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

{beta}-adrenoceptors ({beta}-ARs) regulate cardiac function during sympathetic nerve stimulation. {beta}-ARs are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts on their function is unknown. Here, we developed a technology based on size exclusion to explore the function of {beta}-ARs located in the OSM. We synthetized a PEG-Iso molecule by covalent linking isoprenaline (Iso) to a 5000 Da PolyEthylene-Glycol (PEG) chain to increase the size of the {beta}-AR agonist and prevent it from accessing the TT network. The affinity of PEG-Iso and Iso on {beta}1- and {beta}2-ARs was measured using radioligand binding. Molecular dynamics simulation was used to assess PEG-Iso conformation and visualise the accessibility of the Iso moiety to water. Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network due to the presence of the extracellular matrix. {beta}-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP]i than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]I and similar effects on excitation-contraction coupling parameters. However, PEG-Iso produced a much lower stimulation of nuclear PKA than Iso. Thus, OSM {beta}-ARs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM {beta}-ARs control mainly nuclear PKA and nuclear protein phosphorylation. Size exclusion strategy using ligand PEGylation provides a unique approach to evaluate the respective contribution of T-tubule vs. outer surface membrane proteins in cardiac cells. Significance Statement{beta}-adrenoceptors ({beta}-ARs) regulate cardiac function during sympathetic nerve stimulation. They are present in both cardiac T-tubule and outer surface membranes, but how their location impacts on their function is unknown. By linking the {beta}-AR agonist isoprenaline (Iso) to a PolyEthylene-Glycol (PEG) chain, we increased the size of the agonist to prevent it from entering the T-tubules. Thus, PEG-Iso is only able to activate {beta}-ARs in the outer surface membrane. With this size exclusion strategy, we show that {beta}-ARs located in the outer surface membrane control mainly cytosolic cAMP/PKA pathway and contractility, while those located in the T-tubule membrane control mainly nuclear PKA and nuclear protein phosphorylation.