The KcsA channel was expressed in Escherichia coli BL21(DE3)pLysS cells, solubilized with n-dodecyl β-d-maltopyranoside (DDM; Dojindo), and purified as previously described (54). We genetically deleted the cytoplasmic domain (126 to 160) to reduce lateral fluctuation of the channel on the AFM substrate. To mimic the toxin-binding surface of the Shaker K+ channel, we introduced three mutations (Q58A, T61S, and R64D). All the channels used in this study carried this Shaker K+ channel–mimicking mutation, and thus, we refer to this triple mutant as “WT” herein for simplicity. To immobilize the channel onto a Ni2+-coated mica surface (41, 55), a hexahistidine tag was introduced at the C terminus (as residues 126 to 131). AgTx2 and DMPC were purchased from Smartox Biotechnology (Saint-Egrève, France) and Avanti Polar Lipids (Alabaster, Alabama), respectively.