Supplemental Information Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor Biophysical Journal

To determine optimal live-cell parameters for Indo-1 and TN-L15 in myocytes, we utilized a sequential approach that refined initial in vitro and cell-permeabilized measurements with those obtained from twitching cardiocytes. Specifically, Supplemental Table 1 summarizes the initial starting values for Ca2+ sensor parameters. Here, we utilized a well-accepted Indo-1 Kd of 800 nM from prior work in cardiac myocytes (1), and a TN-L15 Kd of 815 nM (2). The latter value was determined from an apparent in vitro apparent Kd value of 1200 nM (2), which was then divided by an α value of 1.47 for TN-L15 (α ≡ ratio of SCFP signals from TN-L15 under 440 nm excitation, under Ca2+-free and bound conditions). We determined this α value via permeabilized HEK293 cells transfected with TN-L15. Initial estimates for the remaining Indo-1 (Rmin, Rmax, and β) and TN-L15 parameters (Rmin and Rmax) were largely determined from measurements of ionomycin-permeabilized cardiac myocytes, bathed in either EGTA or saturating Ca2+. Reassuringly, the ranges of values obtained in cardiac myocytes overlapped with those obtained in HEK293 cells. The one exception to this congruence was the value of α for TN-L15 as determined in myocytes. This is understandable from the technical standpoint, because the determination of α requires the harshest treatment of myocytes, which entail