineering of Ion Channels

Frank Horrigan (Baylor College of Medicine) discussed mechanisms of large conductance voltage- and Ca²⁺-activated K⁺ (BK) channel modulation. Numerous activators of these channels are known, but their sites of action and the mechanisms with which they modulate the channel voltage sensors, calcium sensors, and gate are poorly understood. The Horrigan research group examined the BK activator phloretin and determined that it disrupts coupling between the BK voltage sensor and channel gate. The ability of phloretin to promote channel activation is dependent on the position of the voltage sensor: when the voltage sensor is in the outward position, the compound is less effective. Chimeras between BK channels and a BK channel homologue insensitive to phloretin revealed that phloretin interacts with the S6 transmembrane helix. The current model is that phloretin binds to a pocket between the S5 and S6 helices to stabilize the open state, and alters the conformation of S6 enough to disrupt coupling between S6 and the voltage-sensing and calcium-sensing channel regions.

Saverio Gentile (Loyola University Chicago) discussed the dysregulation of hERG1 channels in cancer. The Gentile research group discovered that stimulation of hERG1 channels by small molecules can lead to arrest of cancer cell proliferation due to an inability of the cell to pass through the G₀/G₁ cell cycle checkpoint. Activation of hERG1 channels leads to calcium-dependent pathways that degrade both E1 and E2 cyclins. Notably, the pathways and intermediates for E1 and E2 cyclin degradation are different, despite a high level of homology between the two cyclins. Activation of hERG1 channels can also stimulate transcription of p21 even in cancer cells that have downregulated the p53 tumor suppressor that normally controls p21 expression. These results suggest novel mechanisms wherein treatment of cancer cells with hERG1 channel activators generates calcium-dependent biochemical cascades, leading to cell cycle arrest.

Eric Accili (University of British Columbia) highlighted negative cooperativity in cAMP binding to the C-terminal region of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The binding of cAMP can significantly depolarize the V_1/2 value of HCN2 channels, but has only a minimal effect on HCN1 channels. Isothermal titration calorimetry on key components of the HCN2 C-terminal region suggests that interaction of cAMP with the tetrameric C-terminal region is comprised of two binding events with different affinities. Cyclic AMP appears to bind to the first subunit in the HCN2 tetramer with high affinity, and this binding induces conformational changes in the C-terminal region that both lower the affinity for subsequent cAMP binding and facilitate channel activation. Dr. Accili suggests that the conformation of HCN1 before cAMP binding resembles that of HCN2 after the first cAMP binding event, which would explain why the “pre-activated” HCN1 channel is less responsive to cAMP than the HCN2 channel.

Geerten Vuister (University of Leicester) presented research on calmodulin regulation of the transient receptor potential (TRP) channel TRPV subfamily, which comprises nonselective cation channels that contribute to neuronal depolarization and undergo calcium-based inactivation. Evidence suggests that calmodulin regulates TRPV5 and TRPV6 channels through two binding sites within the intracellular C-terminal tail of the channel. Two TRPV subunits can bind to a single calmodulin molecule in a calcium-dependent manner. The Vuister group has identified a new mechanism in which calmodulin can bind to the channel even in low levels of intracellular calcium.

The characterization of an ethanol-sensitive benzodiazepine binding site on gamma-aminobutyric acid (GABA)-A receptors was presented by Richard Olsen (University of California, Los Angeles). Millimolar concentrations of ethanol can enhance chloride currents of GABA receptors containing the delta subunit. His group discovered that imidazobenzodiazepine binding site ligand Ro15-4513 can inhibit the effect of ethanol on GABA-A receptors. Point mutations and chimera receptors revealed an ethanol-sensitive benzodiazepine binding site on the extracellular domain α+/β− interface, separate from the well-known benzodiazepine binding sites in the extracellular loops of the α and δ subunits. These findings help to characterize the modes of action of alcohol in low doses.

