Mechanosensitivity of TRPC6 Ion Channel Reconstituted in the Liposomes

Abstract

The transient receptor potential ion channel family is a diverse group of channels gated by various physical and chemical stimuli. One of the members, TRPC6, is a calcium permeable cation channel located in the ventricular cardiomyocytes. TRPC6 has been reported to be activated by mechanical force, however it is still unclear if TRPC6 is activated directly by membrane tension from the lipid bilayer or whether activation requires additional membrane components including cytoskeleton and/or extracellular matrix for its activation. Thus, it is important to elucidate the gating mechanism of TRPC6.In this study, we purified TRPC6 protein and investigated the mechanosensitivity of TRPC6 reconstituted into the liposomes using the patch clamp technique. We have transfected C-GFP TRPC6 construct into HEK293 cells and demonstrated that the channel is localised in the plasma membrane. Purification were performed using immobilized metal-affinity chromatography. To assess the integrity of the TRPC6 protein, the elution fraction was analysed by fluorescence sized exclusion chromatography (FSEC) and SDS/PAGE gel. GFP fluorescence peak using FSEC showed that TRPC6 channel is found mostly in tetramer state. Fluorescent scan of the gels demonstrated a single fluorescent band at the correct TRPC6 - GFP size. However, Coomassie staining of the same gel revealed additional bands in the gel showing that purification needs optimisation. Using isolated protein, liposome reconstitution was carried out using DR method. We recorded TRPC6 single channel activity in liposomes by patch clamping. TRPC6 ion channel activity demonstrated outward rectification and calculated conductance was ∼44 pS. By applying negative pressure to the patch pipette, we did observe multiple channel activity. TRPC6 was spontaneously active without any negative pressure applied to the patch pipette, which suggests that the membrane tension resulting from the gigaohm seal formation was sufficient to activate the TRPC6 channels.