PEGylated isoprenaline reveals distinct functions of cardiac β-adrenergic receptors located in the T-tubule vs. outer surface membrane

Membrane proteins are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), although at a different density. Classical pharmacology does not allow to explore the function of a membrane protein separately in OSM vs. TTM. Here, we developed a technology based on size exclusion to explore the function of {beta}-adrenergic receptors ({beta}-ARs) located in the OSM. We synthetized a PEG-Iso molecule by covalent linking between isoprenaline (Iso) and a 5000 Da PolyEthylene-Glycol (PEG). Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network. PEG-Iso produced similar effects as Iso on Ica,L, sarcomere shortening and Ca2+ transients. However, PEG-Iso increased [cAMP]i with a lower efficacy than Iso, produced a much lower stimulation of nuclear PKA activity than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]i. Our results show that activation of OSM {beta}-ARs is sufficient to activate cytosolic PKA and excitation-contraction coupling, but insufficient to activate nuclear PKA or nuclear protein phosphorylation for which additional activation of TTM {beta}-ARs is needed.