Yu Zhou (Washington University School of Medicine) discussed calcium-dependent inhibition of BK-type channels by the fungal toxin paxilline. Paxilline inhibition is inversely proportional to the open probability of the channel; the binding affinity of paxilline is over 500-fold greater to the closed channel than the open channel. Paxilline does not change voltage sensor movement; rather it allosterically stabilizes the closed state without blocking ion passage through the channel. It was also discovered that paxilline inhibition is dependent on an inner pore glycine in the BK channel. In future work, mutagenesis scanning of the BK pore region will be employed to better understand the mechanism of action of this toxin.

Rajnish Ranjan (Blue Brain Project, École Polytechnique Fédérale de Lausanne) introduced the online database Channelpedia (www.channelpedia.net), a platform where researchers can compile ion channel information on a Wikipedia-like interface. The platform houses results from the Blue Brain Project, which uses supercomputer modeling to digitally simulate brain tissue. Although the presentation centered on voltage-gated potassium (Kv) channel data housed in Channelpedia, the database currently includes over 180 annotated ion channels and is updated weekly to include new findings from literature. The work of the Blue Brain Project and its amalgamation on Channelpedia are resources for generating new neuronal models of ion channels expressed in the brain.

Transporters and Ion Pumps

Larry Fliegel (University of Alberta) discussed the molecular characterization of the Na⁺/H⁺ exchanger (NHE) and its role in two diseases: “triple negative” metastatic breast cancers and Lichtenstein–Knorr syndrome. In aggressive triple negative breast cancer, the NHE is often hyperactive in cells undergoing tumorigenesis and this hyperactivity can promote metastasis. NHE1 knockout breast cancer cells had decreased rates of migration, reduced invasive potential, and a higher susceptibility to chemotherapy from the drug paclitaxel. Thus NHE inhibition may be useful as an adjuvant to chemotherapy. Study of a family with the autosomal recessive condition Lichtenstein–Knorr syndrome, which can cause ataxia and deafness, revealed a common genetic abnormality in the NHE1 gene. The missense mutation G305A led to defective targeting of NHE1 to the plasma membrane, deglycosylation, and reduced activity of the protein. Thus, loss of NHE1 function can be considered causal in this rare disease.

Chris Triggle (Weill Cornell Medical College in Qatar) discussed newly discovered cellular roles for the type II diabetes drug metformin. On the market since 1958, metformin currently treats 150 million patients through inhibiting gluconeogenesis in the liver. Metformin also has multiple pleiotropic effects outside the liver, such as enhanced GLUT4-mediated glucose uptake in striated muscle. Of note, it can induce mTOR inhibition and cell cycle arrest, promote or inhibit autophagy, inhibit glucose-starvation-induced endoplasmic reticulum (ER) stress, enhance expression of cation transporters, and regulate expression of enzymes enhancing vascular function. Concentration-dependent pleiotropic effects of metformin may thus protect against both cancer and cardiovascular disease.

Nathan Zahler (XRPro Sciences) presented a label-free high-throughput transporter assay. The ion flux technology has been applied to study of the TRP channel subfamily TRPA, which contributes to inflammatory pain. TRPA1 monovalent ion efflux with Rb⁺ and divalent ion influx with Sr²⁺ were measured in the presence of both channel agonists and antagonists in human serum, enabling the acquisition of functional shift measurements in 100% human serum. Additionally, Douglas Krafte (Icagen) announced that XRPro recently acquired Icagen, whose portfolio of assays and cell lines for channel drug discovery will be combined with XRPro's label-free X-ray fluorescence technology.

Paul Li (Simon Fraser University) introduced a novel microfluidic biochip designed to quickly isolate single circulating tumor cells released into the blood from a solid tumor based on their larger size than blood cells. Circulating tumor cells are rare but are often multidrug-resistant, so their identification and eradication are important to the success of cancer treatments. Once a tumor cell is isolated in a chamber on the biochip, fluorescence signal intensities can be used to measure the effects of inhibitors specific to multidrug-resistant cells. The process of isolating and testing cells is complete in ∼2 h. This biochip may lead to earlier detection of solid tumors and a better understanding of chemotherapy resistance mechanisms in circulating tumor cells.

