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.
|Original language||English (US)|
|State||Published - 2012|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Physics and Astronomy(all)