title: Structure and permeability of ion-channels by integrated AFM and waveguide TIRF microscopy.
abstract: Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors.
authors.affiliation: 1] Department of Bioengineering; Department of Mechanical & Aerospace Engineering, University of California San Diego, La Jolla, CA 92093 [2].
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: 1] Department of Bioengineering; Department of Mechanical & Aerospace Engineering, University of California San Diego, La Jolla, CA 92093 [2].
authors.forename: Fernando Teran
authors.initials: FT
authors.lastname: Arce
authors.affiliation: Department of Bioengineering; Department of Mechanical & Aerospace Engineering, University of California San Diego, La Jolla, CA 92093.
authors.forename: Nirav R
authors.initials: NR
authors.lastname: Patel
authors.affiliation: RC Nano Corporation, 2210 Midwest Road, Oak Brook, IL 60523, USA. Current Address: Innovation and New Ventures Office, Northwestern University, 1800 Sherman Ave., Evanston IL 60201.
authors.forename: Arjan P
authors.initials: AP
authors.lastname: Quist
authors.affiliation: Department of Materials, University of California Santa Barbara, CA 93106.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Cohen
authors.affiliation: Department of Bioengineering; Department of Mechanical & Aerospace Engineering, University of California San Diego, La Jolla, CA 92093.
authors.forename: Ratnesh
authors.initials: R
authors.lastname: Lal
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D018031:Connexin 43 / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D010539:Permeability; D016062:Porosity; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D018031:Connexin 43; D005456:Fluorescent Dyes; D007546:Isoquinolines; D008051:Lipid Bilayers; C017475:lucifer yellow; D002118:Calcium