In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of β-AR and adenylyl cyclase, β-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2− gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated β-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of β-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled β-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted β-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated β-AR via Gs.

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