Macroscopic whole-cell current and inside-out patch recording under isotonic K+ conditions from tsA-201/HEK cells transiently expressing homomeric rSK2 and hSK3 channels was used to study block by apamin and extracellular acidosis. Inside-out patches from cells expressing rSK2 channels were bathed in a solution containing 1 mM Ca2+ and displayed two amplitude levels corresponding to conductances of approximately 10 and 18 pS, with the larger amplitude being more common. Rare transitions between amplitude classes were apparent, suggesting that the lower amplitude was a sub-conductance state. Channels exhibited bi-exponential open time distributions with time constants of ~ 1.8 and 12 ms and closed time distributions best described by 3 exponentials (t’s ~1.5, 18 and 100 ms). The prevalence of low amplitude openings was enhanced by extracellular acidosis suggesting that protonation of a residue in the outer pore reduced conduction through the channel. Inclusion of a supra-maximal concentration of apamin (100 nM) in the electrode solution produced patches that displayed a lower activity, which resulted from the recruitment of an additional closed state (t~750-1000 ms). In addition, remaining channel openings were predominantly of low amplitude (10 pS) that displayed prolonged open times. Mutation of a residue in the outer pore of rSK2 produced a channel that displayed only low amplitude openings of approximately 10 pS conductance. This corresponded to the low conductance state of the wild type rSK2 channel promoted by apamin, implying an impairment of the high amplitude conductance pathway that mimicked the effect produced by acidosis. Whole cell currents evoked from cells expressing mutant rSK2 were insensitive to acidosis or apamin indicating this residue was critical for block of macroscopic current. hSK3 inside out patches displayed openings of similar conductance levels to rSK2. Comparison of homomeric hSK3 and rSK2 whole cell currents revealed a similar level of block by apamin (~55%), but significant differences in the block by acidosis. This suggested that SK3 may possess an additional interaction site. Apamin and extracellular protons reduced the macroscopic current by interacting with an outer pore residue in rSK2 and hSK3, which is proposed to be involved in stabilising the conduction pathway. In addition, data is presented suggesting that SK3 has an extra block under acidosis due to an additional interaction site.