Selective inhibition and quantification of endogenous cAMP binding sites in supported native membranes for single-molecule studies

Abstract

Single membrane proteins are often studied in solubilized or reconstituted form to avoid endogenous background signals. However, such an artificial experimental environment, generated by the required purification protocols, might alter functional properties of the proteins investigated. Here we demonstrate on the example of the HCN2 pacemaker channel in supported membranes, that the endogenous background can be reduced by a suitable inhibitor protocol. Additionally, quantitative insight into the density of endogenous cAMP binding sites in the membrane can be obtained.