GoSlo-SR-5-6 is a novel, potent BK channel activator 1 that does not require functional Ca2+ sensors to mediate its effect 2. A recent study 3 has shown that the BK channel opener Cym04 was less effective in the Slo1_9a splice variant, which is expressed in the brain. The purpose of this study was to examine if the effects of GoSlo-SR-5-6 were reduced in the Slo1_9a variant. All experiments were carried out on BK channel α subunits cloned from rabbit bladder or human brain and expressed in human embryonic kidney cells (HEK293). Site-directed mutagenesis on the resulting cDNA was carried out 4) 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 the excised inside/out patch configuration with symmetrical 140 K+ solutions containing either 1mM EGTA or 1 mM HEDTA. Excised patches were held at -60 mV and BK channel currents were evoked using voltage steps from -100 mV up to 200 mV before repolarising back to -80 mV. Peak currents were measured during the voltage steps, corrected for driving force and activation curves constructed. In cells expressing the BKα subunit and exposed to 100 nM Ca2+ at the cytosolic face of the patch, the half maximal activation voltage (V1/2) was 171±2 mV and this was shifted to 57±3 mV (n=12) in the presence of 1 μM Ca2+. Application of GoSlo-SR-5-6 (10 μM) shifted the mean activation voltage (ΔV1/2) by -121±3 mV (n=12). When we examined the effect of GoSlo-SR-5-6 on the Slo1_9a splice variant we found that although a tenfold increase in Ca2+ (from 100 nM to 1 μM) shifted V1/2 from 158±2 mV to 63±3 mV, GoSlo-SR-5-6 application (10 μM) only changed ΔV1/2 by -38 ± 8 mV compared to normal BK channel, (p<0.001 unpaired t test, n=10). To assess the contribution of the proximal linker, we generated a 9A9 chimaera, which contained the alternative proximal linker and found that GoSlo-SR-5-6 shifted the V1/2 by -106 ± 5 mV (n=5). The ΔV1/2 of the 9AA chimaera was -88 ± 7 mV (n=6), but this was not significantly different from the 9A9 chimaera, suggesting that the distal linker contributed little to the response. When we investigated the contribution of the S6 segment using the A99 chimaera, the ΔV1/2 was -53 ± 11 mV (n=6, p<0.05), but was not further reduced in the A9A chimaera, (-51 ± 9 mV, n=7). These data suggest the effect of GoSlo SR5-6 is reduced in the Slo1_9a variant and that the majority of this effect appears to be mediated via differences in the S6 segment of this channel.