Measurement of sarcolemmal vesicle orientation by beta-adrenergic receptor binding

To assess the orientation (inside-out vs. outside-out) of purified cardiac sarcolemmal vesicles, we developed a new method utilizing the known outward-facing binding site of the beta-adrenergic receptor. We compared the binding of the lipid-insoluble ligand 3H-CGP-12177, which binds to beta-adrenergic receptors on outside-out sarcolemmal vesicles only, to the binding of the lipid soluble ligand 125I-iodocyanopindolol, which binds to beta-adrenergic receptors in sarcolemmal vesicles of either orientation. The ratio of CGP to ICYP binding is equal to the fraction of outside-out sarcolemmal vesicles. Sidedness measurements by beta-adrenergic receptor-binding showed similar mean values but less scatter than sidedness assessments by measurement of 3H-ouabain-binding or Na+,K(+)-ATPase activity in the presence or absence of membrane permeabilizing agents.