Electron microscopic localization of the beta-adrenergic receptor using a ferritin-alprenolol probe

This report describes electron microscopic localization of the beta-adrenergic receptor using a beta-adrenergic receptor antagonist conjugated to ferritin. The conjugate was synthesized by reacting a carboxylic acid derivative of alprenolol with ferritin. The ferritin-alprenolol compound was shown to be effective in displacing specific [3H]dihydroalprenolol binding from rat erythrocyte membranes with a dissociation constant (Kd) of 25 nM. Rat erythrocyte ghosts were incubated with the compound and quantitative electron micrographic analysis yielded total binding of 1367 +/- 129 ferritin particles and nonspecific binding of 688 +/- 111 (six experiments). Thus, specific binding was 680 +/- 60 ferritin particles per red cell profile. Qualitative observations suggested that the particles were distributed randomly on the surface of the erythrocyte, although an occasional cluster was seen. A compound from another synthesis was shown be to effective in displacing specific [125I]iodocyanopindolol binding from neonatal rat cardiac myocyte membranes, with a dissociation constant of 13.8 nM, whereas native alprenolol had a dissociation constant of 1.3 nM. Neonatal rat cardiac myocytes were incubated with the compound and processed for electron microscopy. Total binding along the sarcolemmal membrane was 504 +/- 38 ferritin particles/100 micron of membrane and nonspecific binding was 301 +/- 26 ferritin particles/100 micron of membrane (seven experiments), yielding specific binding of 203 ferritin particles/100 micron of membrane. In additional studies, specific binding was inhibited 95% with 10(-5) M l-isoproterenol and 29% with d-isoproterenol, indicating stereoselectivity (seven experiments). The probe was distributed randomly along the sarcolemma with no preferential localization to coated pits or other membrane specializations. From measurements of the surface area of the average cardiac myocyte (732 micron 2), the specific binding of ferritin-alprenolol per 100 micron of membrane (203), and section thickness (0.08 micron), we calculated that cardiac myocytes had 18,575 beta-adrenergic membrane receptor sites. Thus, we have described a method for synthesizing and applying an electron-dense probe for electron microscopic localization of beta-adrenergic receptors. In these studies we determined the distribution of these receptors on rat erythrocyte ghosts and neonatal rat cardiac myocytes.