Large conductance Ca2+-activated K+ (BK) channels modulate many physiological processes including neuronal excitability, synaptic transmission and regulation of myogenic tone. A gain-of-function (E/D) mutation in the pore-forming α subunit (Slo1) of the BK channel was recently identified and is linked to human neurological diseases of coexistent generalized epilepsy and paroxysmal dyskinesia. Here we performed macroscopic current recordings to examine the effects of the E/D mutation on the gating kinetics, and voltage and Ca2+ dependence of the BK channel activation in the presence of four different β subunits (β1-4). These β subunits are expressed in a tissue-specific pattern and modulate BK channel function differently, providing diversity and specificity for BK channels in various physiological processes. Our results show that in human (h) Slo1-only channels, the E/D mutation increased the rate of opening and decreased the rate of closing, allowing a greater number of channels to open at more negative potentials both in the presence and absence of Ca2+ due to increased Ca2+ affinity and enhanced activation compared with the wild-type channels. Even in the presence of β subunits, the E/D mutation exhibited these changes with the exception of β3b, where Ca2+ sensitivity changed little. However, quantitative examination of these changes shows the diversity of each β subunit and the differential modulation of these subunits by the E/D mutation. For example, in the presence of the β1 subunit the E/D mutation increased Ca2+ sensitivity less but enhanced channel activation in the absence of Ca2+ more than in hSlo1-only channels, while in the presence of the β2 subunit the E/D mutation also altered inactivation properties. These findings suggest that depending on the distribution of the various β subunits in the brain, the E/D mutation can modulate BK channels differently to contribute to the pathophysiology of epilepsy and dyskinesia. Additionally, these results also have implications on physiological processes in tissues other than the brain where BK channels play an important role.