GoSlo-SR-5-6 is a novel BK channel agonist that is potent (EC50 ~ 3 uM) and efficacious (shifts V1/2 by >-100 mV), but little is known about the molecular mechanism underlying its effect. In HEK cells expressing wild type BKα subunits, the half maximal activation voltage (V1/2) was 171 mV in 100 nM Ca2+ at the cytosolic face of the patch and 57 mV in 1 μM Ca2+ (37OC). GoSlo-SR-5-6 (10 μM) shifted the mean activation voltage (ΔV1/2) by -121 mV. When the cytoplasmic RCK domains at the C-terminus of the BK channel were deleted and the truncated channels expressed in HEK, we found that the channels were insensitive to 100 nM Ca2+ and 1 μM Ca2+, but GoSlo-SR-5-6 (10 μM) shifted the V1/2 to -105 mV, suggesting that GoSlo-SR-5-6 interacts with the remaining transmembrane domain. We next examined the effect of GoSlo-SR-5-6 on the Slo1_9a splice variant, which differs at the C-terminal end of S6 (S6C) and in the linker region. In this construct, the drug induced ΔV1/2 was only -38 mV. To assess the contribution of S6C, the proximal linker, and the distal linker, we generated a series of chimaeras and found that ΔV1/2 was significantly reduced only when the S6C from Slo1_9a was present. For example, ΔV1/2 was -53 mV in the chimaera containing Slo1_9a S6C. These data suggested that GoSlo-SR-5-6 mediated its effects via S6C. We subsequently identified two residues in S6C (S317R, I326A), and one in the S4/S5 linker (L227A), in which the effects of GoSlo-SR-5-6 were reduced. In the S317R:I326A double mutant, ΔV1/2 was -56 mV, and this was reduced to only -15 mV in the L227A:S317R:I326A triple mutant. These residues reside at a co-evolved intersubunit interface important for electro-mechanical coupling in K+ channels and are important for mediating the effect of this compound.