Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes.
Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.
Department of Neurobiology and Behavior, University of California, Irvine, CA 92697-4550, USA.
Angelo
A
Demuro
Ian
I
Parker
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000502:physiology*; D004594:Electrophysiology; D005260:Female; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008839:Microelectrodes; D008845:Microinjections; D018613:Microscopy, Confocal; D009865:Oocytes / Q000502:physiology*; D055095:Optics and Photonics; D014981:Xenopus / Q000502:physiology*
D020864:Calcium Channels, N-Type; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D002118:Calcium