We have recently developed a new family of BK channel openers called the GoSlo-SR family [1]. One of these, GoSlo-SR-5-6, shifts the voltage required for half maximal activation (V1/2) by more than -100 mV when applied at a concentration of 10 μM. The focus of this study was to examine the molecular site of action of this compound on BK channels. A splice variant (SV) of the BK channel called “Slo1_9a”, has recently been demonstrated in human brain and was found to be less responsive to the BK channel opener NS1619 [2]. The SV has an alternative exon 9, which encodes for the linker region connecting transmembrane segment S6 to RCK1 domain. All channels were transiently expressed in HEK cell lines and studied using the excised inside/out patch configuration at 37oC with symmetrical 140 mM K+ solutions. Application of GoSlo-SR-5-6 (10 μM) to the intracellular side of patches containing rBK channels shifted V1/2 (ΔV1/2) by -121 ± 3 mV (n=14) and by -105 ± 4 mV (n=13) in hBK channels. In the SV, 10 μM GoSlo-SR-5-6 reduced ΔV1/2 to -43 ± 6 mV (n=9), suggesting that the S6-RCK1 linker region contributes to the effects of GoSlo-SR-5-6. We used a combinatorial cloning approach to construct various chimeras of the normal rBK and the SV. The effect of GoSlo-SR-5-6 on BK channels with an altered proximal and distal linkers (9AA) was only reduced by ~10% (compared to the hBK) to -88 ± 7 mV (n=5), suggesting that the linker region does not contribute significantly. However, when we examined the effects of the alternative S6 segment with chimera A99, the ΔV1/2 was significantly reduced to -61 ± 6 mV (n=11) respectively (p<0.05, ANOVA). We carried out site-directed mutagenesis [3] to change the wild type amino acid in rBK to the corresponding amino acid in the SV. Interestingly only the S317R mutant significantly reduced the ΔV1/2 to -80 ± 7 mV (p<0.05, ANOVA) compared to the rBK. We next used a homology model of the BK channel, based on the structure of Kv1.2, to examine other amino acids in this region and found that I326 of adjacent α subunits was close to S317. When we produced an I326A mutation, we found that the ΔV1/2 was also significantly reduced to -73 ± 6 mV (n=6 p<0.05, ANOVA). Interestingly, in the double S317R:I326A mutant, the ΔV1/2 was further reduced to -56 ± 6 mV (n=5, p<0.05, ANOVA). In conclusion, the results of these experiments suggest that GoSlo-SR-5-6 may mediate it effects by interacting with S317 and I326 on adjacent α subunits.