Figure 5. Ca_V2.2/TN-XL sensors respond to channel nanodomain Ca²⁺ signals.

Top cartoon, Ca²⁺ nanodomains under elevated Ca²⁺ buffering, where Ca²⁺ elevation is restricted to nanodomains, each affiliated with individual channel. 40 nm nanodomain calculated with Equation 1, f = 1 (classical assumption), unitary current i = 0.085 pA, and conservatively requiring [Ca²⁺] ~60 nM (the approximate resting physiological Ca²⁺ concentration). a, Simultaneous records of whole-cell current (top) and TIRF fluorescence signals (middle and bottom) from HEK293 cell expressing Ca_V2.2/TN-XL, using 10 mM external Ca²⁺ and 10 mM internal EGTA Ca²⁺ buffering. Shown are data from a single trial, evoked by the first step depolarization in the cell. Top, Voltage and current waveforms, with moderately-sized current chosen to minimize buffer consumption. Middle, Corresponding CFP fluorescence signal after normalization to baseline level (S_C, noisy trace). Black curve, approximate exponential fits (initial phase: 200 ms (10%), 880 ms (90%); recovery phase: 2000 ms (100%)). Vertical ticks, 0.1 increment. Bottom, Corresponding FRET-ratio signal (R_F/C, noisy trace). Black curve, approximate exponential fits (initial phase: 200 ms (10%), 880 ms (90%); recovery phase: 400 msec (70%), 2000 ms (30%)). Vertical ticks, 0.1 increment. b, Average Ca_V2.2/TN-XL responses to nanodomain Ca²⁺ signals, as isolated with 10 mM internal EGTA Ca²⁺ buffering. Protocol and format as in panel a. n = 4 cells, with records taken from the first step depolarization delivered in cells. CDI sometimes increased with repeated depolarizations, reflecting buffer depletion with attendant slowing of R_F/C waveform decay. Cells exhibiting peak currents ≤500 pA were not considered, as these cells demonstrated negligible R_F/C transients, presumably reflecting insufficient trafficking of active channels. Solid-black-curve exponential fits were similar, with S_C exponential fit (initial phase: 200 ms (10%), 880 ms (90%); recovery phase: 500 ms (80%), 2500 ms (20%)), and R_F/C fit (initial phase: 200 ms (10%), 880 ms (90%); recovery phase: 400 msec (70%), 2000 ms (30%)). Dashed black curve, continuation of initial phase S_C exponential fit, which asymptotes at lower horizontal black line in middle. c, Schematic of various classes of Ca_V2.2/TN-XL species in TIRF volume. Blue shading indicates region of excitation by evanscent wave in TIRF volume.