Sikander Gill (Aurora Biomed) discussed the identification of drugs that target co-transporters through tracer ion flux technology. There are an increasing number of studies on neuronal cation-chloride co-transporters (CCCs); however, because they are electroneutral they can be difficult to patch clamp. Aurora Biomed has developed a high-throughput screen using the Ion Channel Readers (ICR) 8000 and 12000. These readers use nonbiological tracer ions, such as Rb⁺ and Li⁺, couple microsampling with atomic absorption spectroscopy, and can be used with mammalian cell lines, Xenopus oocytes, Caenorhabditis elegans, or yeast in 96- or 384-well plates. ICR 8000 reads up to 5,000 wells per day, while the ICR 12000 reads 60,000. Three compounds were found to block CCC activity as measured by Rb⁺ uptake, and potency was ranked by determination of IC₅₀ values.

Ion Channel Screening Technologies

Richard Kondo (Sophion Biosciences) discussed Qube, an automated patch clamp high-throughput screening technology for both voltage- and ligand-gated ion channels that provides continuous voltage clamp and gigaohm seals and can read over 10,000 wells per day. Moreover, Qube allows testing for concentration-response characterization. Dr. Kondo highlighted the use of Qube for screening for both agonists and antagonists of Nav1.7. Qube allows for direct recordings of ion channels, reducing the number of false responses compared with indirect methods such as calcium influx or displacement binding.

David Dalrymple (SB Drug Discovery) introduced the SB Ion Channels sector and its progress in producing and validating cell lines that express TRP channels, for example TRPC6, which has been linked to cardiac hypertrophy. The TRPC6 cell line was validated by assessment of the responses to inhibitors and activators using QPatch. The TRPC6 cell line has been used in the DISCO program, which aims to identify rare plant-derived compounds for pharmaceutical development. Screening of over 2,000 compounds identified 38 putative hits on this channel.

The identification of subtype-specific compounds that target voltage-gated sodium (Nav) channels using Qube technology was presented by Chris Chambers (Pfizer). Nav1.7 is a target for chronic pain therapeutics, so the goal of the study was to identify Nav channel inhibitors that were selective for Nav1.7 over Nav1.5. Using 15 Qube Qchips, 160,000 compounds were screened at a rate of 5,760 per day, and 1,633 compounds of interest were identified, representing a 1% hit rate. IC₅₀ values generated by Qube were found to be similar to those generated using Qpatch. Thus, Qube may be used for both primary and secondary testing, facilitating fast and reliable identification of hits.

Joseph McGivern (Amgen) discussed the benefits of the SyncroPatch 384PE automated patch clamp system, which generates 20,000 fully automated data points per day with a gigaohm seal while matching the robustness of results from low-throughput electrophysiology methods. Validation with several cell lines, including CHO and HEK, demonstrated a completion success rate of over 85%. The SyncroPatch 384PE was combined with a CellCube automated cell harvester on a CyBi FeliX platform to provide a continual supply of fresh cells for screening. Screening of a Nav channel on a multihole chip was validated by determining the effects of choice of recording chip, dimethyl sulfoxide concentration, and cell confluence.

George Okeyo (Nanion Technologies) summarized the CiPA program and the progress Nanion has made in high-throughput screening of ion channels selected for CiPA such as voltage-gated calcium channel Cav1.2, Nav1.5, hERG, and Kv7.1. Several automated technologies are useful for laboratory cardiac safety assays under the new CiPA guidelines, namely the Patchliner, the CardioExcyte96, and the SyncroPatch 384/768PE. The Patchliner performs automated patch clamping, with features such as rapid solution exchange, a heatable pipette, and short compound exposure times. The CardioExcyte96 measures contractility in intact stem cell-derived cardiomyocytes in 96-well plates with an optional incubation chamber. This device allows for combined measurements of extracellular field potential and impedance at a 1 ms resolution. The SyncroPatch 384/768PE allows for high-throughput automated patch clamping with an 85% completion success rate.

