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 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: 2,000 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%), 2,000 ms (30%)). Vertical ticks, 0.1 increment. (b) Average 2/TN–XL responses to nanodomain Ca²⁺ signals, as isolated with 10 mM internal EGTA Ca²⁺ buffering. Protocol and format as in (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%), 2,500 ms (20%)), and R_F/C fit (initial phase: 200 ms (10%), 880 ms (90%); recovery phase: 400 msec (70%), 2,000 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 2/TN–XL species in TIRF volume. Blue shading indicates region of excitation by evanscent wave in TIRF volume.