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08. - 11.11.2021 | Neuroscience 2021

211108 Event image SFN Neuroscience 2021
Conference Venue: Virtual Event 

Go to the Conference website here.

Poster Presentations 

Dr. Alison Obergrussberger (Nanion Technologies GmbH): Pharmacology of Transient receptor potential cation M8 (TRPM8) channels using different activation stimuli

Abstract:
Transient Receptor Potential (TRP) channels are widely distributed throughout the mammalian central and peripheral nervous systems. The TRP channel subfamily M (melastatin) member 8 (TRPM8) is a nonselective cation channel, proposed to be the predominant thermoreceptor for cellular and behavioral responses to cold stimuli. TRPM8 channels can be found in multiple organs and tissues, and are involved in regulation of various important processes such as inflammatory reactions, immunomodulatory effects, pain, cell proliferation, migration and apoptosis, and vascular muscle tension. Disorders ranging from migraine, dry eye disease and cancer, amongst others, have been attributed to TRPM8 rendering this channel a promising target for drug discovery. Here, we studied the responses of TRPM8 expressed in CHO cells activated using different stimuli on automated patch clamp (APC) systems.

TRPM8 is activated by chemical cooling agents including menthol and icilin or by temperatures lower than ∼26 °C. Using a high throughput APC device, TRPM8 was activated by increasing concentrations of menthol with an EC50 of approximately 20 µM. In addition, TRPM8 was activated when the system was cooled to 18°C and further activated at 12°C. A small, lower throughput APC device was also used to activate TRPM8 repetitively using solution at 10°C. This was subsequently blocked by increasing concentrations of capsazepine applied at 10°C with an IC50 of 12.9 µM. External solution warmed to 35°C also blocked the TRPM8-mediated current.

To this end, TRPM8 could be reproducibly activated by different stimuli, and blocked by pharmacological agents or heated solution. Therefore, the use of TRPM8 expressed in cell lines coupled with high throughput APC provides a promising tool for basic research and may have important implications in drug development for compounds acting on TRPM8.

When: Tuesday November 9th, 2021 - 8:45 AM - 9:45 AM CST | 3:45 PM – 4:45 PM CET  

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Narrated Poster Presentation
 


Rocco Zerlotti (Nanion Technologies GmbH): Solid supported membrane-based electrophysiology (SSME) electrophysiology meets SGLT1 and GAT1

Abstract:
Transporter assays are often limited by the availability of labeled substrates and lack real-time data. Here, we developed functional assays to characterize the human Na+/Cl−/γ-aminobutyric acid (GABA) and Na+/glucose co-transporters - GAT1 and SGLT1 - using solid supported membrane-based electrophysiology (SSME). This approach overcomes several bottlenecks of other techniques and provides new insights into GAT1 and SGLT1 transport mechanisms.

In conventional electrophysiology voltage steps are used to trigger pre steady-state (PSS) and transport currents, which are commonly recorded in whole cells at a defined holding potential. Transport and PSS electrogenicity in SGLT1 and GAT1 triggered by voltage steps is postulated to be a result of transitions within the sugar and GABA-free carriers, e.g. the alternating access of the charged sodium binding sites within the empty carrier. In contrast, SSME utilizes membrane vesicles at 0 mV and the transport cycle is triggered by applying a substrate concentration gradient as the main driving force. Using SSME, we observed substrate-induced PSS currents, most likely representing conformational transitions within the substrate-loaded carrier, which are not observed with conventional electrophysiology. We examined the impact of different driving forces on influx, efflux, and PSS currents, focusing on sodium gradients and membrane voltage. We found that internal accumulation of sodium strongly reduces Vmax, rendering sodium release rate limiting at 0 mV. Application of membrane voltage only affected the apparent KM in SGLT1, but Vmax in GAT1. We also found that transport properties in GAT1 mediated influx and efflux modes are highly asymmetric, while SGLT1 has similar properties for influx and efflux.

When: Wednesday November 10th, 2021 - 1:30 PM - 2:30 PM CST | 8:30 PM – 9:30 PM CET   

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