Large conductance, voltage and calcium activated K+ (BK) channels are formed by four pore forming α subunits. These channels are modulated by regulatory β (β1-4) and γ (γ1-4) subunits, which alter the biophysical and pharmacological properties of the channel. The β1 subunit, expressed in smooth muscle, shifts the voltage sensor activation to more negative membrane potentials1. Recently, we identified a BK channel openor, GoSlo-SR-5-130 (GoSlo130) that required β1 subunits to mediate its full effects2, but little is known about which β1 residues contribute to this enhanced effect. Currents from I/O patches were recorded using the patch clamp technique from HEK cells expressing BKα and mutant BKβ1 subunits. Experiments were performed at 37oC and the cytosolic surface of patches bathed in symmetrical (140 mM) K+ and 100 nM free Ca2+. Patches were held at -60 mV and stepped from -100 mV to +200 mV in 20 mV increments. Mutant β1 subunits were transiently transfected into HEK cells along with BKα subunits using the Ca2+ phosphate technique. GoSlo130 (10 μM) shifted the voltage required for half maximal activation by -28±3 mV (ΔV1/2, n=6) and this was increased to -94±6 mV (n=5, p<0.05) when applied to patches coexpressing WT BKαβ1 subunits. The BKβ1 subunit consists of two transmembrane domains, a large extracellular loop and small intracellular N and C termini. To investigate if the intracellular termini were essential for the effects of GoSlo130, we deleted the N terminus alone (BKβ1DNT2-15) and in combination with the C terminus (BKβ1DNT2-15&CT179-191). When GoSlo130 was applied to the BKαβ1DNT construct, the ΔV1/2 was significantly reduced to -25±7 mV (n=5, p<0.05). Deletion of both termini had little further effect (ΔV1/2 = -20±4 mV, n=6), suggesting that the NT of the β1 subunit played a role in mediating the effects of GoSlo130. To investigate which NT residues were important, we utilized a sequential deletion approach3. The effects of GoSlo130 were not significantly altered when residues 2-12 were deleted (ΔV1/2 -105±8 mV, n=6). However, deletions of membrane flanking residues significantly reduced the effect of GoSlo130 (ΔV1/2 -78±6 mV, n=6; -47±9 mV, n=6) in the BKαβ1ΔNT2-13, BKαβ1ΔNT2-14 constructs, respectively. These data suggested that residues 13-15 of the NT were essential for mediating the effects of GoSlo130. We next mutated residues 13-15 from ETR to GGG on the full length BKβ1 construct and found that GoSlo130 still shifted the activation V1/2 by -87±2 mV (n=6) compared to -94±6 mV in the WT BKβ1 construct. These data suggest that the backbone of residues E13, T14 and R15 in BKβ1 may contribute to the effects of GoSlo130 in BKαβ1 channels.