Abstract
Although there have been several reports pertaining to the existence of the cGMP-gated channel in the disk membrane of rod photoreceptors, its density there relative to that of the photoreceptor plasma membrane is unknown. Using immunoblotting, immunohistochemical, and reconstitution techniques on purified disk and plasma membrane preparations, we found that the density of channels in the plasma membrane was at least 50-fold higher than that of the disk membrane. Purification of membrane fractions without prior digestion of cytoskeletal components by mild trypsinization was found to increase the amount of channel protein present in disk membrane preparations. We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptor cell.
Highlights
From the $Max-Planck Institut fur Biophysik,AbteilungMolekulare Membranbiologie,Heinrich-Hoffmannstrasse7,D6000 Frankfurt amMain 71,Federal Republic of Germany, the TDepartment of Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada, and the IIAbteilung Biophysik,Universitat Osnabriick, Postfach 4469, D
Purification of membranefractions without prior digestion of cytoskeletal componentsby mildtrypsinization was found to increase the amount of channel protein present in disk membrane preparations
We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptorcell
Summary
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No 12, Issue of April 25, pp. 6996-6999 1989 Printed in il.S.A. 6996-6999 1989 Printed in il.S.A. The cGMP-gated Channeolf Bovine Rod Photoreceptors Is Localized Exclusively in the Plasma Membrane*. We propose that the presence of the channel protein in rod disk membrane preparations is an artifact arising from fusion of plasma membrane components during permeabilization of the photoreceptorcell. The channel protein responsible for this conductance has been purified and functionally reconstituted [4,5,6] This channel presumably exerts its role in phototransduction in the photoreceptor plasma membrane, cGMP-gated conductances in these cells were first demonstrated to occur across the disk membrane [7], an observation which has subsequently been confirmed in several other studies [8,9,10,11, 19]. The abbreviations used are: ROS, rod outer segment; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonatHee;pes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonaicid
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