The BK channel opener GoSlo-SR-5-6 shifts the half maximal activation voltage (V1/2) of BKα subunits by >100 mV at 10 μM1. To investigate how it activates BK channels, we examined if its effects were altered in the Slo1_9a splice variant3. Experiments were carried out on either BK Slo1_9a α subunits cloned from the human brain or BK α subunits cloned from the rabbit bladder and transiently expressed in human embryonic kidney cells (HEK293). Site-directed mutagenesis on the resulting cDNA was carried out as previously described2 and confirmed by sequencing. HEK cells were grown in DMEM medium supplemented with 10% FCS, penicillin and streptomycin. HEK cells were dissociated with trypsin (1%), plated onto 35 mm Petri dishes and maintained in culture at 37oC in 5% CO2 prior to use. All experiments were performed at 37oC using excised inside/out patch configuration with symmetrical 140 mM K+ solutions containing either 1mM EGTA or 1 mM HEDTA. Excised patches were held at -60mV and BK channel currents were evoked using voltage steps from -100mV to 200mV before repolarising back to -80 mV. Peak currents were measured during the voltage steps, corrected for driving force and activation curves constructed. Slo1_9a is expressed exclusively in the brain and differs from the normal BK channel in 13 residues between the end of the S6 domain and the RCK1 linker. Application of GoSlo-SR-5-6 to A99 chimaera (which contains the Slo1_9a S6 sequence but normal BK channel proximal and distal linkers) shifted V1/2 by only -53±11 mV (n=6). We hypothesised that the majority of the reduction in the response to GoSlo-SR-5-6 was mediated by the amino acids in the S6 domain of Slo1_9a and used a combination of mutagenesis and electrophysiology to test this. The V1/2 of Slo1_9a currents in 100 nM Ca2+ was 158 ± 2 mV and was shifted to 63±3mV (n=10) in 1 μM Ca2+. Application of GoSlo-SR-5-6 (10 μM) changed activation voltage (ΔV1/2) by -38± 8mV in the Slo1_9a variant compared to -121±4 mV (n=12) in the normal BK channels. When A323I was mutated in Slo1_9a, application of GoSlo-SR-5-6 (10 μM) significantly changed ΔV1/2 to -61±4 mV compared to the Slo1_9a variant (n=6, p<0.05, unpaired t-test). When we mutated R317S in Slo1_9a, application of GoSlo-SR-5-6 to the this mutant increased ΔV1/2 to -90±6 mV (n=7, p<0.001). GoSlo-SR-5-6 also significantly increased ΔV1/2 in the R317I mutant to -69±9 mV. The effect of GoSlo-SR-5-6 was significantly reduced in the when the S317R mutation was performed, with a ΔV1/2 of -80±8 mV compared to -121±4 mV (p<0.001) in normal BK channels. These results suggest that R317 contributes significantly to the reduced response to GoSlo-SR-5-6 in the BK Slo1_9a variant.