nicotinic receptor channels
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2007 ◽  
Vol 71 (5) ◽  
pp. 1407-1415 ◽  
Author(s):  
Diego Rayes ◽  
Marina Flamini ◽  
Guillermina Hernando ◽  
Cecilia Bouzat

2005 ◽  
Vol 126 (3) ◽  
pp. 179-192 ◽  
Author(s):  
Angelo Demuro ◽  
Ian Parker

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 αβγδ 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.


2002 ◽  
Vol 540 (2) ◽  
pp. 425-434 ◽  
Author(s):  
Serguei S. Khiroug ◽  
Patricia C. Harkness ◽  
Patricia W. Lamb ◽  
Sterling N. Sudweeks ◽  
Leonard Khiroug ◽  
...  

2002 ◽  
Vol 25 (3) ◽  
pp. 433-437 ◽  
Author(s):  
Klaus Krampfl ◽  
Reinhard Dengler ◽  
Johannes Bufler

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