Figure S1 - Effect of Ca_vβ Subunits on Structural Organization of Ca_v1.2 Calcium Channels

FRET efficiency and donor/acceptor ratio of FRET standards. Shown are bar graphs summarizing the mean FRET efficiency (A) and the D/A ratio (B) for the indicated FRET calibration constructs. Data are presented as mean±SEM. (A) FRET efficiency values were: C4R (0.110±0.005), R39C (0.081±0.003), CTV (0.023±0.004), V4R (0.433±0.011), RTV (0.191±0.009), C5V (0.474±0.014), C39V (0.266±0.014) and CTV (0.179±0.006). (B) D/A ratios were: C4R (0.99±0.009), R39C (1.00±0.10), RTC (1.00±0.06), CRC (1.96±0.10), RCR (0.48±0.03), V4R (1.00±0.07), RTV (1.00±0.10), RVR (0.53±0.09), C5V (1.00±0.04), C39V (0.95±0.09), CTV (1.00±0.06), CVC (2.00±0.10) and VCV (0.54±0.02). The number of tested cells is shown in the bars. As one can see, increasing the length of the linker between the fluorophores significantly reduced FRET efficiency consistent with an increased distance between donor and acceptor. The measured mean D/A ratio corresponds well to the expected values of 1.0 (1∶1), 2.0 (2∶1) and 0.5 (1∶2). D/A ratios were also determined for the three-color construct CRV. D/A ratio and FRET efficiency were calculated in CRV independently for each pair of fluorophores. D/A ratio (EFRET) are: for CV, 1.00±0.07 (0.45±0.02); for VR, 1.10±0.10 (0.41±0.02), and for CR, 0.99±0.07 (0.20±0.01), n = 11. Thus, the D/A ratio of three-color standards well corresponds to the expected 1∶1 ratio. (0.29 MB EPS)