Nanodomain Ca²⁺ of Ca²⁺ channels detected by a tethered genetically encoded Ca²⁺ sensor

Coupling of excitation to secretion, contraction and transcription often relies on Ca²⁺ computations within the nanodomaing-a conceptual region extending tens of nanometers from the cytoplasmic mouth of Ca ²⁺ channels. Theory predicts that nanodomain Ca²⁺ signals differ vastly from the slow submicromolar signals routinely observed in bulk cytoplasm. However, direct visualization of nanodomain Ca²⁺ far exceeds optical resolution of spatially distributed Ca²⁺ indicators. Here we couple an optical, genetically encoded Ca²⁺indicator (TN-XL) to the carboxy tail of Ca V 2.2 Ca²⁺ channels, enabling near-field imaging of the nanodomain. Under total internal reflection fluorescence microscopy, we detect Ca²⁺ responses indicative of large-amplitude pulses. Single-channel electrophysiology reveals a corresponding Ca²⁺ influx of only 0.085 pA, and fluorescence resonance energy transfer measurements estimate TN-XL distance to the cytoplasmic mouth at ∼55Å Altogether, these findings raise the possibility that Ca²⁺ exits the channel through the analogue of molecular portals, mirroring the crystallographic images of side windows in voltage-gated K channels.