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27. - 28.03.2023 | 7th RSC-BMCS / SCI Symposium on Ion Channels as Therapeutic Targets

7th RSC SCI Symposuim on Ion Channels as Therapeutic Targets

Venue: Hinxton Hall, Wellcome Genome Campus, Cambridge.
Go to the Conference website here


 

Meet Dr. Alison Obergrussberger and Dr. Elena Dragicevic

 

Join Alison Obergrussberger (Scientific Communication Manager), and Elena Dragicevic (Senior Sales and Alliance Manager) at booth #2 to learn more about our technology and how can they meet all you Patch Clamp needs. 

 

 


 

Nanion will be present with three posters:

Poster 1:

Presenter: Alison Obergrussberger (Nanion)
Date: TDB
Time slot: TBD
Title: Pharmacology of Transient receptor potential cation (TRP) channels using different activation stimuli


Abstract: 

Transient Receptor Potential (TRP) channels are widely distributed throughout the mammalian central and peripheral nervous systems. They can be directly activated by ligands, heat or cold and mechano-stimulation, and are important targets in drug discovery for the treat¬ment of pain, respiratory diseases, cancer, immune disorders and others. Here, we studied the responses of TRPA1, TRPV1, TRPV3, TRPV4 and TRPM8 assay-ready and cultured cells activated using a variety of stimuli on automated patch clamp (APC) systems.
TRPA1 and TRPM8 are crucial in sensing noxious cold and inflammatory pain, responding to irritant environmental and food compounds, and metabolites produced during oxidative stress. TRPA1 is expressed in sensory neurons of the dorsal root ganglion (DRG) and trigeminal ganglion. Thus, TRPA1, and possibly TRPM8, antagonists are considered a promising approach for the treatment of acute and chronic pain. Desensitization is one of the biggest challenges for drug screening of TRPA1 channels. Here, we obtained IC50s obtained with short ligand exposure using a high throughput device, the SyncroPatch 384 (Figure 1). TRPM8 was activated repetitively activated using solution at 10°C on the Port-a-Patch using the temperature-controlled perfusion system at 10 °C. Capsazepine (10 µM) was used to block the activated current. TRPM8 was also activated by solution <18°C on the SyncroPatch 384 (Figure 2).
The TRPV1, 3 and 4 channels are ligand-gated, non-selective cation channels involved in nociception, and respond to elevated temperatures and compounds. Identifying compounds with differential effects on ligand vs heat activation may be crucial in the discovery of new treatments for pain with fewer side effects. Here, we developed robust methods for temperature and pharmacological activation to study differential effects of blockers. We used ligand activation (capsaicin, 2-APB, GSK1016790) and repeated heat activation of TRPV1, 3, 4 (Patchliner heated pipette, 37-45°C) followed by the addition of blockers (Figure 3). TRPV4 was activated by heat (38°C) and partially blocked by ruthenium red on the Port-a-Patch. In summary, we established a range of reliable automated approaches to study temperature vs pharmacological influences on TRP channels.

Poster 2:

Presenter: Elena Dragicevic (Nanion)
Date: TDB
Time slot: TBD
Title: Identification of novel TMEM175 modulators using a high-throughput automated patch-clamp and solid-supported membrane- (SSM-) based electrophysiology platforms


Abstract: 

