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Biophysical Journal 86:145-151 (2004)
© 2004 The Biophysical Society

Diffusion of Single Cardiac Ryanodine Receptors in Lipid Bilayers Is Decreased by Annexin 12

S. Peng *, N. G. Publicover {dagger}, J. A. Airey *, J. E. Hall {ddagger}, H. T. Haigler {ddagger}, D. Jiang §, S. R. Wayne Chen § and J. L. Sutko *

* Department of Pharmacology and the {dagger} Biomedical Engineering Program, University of Nevada, Reno, Nevada; {ddagger} Department of Physiology and Biophysics, University of California, Irvine, California; and § Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, 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.

Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca2+ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (DRyR) of 1.2 x 10-8 cm2 s-1 and a mean path length of 5.0 µm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca2+-dependent manner. Addition of annexin 12 (0.25–4.0 µM) to the trans side of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2 µM) decreased the apparent DRyR 683-fold from 1.2–10-8 to 1.8 x 10-11 cm2 s-1 and the mean path length 10-fold from 5.0 to 0.5 µm without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca2+ or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.




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