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.

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

“…Information on the molecular structure and organization of gap junctions in the cell membranes are available in the literature. However, high-resolution AFM imaging of the gap junctions are typically studied in purified membrane fractions, or reconstituted in lipid bilayers [76][77][78]. Here, the Cx43 gap junctions at the cell-cell contacts were inaccessible to the AFM probe due to their location between the cell membranes.…”

Assembling the puzzle: Oligomerization of α-pore forming proteins in membranes

“…To enhance spatial and temporal resolution simultaneously, these structural and dynamic techniques are often combined on the same device. FLIM/FRET [113], TIRF/AFM [114] and single-molecule imaging/single-channel recording [115] are only few examples of the advances made in this direction. Big efforts have also been done in order to improve existing techniques, like in the case of high speed AFM [36].…”

Review: Cantilever-Based Sensors for High Speed Atomic Force Microscopy

“…AFM provides a technology that can also be integrated with other microscopic and spectroscopic techniques such as laser scanning confocal microscopy (LSCM) [ 23 , 24 ], total internal reflection fluorescence microscopy (TIRFM) [ 25 , 26 , 27 ], aperture correction microscopy (ACM) [ 28 ], correlative stimulated emission depletion microscopy (STEDM) [ 29 , 30 , 31 ], fluorescence lifetime imaging microscopy (FLIM) [ 32 , 33 ], stochastic optical reconstruction microscopy (STORM) [ 34 , 35 ], super-resolution fluorescence microscopy (SRFM) [ 36 ], tip-enhanced raman spectroscopy (TERS) [ 37 , 38 , 39 ], scanning near-field optical microscopy (SNOM) [ 40 , 41 , 42 ], and Förster resonance energy transfer (FRET) [ 43 ]. These correlative approaches offer a good spatial (nm) and high temporal (ms) resolution to study cellular and molecular biophysics.…”