Intracellular Mg2+ at physiological concentrations activates mSlo1 BK channels by binding to a metal-binding site in the cytosolic domain. Previous studies suggest that residues E374, Q397, and E399 are important in Mg2+ binding. In the present study, we show that mutations of E374 or E399 to other amino acids, except for Asp, abolish Mg2+ sensitivity. These results further support that the side chains of E374 and E399 are essential for Mg2+ coordination. To the contrary, none of the Q397 mutations abolishes Mg2+ sensitivity, suggesting that its side chain may not coordinate to Mg2+. However, because Q397 is spatially close to E374 and E399, its mutations affect the Mg2+ sensitivity of channel gating by either reducing or increasing the Mg2+ binding affinity. The pattern of mutational effects and the effect of chemical modification of Q397C indicate that Q397 is involved in the Mg 2+-dependent activation of BK channels and that mutations of Q397 alter Mg2+ sensitivity by affecting the conformation of the Mg 2+ binding site as well as by electrostatic interactions with the bound Mg2+ ion.