TMEM175 is a novel, constitutively active ion channel involved in regulating lysosomal pH and autophagy. Mutations in this gene impair normal lysosomal and mitochondrial function, thereby increasing aggregation of insoluble proteins such as phosphorylated α-synuclein, leading to symptoms typical of Parkinson’s Disease (PD). Consequently, TMEM175 demonstrates significant potential as a key player in the treatment of PD. The lack of specific pharmacological tools has hampered further investigation into the exact role of TMEM175 in normal lysosomal function and pathological processes.
Advancements in high-throughput screening technologies have allowed rapid assessment of large numbers of compounds against ion channel drug targets using automated patch-clamp. We have successfully developed recombinant cell lines expressing wild-type (WT) TMEM175, a gain of function (Q65P), and loss of function (M393T) mutants using stably transfected HEK cells. Here, we report the characterization of the WT TMEM175 cell line performed using high-throughput automated patch-clamp electrophysiology and show reproducible concentration-response curves with the potassium channel inhibitor 4-aminopyridine (4-AP). A rapid and robust, automated high-throughput electrophysiology screening assay was subsequently developed to enable the identification of both activators and inhibitors of TMEM175.
Additionally, we have used solid-supported membrane- (SSM-) based electrophysiology (SSM-E) for TMEM175 recordings from lysosomal membranes purified and prepared from the WT TMEM175 cell line. Dose-dependent signal enhancement and inhibition of TMEM175 currents were successfully characterized. The SSM-E approach allows for stable and robust recordings from proteins residing in organellar membranes with a throughput of up to 10’000 data points per day.
In summary, the TMEM175 stable cell line was characterized using an automated patch-clamp and SSM-based electrophysiology. We developed and executed robust, high-throughput, and high-content direct electrophysiological intracellular screening assays, with unusually high success rates. As the TMEM175 channel has recently been suggested as an H+ channel at low pH, we are now developing H+ assays to be used in conjunction with K+ assays. The successful development of TMEM175 electrophysiology assays capable of identifying novel pharmacological tools will enable investigation of the role of this exciting target in normal physiology and in disease.

 

Poster 3:

Presenter: Marc Rogers (on behalf of Anatomic)
Date: TDB
Time slot: TBD
Title: Human Induced Pluripotent Stem Cell-Derived Nociceptors suitable for Automated Patch Clamp High Throughput Pain Drug Discovery

Abstract: 

There is an unmet need for novel non-addictive pain analgesics as the opioid epidemic continues. The ability to screen compounds on human sensory neuron nociceptors with high throughput would increase the efficiency and pace of preclinical pain drug discovery and improve translational success of new pain drug candidates. We have previously demonstrated that human nociceptors can be generated in an accelerated, scalable method from human induced pluripotent stem cells (hiPSCs), and that the hiPSC-derived nociceptors share similarities to human dorsal root ganglia based on whole-transcriptome profiling and expression of functional voltage- and ligand-gated channels important for nociception.
In this study, we utilise a novel dissociation method to enable automated patch clamp electrophysiological recordings of multiple ion channels in RealDRG™ cultures on the Patchliner and SyncroPatch 384 systems. The functional expression and biophysical and pharmacological properties of voltage-gated sodium (Nav) and potassium ion channels (Kv), and ligand-gated ionotropic GABA and P2X receptors were studied over 14, 21 and 28 days in culture, along with excitability properties and action potentials in current clamp.
The percentage of cells with at least one evoked action potential increased from 42% to 77% over the course of maturation, with success rates decreasing from 56 to 35% as the cells matured. There was also an increase in voltage-gated sodium (Nav) and potassium (Kv) currents over time in culture, with success rates ranging from 94-98% and 93-100%, respectively. Most neurons had tetrodotoxin-resistant (TTXr) sodium currents, with a trend of increasing number of cells with Nav current and fraction of TTXr current per cell. A ligand puff protocol was developed to reproducibly evoke ligand-gated GABA-A and P2X receptor ion channels without desensitisation. These findings demonstrate the ability to functionally screen multiple analgesia drug targets in human iPSC nociceptors using high throughput automated patch clamp systems.


Synopsis of the Sympusium 


Ion channels are important targets for therapeutic intervention due to their extensive roles in human physiology and the pathophysiology of disease. Many successful drugs targeting this gene family have been discovered for diseases such as hypertension, epilepsy and neuropathic pain.  This symposium, the 7th in an ongoing series, will showcase the most recent advances to aid the design of new ion channel therapeutics and promote interaction between scientists with a shared interest in the field of ion channel drug discovery.

Who should attend
Scientists from industry and academia interested in the therapeutic potential of ion channels. The organising committee particularly welcomes attendance and participation of students or early career researchers working in the field of ion channels.

Find more information about the symposuim on their website.

 


 

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