Identification of the cardiac ryanodine receptor channel in membrane blebsof sarcoplasmic reticulum

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells wer e generated after saponin perforation of the plasma membrane followed by co mplete hypercontraction of the cell. Although characteristic proteins of th e plasma membrane, namely the beta1-adrenoreceptor and G alphai, were stain ed by monoclonal antibodies in the hypercontracted cells, these proteins co uld not be detected in the adjacent blebs. Monoclonal antibodies to the car diac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hyperc ontracted cells. From the immunofluorescence signals we conclude that the b lebs are mainly constituted of corbular and junctional SR membrane, and onl y to a lesser extent of network SR membrane. Deconvolution microscopy revea led that the membrane location of RyR2, calsequestrin and SERCA2 in the ble b is comparable to native SR membrane. At the bleb membrane gigaohm seals c ould be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified. ( C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.