Large conductance, voltage and Ca2+ activated KC channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BK alpha and BK alpha beta channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BK alpha gamma channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BK alpha channels coexpressed with 4 different LRRC (gamma(1-4)) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of gamma(1-4) subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different gamma subunits. In cells expressing BKa channels, the shift in V-1/2 (DV1/2) induced by SR-5-44 (3 mu M) was -76 +/- 3 mV, whereas it was significantly reduced by 70 % in BKag1 channels (DV1/2D -23 +/- 3, p < 0.001, ANOVA). In BK alpha gamma channels the DV1/2 was -36 +/- 1 mV, which was less than that observed in BK alpha gamma and BK alpha gamma(1) channels where the DV1/2 was -47 +/- 5 mV, and -82 +/- 5 mV, respectively. Additionally, the excitatory effects of alpha'beta b specific' BK channel opener, SR-5-130 were only partially restored in the patches containing BK alpha gamma(1-4) channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory g subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.