High-level expression of voltage-gated sodium channels in thymidine-arrested human embryonic kidney cells.

Abstract

The benefit of using heterologous mammalian cell lines such as human embryonic kidney (HEK293) cells for expressing voltage-gated ion channels includes efficient translation and processing of such large membrane proteins. Still, the biophysical assessment of these channels is hindered by their low expression and reduced localization on the surface of a cell. Having a higher expression level of ion channels is crucial for investigating mutations that cause a decrease in the current amplitude as well as for measuring tiny currents such as gating currents and gating pore currents that are in the range of 0.1 to 1% of the peak current. We aimed at improving the efficiency of ion channel expression and enhancing their localization in cell membranes by modifying the protocol of cell cycle arrest at the G1/S boundary using thymidine to direct the cells into increasing the expression machinery during viral transduction of mammalian voltage-gated sodium channels. The combination of cell arrest at the G1/S boundary and mammalian baculovirus system (BacMam) resulted in a 5-fold increase in the current density of different mammalian voltage-gated sodium channels in HEK293 cells compared to the current literature. By applying this modified protocol for expression, we succeeded to measure a pathogenic gating pore current in HEK293 cells of ∼0.5% of the central pore current induced by an autism-related mutation in the R2 gating charge in Domain II (R853Q) of Nav1.2, which is blocked in a voltage-dependent manner when the voltage sensors activate. These results show that our protocol is a universal one that can be applied on different voltage-gated ion channels and possibly other membrane proteins. This ultimately will pave the way to decipher the structure and function of ion channels and their association with ion channelopathies and other diseases.