Figure 7. Potential fenestrated Ca²⁺ egress within Ca_V2.2 channel nanodomain.

a, On-cell single-channel ramp experiments, except 10 mM Ca²⁺ is charge carrier to accord with whole-cell experiments; 700-Hz lowpass filtering. Voltages adjusted to align single-channel activation with whole-cell activation curves. Exemplars from single patch, with openings to GHK relation (gray curve). Unitary current i at 30 mV (green circles), 0.085 pA in 5 patches. b, Gain factor A, slope of Ca_spike versus i relation. Shading, various estimates of A over confidence range of Ca_spike. Sloped dashed line, lower-limit estimate of A using uncorrected data. c, Schematic, FRET construct to estimate r_sensor. Substituting circularly permuted amber for circularly permuted Citrine of TN-XL maintains sensor-channel configuration. d, Estimating r_sensor via FRET. Each black symbol, individual HEK293 cell expressing construct in c. Two outputs for each such cell obtained via 3³-FRET analysis⁵⁴ of fluorescence images: FRET efficiency and ratio of donor (CFP) to acceptor (YFP) molecules (D/A ratio), both plotted versus CFP intensity (∝ construct expression level). Data from cells exhibiting D/A ratio ~1 (as expected from c) are displayed and analyzed (bottom subpanel, black line at unity). Invariant FRET efficiency with increasing CFP intensity (top subpanel) excludes spurious FRET, facilitating interpretation of FRET efficiency (~0.35, blue line, top subpanel, left axis) as indicative of r_sensor ~55 Å (top subpanel, right axis). Gray symbols, control cells expressing YFP and CFP separately, yielding efficiencies ~0. e, A versus sensor distance from channel mouth (r_sensor), calculated with Neher-Stern equation (Eq. 1), using parameters^13,60 D_Ca = 0.4 μm² ms⁻¹ and k_Bon = 10⁻³μM⁻¹ ms⁻¹ for EGTA. Gray curves, plots with f values as labeled. Mapping A from b, with r_sensor from d, yields f ~0.53 (black curve) and range (0.4–0.7, shading). f, Schematic, idealized fenestrated Ca²⁺ egress from inner channel mouth. Green cones, permissive radial diffusion paths from source (green dot), summing to f ~ 0.53. Target molecule (gray ball) within/near portal enjoys preferential signaling.