title: Nanodomain Ca²⁺ of Ca²⁺ channels detected by a tethered genetically encoded Ca²⁺ sensor.
abstract: Coupling of excitation to secretion, contraction and transcription often relies on 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 carboxy tail of Ca(V)2.2 Ca(2+) channels, enabling near-field imaging of the nanodomain. Under total internal reflection fluorescence 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 fluorescence resonance energy transfer 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 analogue of molecular portals, mirroring the crystallographic images of side windows in voltage-gated K channels.
authors.affiliation: Department of Biomedical Engineering, Calcium Signals Laboratory, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205-2196, USA.
authors.forename: Lai Hock
authors.initials: LH
authors.lastname: Tay
authors.forename: Ivy E
authors.initials: IE
authors.lastname: Dick
authors.forename: Wanjun
authors.initials: W
authors.lastname: Yang
authors.forename: Marco
authors.initials: M
authors.lastname: Mank
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
authors.forename: David T
authors.initials: DT
authors.lastname: Yue
mesh_terms: D002118:Calcium / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics
chemical_list: C500545:CACNA1B protein, human; D020864:Calcium Channels, N-Type; D002118:Calcium