Electrophysiology characterization of human CLC-1 and CLC-2 ion channels stably expressed in HEK cells using the SynchroPatch 384i automated electrophysiology platform

The CLC family comprises voltage-dependant cell surface chloride (Cl) ion channels and intracellular Cl/H+ transporters which perform an array of physiological functions. Two members of the CLC family, CLC-1 and CLC-2, are inward-rectifying ion channels with crucial roles in physiology. CLC-1 is predominantly expressed in skeletal muscle and performs a vital role in homeostasis of muscle excitability by repolarisation of skeletal muscle in response to depolarisation of an action potential. Variants of the CLCN1 gene are associated with a rare disorder of muscle membrane hyperexcitability, myotonia congenita. In contrast, the widely expressed CLC-2 is activated upon hyperpolarisation of the membrane and is involved in many physiological functions, including epithelial transport. Variants of the CLCN2 gene have been linked to rare disorders such as leukodystrophy and hyperaldosteronism.

 

Despite the channelopathies caused by CLC channel dysfunction, only a small number of selective CLC channel modulators have been reported, and the pharmacology of these channels is poorly characterized compared to cation or other ligand-gated Cl channels. The non-selective nature of currently defined Cl- channel modulators provide an obstacle in developing a therapeutic strategy targeting CLC channelopathies. This highlights the importance of developing novel routes to identify selective compounds in pursuit of therapeutic remedies to CLC channel dysfunction.

 

Advancements in 384-well automated high throughput electrophysiology allows accelerated evaluation of large numbers of molecules against ion channel targets such as CLC-1 and CLC-2. We have developed robust, high quality recombinant cell lines expressing wild-type CLC-1 and CLC-2 ion channels. Electrophysiological characterization of these ion channels, including current-voltage relationship and assessment of known CLC modulators was performed using automated voltage-clamp electrophysiology. Reproducible and selective inhibition of 9-AC for CLC-1 (~1 µM IC50) over CLC-2 (no effect) and CdCl2 inhibition of CLC-2 (~55 µM IC50) currents over CLC-1 (~350 µM IC50), as well as inhibition with known Clchannel blocker ZnCl2 confirm that a successful assay has been developed, capable of rapidly identifying and characterizing novel pharmaceutical tools targeting CLC-1 and CLC-2 channels. This should allow further investigation into the role of these exciting targets in physiological and pathological conditions.

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