Figure 4: Calibration of Ca_V2.2/TN–XL fusion construct.

(a) High-amplitude Ca²⁺ influx into HEK293 cell expressing Ca_V2.2 channels, observed with 10 mM external Ca²⁺ and minimal internal Ca²⁺ buffering of 1 mM EGTA. (b) Simultaneous [Ca²⁺] measurement (dark trace) deduced from 10 μM Fluo-4FF Ca²⁺-indicator fluorescence from entirety of same cell (514 nm excitation, 545 nm emission). Ca²⁺ determination rendered ratiometric by inclusion of 2.5 μM red Alexa 568 dye (514 nm excitation, 580 nm long-pass emission). Inset, cartoon of spatial Ca²⁺ gradients in this cell. Red traces, projected [Ca²⁺] waveforms from Ca²⁺ diffusion mechanism constrained by Ca²⁺ input in (a) and aggregate [Ca²⁺] (dark trace): lower red trace, aggregate [Ca²⁺]; upper red trace, submembranous [Ca²⁺]. (c), Average FRET-ratio (R_F/C) response of Ca_V2.2/TN–XL channels in TIRF volume (green trace); same protocol as in (a,b). Red trace, output of TN–XL forward transform in (e), when driven by submembranous [Ca²⁺] input (b, upper red trace). (d) Normalized steady-state FRET-ratio response (R_norm=(R_F/C−R_min)/(R_max−R_min) of Ca_V2.2/TN–XL to [Ca²⁺] (experimental green symbols). Red trace, steady-state output of TN–XL forward transform in (e) when driven by differing fixed [Ca²⁺]. (e) State-diagram representation of Ca_V2.2/TN–XL. Red circles, Ca²⁺ ions. UB, conformation with no calcium bound. B, various conformations with calcium bound. (f) System of states and corresponding fluorescence/FRET properties constitutes a forward transform that predicts TN–XL sensor outputs as a function of time-varying Ca²⁺ inputs.1)