Optical single-channel recording: imaging Ca2+ flux through individual ion channels with high temporal and spatial resolution.
Developments in imaging technology now enable visualization of the functioning of individual ion channels in living cells: something previously possible only by the electrophysiological patch-clamp technique. We review techniques that track channel gating via changes in intracellular [Ca2+] resulting from openings of Ca(2+)-permeable channels. Spatial and temporal resolution are optimized by monitoring Ca2+ close to the channel mouth, and we describe the use of two imaging modalities: confocal laser scan microscopy (linescan CLSM) and total internal reflection fluorescence microscopy (TIRFM). Both currently achieve a kinetic resolution of <10 ms, provide a simultaneous and independent readout from many channels, and enable their locations to be mapped with submicrometer resolution. TIRFM provides 2-D images from a very thin (approximately 100 nm) optical section, but it is restricted to channels in the plasma membrane of cells adhering close to a cover glass. In contrast, CLSM can image channels in intracellular membranes but, to achieve good temporal resolution, has been utilized only in a linescan mode with limited spatial information. We anticipate that imaging techniques will develop as a useful adjunct to patch-clamping for single-channel studies, with capabilities including simultaneous readout from multiple channels, high-resolution mapping of channel location, and mobility that is inaccessible by electrophysiological means. Optical single-channel recording is applicable to diverse voltage- and ligand-gated Ca(2+)-permeable channels and has potential for high-throughput functional analysis.
Demuro
Angelo
A
University of California, Irvine, Department of Neurobiology and Behavior, Irvine, California 92697-4550, USA.
Parker
Ian
I
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D004867:Equipment Design; D015640:Ion Channel Gating; D018613:Microscopy, Confocal / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods
D015220:Calcium Channels; D002118:Calcium