Glenn Kirsch (ChanTest/Charles River) discussed the NicScreen project, a joint endeavour between the Center for Tobacco Products and ChanTest/Charles River. NicScreen aims to develop assays that identify tobacco product constituents that generate subtype-specific nicotinic acetylcholine receptor (nAChR) activity. Neuronal nAChR subtypes are classified in part by their α and β subunit composition, and certain subtypes have been implicated in tobacco addiction. Dr. Kirsch discussed high-throughput nAChR assays performed using the IonWorks Barracuda automated patch clamp to screen 100,000 compounds in 7 weeks; the assays identified α7 specific agonists, antagonists, and positive allosteric modulators (PAMs). They also screened α7 antagonists using the fluorescent imaging plate reader FLIPR TETRA, and the percent inhibition of some α7 antagonists differed between IonWorks Barracuda and the FLIPR.

Ion Channels as Disease Targets

Shawn Iadonato (Kineta) highlighted dalazatide, a peptide derived from a sea anemone that is the first known antagonist of the Kv1.3 channel. Dalazatide preferentially targets effector memory T lymphocytes that have high levels of Kv1.3 and does not inhibit central memory, naïve, or regulatory T cells. Effector memory T cells promote inflammation, and subgroups of these cells with high Kv1.3 expression have been implicated in several autoimmune diseases. Dalazatide was effective in animal models of multiple sclerosis, arthritis, and autoimmune glomerulonephritis. Clinical studies with psoriasis patients demonstrated that up to 90% of participants showed improvements in lesions up to 4 weeks after the final drug dose. Dalazatide demonstrates favorable patient safety, supporting its potential as an effective drug against autoimmune disease.

Annarosa Arcangeli (University of Florence) addressed the role of hERG1 as a novel biomarker in cancer. Signaling mediated by hERG1 can promote cell proliferation, angiogenesis, cell invasiveness, and resistance to apoptosis. The hERG1 channels form complexes with β1 integrins and thereby modulate integrin-related signaling. Notably, these hERG1/β1 integrin complexes only form in tumor cells and not in healthy cardiac tissue. Additionally, isoform hERG1B is preferentially blocked by the compound CD 160130 without causing cardiotoxicity or long QT syndrome. CD 160130 does not bind to the canonical hERG1 binding site, and it is cytotoxic to leukemia cells but not normal bone marrow cells in mouse models.

The role of Kv7 channel modulation in depression was presented by Hailin Zhang (Heibei Medical University). The midbrain dopaminergic pathway originating in the ventral tegmental area (VTA) has been implicated in depression. The Zhang group questioned whether the dominantly expressed Kv7.4 channels are involved in modulating VTA dopaminergic neuronal activity. They found dopamine can activate Kv7.4 channels, contributing to dopamine-induced neuronal hyperpolarization. The compound AQ4 reduces action potential frequency in dopaminergic neurons, and its efficacy is abolished in a KCNQ4-knockout mouse in which Kv7.4 is not expressed. Social defeat mice were examined as a model for depression, and showed increased excitability of VTA dopaminergic neurons associated with reduced Kv7.4 function. AQ4 normalized social interaction of social defeat mice, but not KCNQ4-knockout mice. Therefore, activation of Kv7.4 channels may reduce excitability of VTA dopaminergic neurons and serve as a therapy for treating depressive-like behavior.

Hannah Gaunt (University of Leeds) discussed the anticancer potential of (-)-Englerin A, a compound from the plant Phyllanthus engleri. (-)-Englerin A specifically activates TRP channel family members TRPC4 and TRPC5 at nanomolar concentrations, and calcium influx was elicited by the compound in a renal cell carcinoma cell line overexpressing TRPC4. (-)-Englerin A reduced tumor growth after injection into mice, and high levels of TRPC1 and TRPC4 were correlated with (-)-Englerin A-induced cancer cell death.

