200810 blog image special announcement nanion and friends week 2020

Presentation: Measurement of Transient receptor potential cation (TRP) channels using the Patchliner and Port-a-Patch

Speakers:
Dr. András Horváth | Application Scientist - Nanion Technologies

Abstract:
CHO cells expressing transient receptor potential cation channel V (TRPV) members 1, 3 and 4 and subfamily M member 8 were studied using our automated patch clamp systems, the Patchliner Octo (PL) and Port-a-Patch Perfusion (PaPP). During the recordings, heat, cold and/or ligand activation was performed. A classical ramp pulse-protocol (–100 mV to 100 mV) was applied.

Heat activation of TRPV1, 3, 4 channels was performed repeatedly by the heated pipetted (37-45 °C) of the PL. Interestingly ruthenium red (RR, 50 and 200 µM) was not able to prevent heat activation. Experiments involving TRPV4 were also performed on the PaPP. The cannel could be activated by heat and only partially blocked by RR. Ligand activation could be also performed on the PL (10 µM Capsaicin – TRPV1, 200 µM 2-APB – TRPV3, 100 nM GSK1016790 – TRPV4) and TRPV4 on the PaPP. In all cases the effect could be inhibited using  blockers. TRPM8 channel could be repetitively activated using solution at 10°C on the PaPP at 10 °C. Capsazepine (10 µM) was used to block the activated current.

Both the PL and PaPP are powerful tools to study TRP channel physiology (both using heat activation and ligand activation) and could be used to find compounds which block the temperature and ligand response separately.

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Presentation: TRP channels in human corneal cells and their role in ophthalmology

Speakers:
Prof. Dr. Stefan Mergler | Charité - Universitätsmedizin Berlin

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Presentation: Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms.

Speakers:
Dr. Marc Rogers - Chief Scientific Officer | Metrion Biosciences 

Abstract:
Whilst voltage-gated ion channels formed the bulk of academic and industrial effort in developing and utilising APC assays for ion channel drug discovery, recent years have seen increasing interest in ligand-gated receptors. These targets offer specific challenges for APC systems in terms of lower channel expression, rapid application and wash-off of ligands, and loss of responsiveness due to short- and long-term desensitisation. In this presentation I will outline successful development of pipette- and tip-based APC assay formats for the rapidly-activating ASIC1A channel on the Patchliner and SyncroPatch384i.

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Presentation: Pharmacological Characterization of Natural Tobacco Alkaloids in the Presence of Positive Allosteric Modulators Against Humana4b2 and a7 Nicotinic Acetylcholine Receptors

Speakers:
Dr. Omar Alijevic | Philip Morris International

Abstract:
The pharmacological profile of tobacco alkaloids is essential for understanding their physiological effects. Nicotinic acetylcholine receptors (nAChRs) are an important target of tobacco alkaloids with primarily agonistic effects reported for α4β2 nAChRs, but with minimal evidence of α7 activity. In this study, we used a membrane potential assay and automated patch-clamp electrophysiological approaches to functionally characterize distinct groups of tobacco alkaloids in the presence of a subunit-specific positive allosteric modulator (PAM) of human α4β2 and α7 nAChRs. We screened a total of 71 tobacco alkaloids, of which 16 were active against α4β2 and 11 against α7 nAChRs.

The most abundant alkaloids in tobacco leaves—namely nicotine, nornicotine, anabasine, (S)-anatabine, and (R)-anatabine—exhibited potencies (EC50alkaloid+PAM) of 0.02-20 μM against α4β2 and 0.2-10 μM against α7 nAChRs. In the presence of the PAM, nicotine and anabasine, respectively, were found to be the most potent α4β2 and α7 nAChRs agonists. Relative to (S)-anatabine, (R)-anatabine was 5-fold more potent against α4β2; the relationship was found to be inverse in case of α7 nAChRs. In addition, 13 alkaloids demonstrated agonistic effects only in the presence of the PAM and were, therefore, considered to be silent agonists. In conclusion, the data revealed 17 naturally occurring tobacco alkaloids that exhibited a dramatic increase in potency against human α4β2 and α7 nAChRs in the presence of PAMs (relative to that in the absence of the PAM). Our study recognized a subunit-specific enantiomer preference of anatabine and identified several alkaloids with silent agonist properties for human α4β2 and α7 nAChRs.

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