Nanodomain Ca(2+) of Ca(2+) channels detected by a tethered genetically encoded Ca(2+) sensor

Coupling of excitation to secretion, contraction, and transcription often relies upon Ca(2+) computations within the nanodomain—a conceptual region extending tens of nanometers from the cytoplasmic mouth of Ca(2+) channels. Theory predicts that nanodomain Ca(2+) signals differ vastly from the slow submicromolar signals routinely observed in bulk cytoplasm. However, direct visualization of nanodomain Ca(2+) far exceeds optical resolution of spatially distributed Ca(2+) indicators. Here we couple an optical genetically encoded Ca(2+) indicator (TN-XL) to the carboxyl tail of Ca(V)2.2 Ca(2+) channels, enabling nearfield imaging of the nanodomain. Under TIRF microscopy, we detect Ca(2+) responses indicative of large-amplitude pulses. Single-channel electrophysiology reveals a corresponding Ca(2+) influx of only 0.085 pA, and FRET measurements estimate TN-XL distance to the cytoplasmic mouth at ~55 Å. Altogether, these findings raise the possibility that Ca(2+) exits the channel through the analog of molecular portals, mirroring the crystallographic images of side windows in voltage-gated K channels.