title: "Optical patch-clamping": single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels.
abstract: We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of alphabetagammadelta subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel "channel chip" representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine 92697, USA.
authors.forename: Angelo
authors.initials: A
authors.lastname: Demuro
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
mesh_terms: D000109:Acetylcholine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D002794:Choline / Q000031:analogs & derivatives / Q000494:pharmacology; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D007368:Interferometry; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D055095:Optics and Photonics; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014741:Video Recording; D014982:Xenopus laevis
chemical_list: D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C009335:subecholine; D002794:Choline; D000109:Acetylcholine; D002118:Calcium