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Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum
Author(s) -
Böhle Thomas,
Brandt Mathias C.,
Henn Nadine,
Schmidt Annette,
Bloch Wilhelm,
Beuckelmann Dirk J.
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)02862-9
Subject(s) - calsequestrin , endoplasmic reticulum , ryanodine receptor , bleb (medicine) , ryanodine receptor 2 , chemistry , biophysics , membrane , monoclonal antibody , cardiac muscle , cell membrane , microbiology and biotechnology , biology , biochemistry , antibody , anatomy , immunology , trabeculectomy , neuroscience , glaucoma
Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the β1‐adrenoreceptor and Gαi, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga‐ohm seals could be obtained and patches could be excised in a way that single‐channel currents could be measured, although these are not completely identified.