David Hackos (Genentech) discussed activation of N-methyl-D-aspartate (NMDA) receptors to treat schizophrenia. Schizophrenia-like behaviors can manifest in both humans and animal models when NMDA receptors are inhibited. Calcium influx was monitored in an ultra-high-throughput screen of over 2.5 million compounds to identify PAMs of NMDA receptors. A new class of compounds was discovered that is specific to the NR2A subunit and interacts with the receptor within the ligand binding domain. One of these NR2A subunit-specific compounds was shown to shift glutamate potency without altering glycine potency, and to enhance long-term potentiation of NMDA receptors in hippocampal slices. These newly discovered PAMs for NMDA receptors may be the key toward the development of a new approach to treatment of schizophrenia.

Alcohol, Tobacco, and Ion Channels

Andrew Holmes (U.S. National Institutes of Health) discussed how a link between alcohol and NMDA receptors leads to deleterious effects of chronic alcohol use. Mice exposed to ethanol via inhalation in the chronic intermittent ethanol model demonstrated hindered fear extinction following trauma. Effects observed in the mouse prefrontal cortex included remodeling of dendritic arbors and decreased burst firing. Since NMDA receptors regulate bursting, ethanol exposure may lead to a decrease in NMDA receptors in the prefrontal cortex. These results suggest that chronic alcohol use may impair NMDA-mediated neuronal plasticity in the prefrontal cortex and compromise the ability of users to overcome traumas.

Javier Camacho (CINVESTAV) outlined how the estradiol-induced expression increase of ether á go-go 1 (Eag1) channels, which typically have a limited expression pattern, may allow Eag1 channels to serve as an early cervical cancer marker. Estradiol is a form of estrogen associated with an increased risk of cancer, and both estrogens and Human Papilloma Virus oncogenes are known to upregulate Eag1. Given this connection, the Camacho group examined Eag1 expression levels in cervical cancer patients and compared them to Pap smear samples from healthy individuals. They found high Eag1 expression in cancerous samples and in patients taking estradiol when no cervical lesions were present.

Li Zhang (U.S. National Institutes of Health) discussed glycine-gated receptors (GlyR) and their role in alcohol addiction. Glycine functions as an inhibitory neurotransmitter by binding to GlyR. Glycine-bound GlyR allows passage of chloride ions, leading to hyperpolarization of neuronal membranes. Ethanol enhances chloride current passage through GlyR, and this effect depends critically on specific amino acid residues in GlyR. The potentiating effect of ethanol on GlyR in neurons and ethanol-induced behaviors were reduced or abolished in knockin mice harboring mutations in two transmembrane domain residues. These results suggest an important role for GlyR in regulating alcohol sensitivity in neurons and alcohol-based behaviors.

Ion Channels as Pain Targets

Ru-Rong Ji (Duke University Medical Centre) discussed evaluation of resolvins as a treatment for TRP channel-based inflammatory pain. Previous research showed that resolvins, derived from omega-3 polyunsaturated fatty acids, inhibit inflammation and resultant cytokine and chemokine expression in mouse models. In the present work, Dr. Ji showed that resolvins are endogenous inhibitors of TRPA1 and TRPV1, which are mediators of pain in dorsal root ganglion (DRG) neurons. Resolvin RvE1 blocks TRPV1-mediated spinal synaptic transmission and pain; however, it does not affect basal pain levels. These effects of RvE1 are mediated through the G-protein coupled receptor (GPCR) ChemR23, which is co-expressed with TRPV1 in human DRG neurons. In HEK cells, the presence of ChemR23 was shown to be required for RvE1 to block TRPV1 currents. Moreover, RvE1 is unable to block TRPV1 currents in B-arrestin knockout mice. These results suggest that resolvins and their associated GPCRs may provide a novel therapeutic approach to treat chronic inflammatory pain.

