We have synthesised a novel BK channel opener GoSlo-SR5-6, that mimics the effect of Ca2+ by shifting the voltage required for half maximal activation in 100nM Ca2+ by -107±7 mV, (n=12,10 μM) in native rabbit bladder smooth muscle cells. We hypothesised that this molecule interacts with the Ca2+ sensing apparatus of the BK channel. All experiments were carried out on BK channel α subunits cloned from the rabbit urethra and expressed in human embryonic kidney cells (HEK293). Site-directed mutagenesis on the resulting cDNA was carried out using the method of Sawano & Miyawaki (2000) 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 K+ solutions containing 140 mM KCl, 10 mM Glucose, 10 mM HEPES and either 1mM EGTA (for free [Ca2+] 100 nM) or 1 mM HEDTA (for free [Ca2+] > 300 nM). All solutions had a pH of 7.2 and the pipette solution contained 100 nM free Ca2+. The BKα subunit has a large cytoplasmic tail that contains two high affinity Ca2+ binding sites in the RCK1 domain and Ca2+ bowl respectively. When we examined the Ca2+ sensitivity of BKα subunits re-expressed in HEK 293 cells, a 10-fold increase in Ca2+ from 100 nM to 1 μM shifted the activation V1/2 from 177±4 mV to 92±6 mV (ΔV1/2= -85±5, n=6, p<0.05 paired t test). When GoSlo-SR5-6 (10 μM) was applied in the presence of 100 nM Ca2+ the ΔV1/2 was -90 mV (n=6, p<0.05). To examine if abolition of Ca2+ sensing in the RCK1 domain reduced these effects, we mutated D367A (Xia et al., 2002) and found that although the Ca2+ sensitivity of the channels was decreased compared to the normal BKα, the effect of GoSlo-SR5-6 was not diminished, suggested that a functional Ca2+ sensor in the RCK1 domain was not required for these molecules to exert their effects. We carried out a double mutation on M513I in the RCK1 domain and D898A in the Ca2+ bowl (Bao et al., 2004). This double (RCK & BOWL) mutant was practically insensitive to Ca2+ below 100 μM and a hundred-fold increase in Ca2+ from 100 nM to 10 μM only shifted the activation V1/2 from 144±8 mV to 115±10 mV (n=4). Despite this reduction in the channel’s Ca2+ sensitivity, application of 10 μM GoSlo-SR5-6 still shifted the activation V1/2 of the channel to 47±8 mV(ΔV1/2 = -97±10 mV, n=4). These data suggest that GoSlo-SR-5-6 can still activate BKα subunits in the absence of functional high affinity Ca2+ sensors.