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.

Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation.

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells.

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.

Sedation and anesthesia mediated by distinct GABA(A) receptor isoforms.

The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.

Characterizing human ion channels in induced pluripotent stem cell-derived neurons.

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na(+) (Na(v)), Ca(2+) (Ca(v)), and K(+) (K(v)) channels as expected from excitable cells. The Na(v) current was TTX sensitive, IC(50) = 12 ± 6 nM (n = 5). About 50% of the Ca(v) current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the K(v) channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC(50) = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABA(A) receptor antagonist bicuculline, IC(50) = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Na(v) currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching "excitable cells." These results show high potential for pluripotent stem cell-derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Brain-derived neurotrophic factor mitigates chronic ethanol-induced attenuation of gamma-aminobutyric acid responses in cultured cerebellar granule cells.

This study examined the effect of chronic exposure to ethanol and brain-derived neurotrophic factor (BDNF) on the responsiveness of cerebellar granule cells to gamma-aminobutyric acid (GABA). Cerebellar granule cell cultures were chronically exposed to ethanol (100 mM), BDNF (20 ng/ml), or the combination of ethanol and BDNF. Whole-cell current responses of granule cells to exogenously applied GABA were monitored following at least 5 days of chronic exposure. In the ethanol-treated cultures, granule cell responsiveness to GABA was attenuated. Concomitant exposure of cultures to ethanol and BDNF mitigated the ethanol-induced attenuation of GABA response, although BDNF, by itself, did not affect responsiveness to GABA. BDNF increased the expression of the GABA(A) receptor alpha6 subunit, whereas ethanol had no effect, in chronically treated granule cell cultures. In addition, concomitant treatment with BDNF and ethanol did not increase the expression of the GABA(A) receptor alpha6 subunit, so the subunit expression alone could not account for the mitigating effect of BDNF. We propose that different mechanisms regulating responsiveness to GABA underlie the effects induced by ethanol and BDNF, with the former influencing the expression of functional GABA(A) receptors and the latter involving the activation of the TrkB receptor and its downstream signaling pathways.