Figure 3.

Assembly of coiled-coil segments and their functional role. (A) FRET measurement of binding strength comparison between CBDs (left panel) and CBD-CC segments (right panel) in live cells. The binding affinity was evaluated by the same analysis style as in Figure 1D. A negative control (CFP and YFP only) for FRET measurements is shown in the panel for CBD only (gray circles). (B) Crosslinking of CBD-CC fragments. Purified CBD-CC (200 μM) was treated with 0–3 mM EGS crosslinker. Arrows indicate monomer (M, 11.6 kDa), dimer (D), tetramer (T), and oligomer (O) formations of CBD-CC. (C) Size exclusion chromatography results for CBD-CC with and without CaM are shown as red and black traces, respectively. The inset shows the standard curve estimation of the complex sizes of CBD-CC with and without CaM. (D) Crosslinkings of CaM to CBD-CC by EDC/NHS (1 mM) in the presence (1 mM) or absence of Ca²⁺ were separated in SDS-PAGE and were imaged by CBB stain (left), or immunoblotting for CaM (right). The positions of the expected mol wt for single CaM (16.7 kDa) and single or multiple CBD-CC complexes (1:1, 1:2, 1:3, 1:4) are indicated by arrows. (E) Currents from TRPC6 with a truncated coiled-coil (TRPC6_ΔCC), evoked by CCh. Inactivation plots constructed by plotting residual currents against time for TRPC6_WT and TRPC6_ΔCC are shown as black and red circles, respectively. (F) Representative TRPC6 currents trace from the cell cotransfected with the N-lobe (CaM_ΔC) and C-lobe (CaM_ΔN). Residual current plots of CaM_ΔC/CaM_ΔN (red circles) versus TRPC6WT without overexpression of CaM (CaMendo) (black circles). Data from CaM_ΔC or CaM_ΔN only are shown in Supplemental Figure 5.