Rb⁺ Efflux Assay for Assessment of Non-Selective Cation Channel Activities

Abstract

Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.

Targeting Circuit Abnormalities in Neurodegenerative Disease

“…While manual patch-clamp is a high-fidelity and data-intense approach, it is low-throughput and therefore impractical for most drug-discovery purposes. However, fluorescence-based assays (Whiteaker et al, 2001;Beacham et al, 2010), radioisotope flux assays (Terstappen, 1999;Weaver et al, 2004;Liu et al, 2010;Weaver, 2018), and now automated patch clamp platforms (Srinivasan et al, 2022) have emerged as breakthroughs in our ability to screen ion channels. While still early, the development of human-derived stem cell and organoid-based platforms offers a powerful opportunity to evaluate aberrant circuitry and test ion channel modulators in human-based models.…”

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“…After few min, the media is removed, and cells are washed with buffer to remove Rb + present in the extracellular solution 26,27 .…”

Correlational Patch-Clamp Spectrometry of the Ion Channels

“…Данное явление проявляется в том, что координируются ионы других элементов (примеры координационной имплементации -координация Cu ; часто процессы координации неспецифичных ионов порой или каналом носят не тот же стерический характер и обладают не той же кинетикой, что процесс фиксации «специфичного» иона, так как фиксация имеет менее жесткий и / или «эктопический» характер, коррелирующий с её малой селективностью по отношению к данному иону [88]). В классической мембранологии -ещё до возникновения каналомики -была широко развита проблема замещения ионного состава окружающей среды и замещающих ионов, при внедрении которых ионные каналы продолжают работать, несмотря на то что ионы не являются специфичными для данного канала в его нативном состоянии.…”

“…На данный момент имеются десятки статей по: замещающему действию ионов Rb + на калиевые каналы (см., напр. : [88][89][90][91][92][93][94][95][96][97][98][99][100][101][102] [154,[156][157][158][159][160][161][162][163][164][165] и др. металлов.…”