Using an agarose hydration technique for protein incorporation into vesicular bilayers, we elucidate the effects of membrane composition and ordering on G Protein Coupled Receptors (GPRCs). We successfully incorporate GPCRs into model membranes in the form of giant unilamellar proteinvesicles (GUPs). Using this completely in vitro platform we observe that the functional rate of the human serotonin receptor, GPCR 5-HT 1A , and the A 2 a Adenosine GPCR is dependent on membrane composition and ordering. We use BODIPY-GTPgS as our fluorescent marker to track the irreversible exchange between GDP and GTP on G proteins over time in GUPs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), brain sphingomyelin (BSM), and cholesterol (Chol) as well as synthetic lamellar phase diblock copolymer. Furthermore, using this approach we demonstrate that the incorporated receptors display a biased orientation with the N-terminus located on the exterior (extracellular) and the C-terminus on the interior (cytosolic).
channels have been shown to be localized to the intercalated disks along with Cx43 channels. Recent evidence of reciprocity in the co-localized expression of Kir2.1 and Nav1.5 channels in cardiac myocytes suggest that ionic currents due to these two channels are in some way calibrated to each other. In isolated cells, the fast sodium current is much larger than IK1 resulting in a large depolarization reserve. Using simulations of chains of cardiac cells, we show that the depolarization reserve for conducting action potentials is significantly smaller than in isolated cells. We also studied the changes in the depolarization reserve with variations in gap junction channel density and explored the conditions under which propagation slowed or failed. These insights will allow a better understanding of the effects of Na channel blockers as well as regional differences in action potential conduction in the heart.
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