This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peng, S. Articles by Sutko, J. L. Search for Related Content PubMed PubMed Citation Articles by Peng, S. Articles by Sutko, J. L.

Imaging Single Cardiac Ryanodine Receptor Ca²⁺ Fluxes in Lipid Bilayers

S. Peng ^*, N. G. Publicover , G. J. Kargacin , D. Duan ^*, J. A. Airey ^* and John L. Sutko ^*

^* Department of Pharmacology and the Biomedical Engineering Program, University of Nevada, Reno, Nevada; and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Correspondence: Address reprint requests to John Sutko, Dept. of Pharmacology, 318 Howard Bldg., Room 214, University of Nevada, Reno, NV 89557. Tel.: 775-784-4121; Fax: 775-784-1620; E-mail: sutko{at}unr.edu.

In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca²⁺ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca²⁺ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca²⁺ (25–50 mM) that result in saturating RyR2 Ca²⁺ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca²⁺ currents having amplitudes of 0.25–14 pA. Ca²⁺ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca²⁺ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca²⁺ currents, and for addressing other current research questions is discussed.

This article has been cited by other articles:

				A. Demuro and I. Parker "Optical Patch-clamping": Single-channel Recording by Imaging Ca2+ Flux through Individual Muscle Acetylcholine Receptor Channels J. Gen. Physiol., August 29, 2005; 126(3): 179 - 192. [Abstract] [Full Text] [PDF]

				J.M. Cordeiro, R. Brugada, Y.S. Wu, K. Hong, and R. Dumaine Modulation of IKr inactivation by mutation N588K in KCNH2: A link to arrhythmogenesis in short QT syndrome Cardiovasc Res, August 15, 2005; 67(3): 498 - 509. [Abstract] [Full Text] [PDF]

				S.-Q. Wang, C. Wei, G. Zhao, D. X.P. Brochet, J. Shen, L.-S. Song, W. Wang, D. Yang, and H. Cheng Imaging Microdomain Ca2+ in Muscle Cells Circ. Res., April 30, 2004; 94(8): 1011 - 1022. [Abstract] [Full Text] [PDF]

				S. Peng, N. G. Publicover, J. A. Airey, J. E. Hall, H. T. Haigler, D. Jiang, S. R. W. Chen, and J. L. Sutko Diffusion of Single Cardiac Ryanodine Receptors in Lipid Bilayers Is Decreased by Annexin 12 Biophys. J., January 1, 2004; 86(1): 145 - 151. [Abstract] [Full Text] [PDF]