Nikita Gamper (University of Leeds) highlighted the role of anoctamin 1 (ANO1, also known as TMEM16A) channels in inflammatory pain. ANO1 is a calcium-activated chloride channel (CaCC) that causes an efflux of chloride and thus membrane depolarization and neuronal excitation in peripheral nociceptive neurons. ANO1 channels have only recently undergone characterization, and their spectrum of functions is still not entirely defined. ANO1 is activated by calcium release from the ER downstream of proinflammatory GPCRs such as bradykinin B2 receptor and protease activated receptor-2. Therefore, inflammatory mediators such as bradykinin, proteases, and histamines can activate ANO1 in DRG neurons. Knockdown of ANO1 by siRNA reduced BK channel inward currents, and inhibition of CaCCs lessened BK-induced pain in nociceptive nerve fibers. Thus, ANO1 and other CaCCs contribute to BK-based excitation of DRG neurons. ANO1 resides on the DRG neuronal cell membrane in close proximity to proinflammatory GPCRs; both perhaps lie within a lipid raft. ANO1 is also close to inositol 1,4,5-trisphosphate (IP3) receptors in the ER membrane. GPCRs and ANO1 on the plasma membrane, along with IP3 on the ER membrane, may cluster together to create a microdomain signaling complex that encourages only calcium released from the ER to activate ANO1 and lead to inflammatory pain.

Yan Xu (University of Pittsburgh) discussed engineered ion channels and their potential as a treatment for inflammatory pain. As an alternative to developing drugs to match existing channels, novel channels can be designed to respond to a selected compound. The Xu research group generated nonimmunogenic pH-gated “surveillance” chloride channels that are silent under normal conditions but respond rapidly to inflammation-induced cellular changes. These engineered channels respond to inflammatory tissue acidosis and reverse the hypersensitization of DRG neurons that induces pain. In rat models, inflammatory pain was prevented without affecting normal motor function and nociception. This research opens the door to “receptor therapeutics” pain therapy, in which selected compounds are endowed with analgesic properties.

Van Lu (National Institutes of Health) discussed the study of ion channel promoters to determine how variations in ion channel expression contribute to disease. Nav1.8 was of particular interest due to its tissue-specific expression and potential involvement in multiple sclerosis; it is expressed in the dorsal root ganglia at much higher levels in comparison to other tissues. Dr. Lu's group identified a putative promoter for the Scn10a gene that encodes Nav1.8, and they generated a transgenic mouse model in which a 3.7 kb section of the Scn10a promoter was joined to an enhanced green fluorescent protein (EGFP) gene. EGFP levels diminished when DRG neurons are grown for several days in culture without growth factors, whereas EGFP was sustained in the presence of growth factors, indicating that the reporter mouse appropriately expressed Nav1.8. Novel areas of expression of Nav1.8 were discovered in the hypothalamus of the Scn10a-EGFP mouse, suggesting that this model may contribute to an increased understanding of aberrant expression of Nav1.8 in neurological diseases.

Xinzhong Dong (Johns Hopkins University) discussed Tmem100, a regulator of TRPA1/TRPV1 complexes. Tmem100 is a two pass transmembrane protein that interacts with TRPA1, preventing TRPV1-dependent inhibition and enhancing TRPA1 open probability. Dr. Dong's group mutated a series of positively charged residues in the Tmem100 C-terminal region to noncharged residues and drastically altered the function of Tmem100; the mutated protein inhibits TRPA1 function. Fluorescence resonance energy transfer studies suggested wild type Tmem100 weakens the association of TRPA1 and TRPV1, whereas the mutated Tmem100 strengthens the association. A cell-permeable peptide containing the C-terminal region of the mutated Tmem100 protein injected into Tmem100-knockout mice reduced TRPA1 activity and TRPA1-associated pain in a TRPV1-dependent manner. Thus, this peptide may represent a possible treatment option for both inflammatory and neuropathic pain.