In this report, we show an improved method for the simultaneous measurement of optical and electrical properties of single-channel proteins for analysis of the gating mechanism. Large-conductance Ca 2+ -activated potassium (BK) channels were isolated from porcine uterine smooth muscle and labeled with Cy5 via antibody against the Nterminal. These Cy5-labeled BK channels were incorporated into lipid bilayer membranes followed by single channel current measurements. Cy5-labeled BK channels

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... ed Ca 2+ and voltage sensitivity. The orientation of the vesicles was determined to be outside-out. Charybdotoxin applied from the cis side blocked the channel current. For stable observations of ligand and channel binding, BK channel immobilization was also examined. The lateral diffusion coefficient of BK channels decreased over 200 fold in 1 µM annexin V while the open probability did not change. This study is a significant advancement in simultaneous measurements of ligand binding and current change at the single channel level.