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Free keywords:
TARGETED RECOMBINANT AEQUORIN; CALCIUM-CHANNEL; TRANSMITTER RELEASE;
N-TYPE; ELEMENTARY MECHANISMS; PLASMA-MEMBRANE; GATING-CURRENT; LIVING
CELLS; TROPONIN-C; CALMODULIN
Abstract:
Coupling of excitation to secretion, contraction and transcription often relies on Ca2+ computations within the nanodomain-a conceptual region extending tens of nanometers from the cytoplasmic mouth of Ca2+ 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 similar to 55 angstrom. 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.
