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. 2012 Oct 15;590(Pt 24):6257–6266. doi: 10.1113/jphysiol.2012.240911

Figure 1. C terminal cleavage-mediated Panx1 channel activation using a TEV protease system.

Figure 1

A, schematic of a Panx1 (TEV) construct with TEV protease site substituted for the C terminal caspase site. Co-expression of TEV protease results in C terminal cleavage and subsequent Panx1 channel activation leading to YO-PRO-3 dye uptake into the cell. B, when co-transfected with TEVp in HEK293T cells, Panx1(TEV) generated robust whole cell CBX-sensitive currents with I–V properties characteristic of Panx1. C, comparison of Panx1 channel activity assessed by membrane current recording and by dye uptake. HEK293T cells expressing C terminally deleted, constitutively activated (PanxΔC), cleavage activated (Panx1(TEV) + TEVp), and basally inactive Panx1(TEV) were recorded in whole cell voltage clamp mode (left panel), or treated with YO-PRO-3 dye, fixed and fluorescence measured on a plate reader (right panel). YO-PRO-3 dye uptake assay was performed in a 96-well plate format and can reflect differences in Panx1 channel activity as measured by whole cell voltage clamp recordings.