An inner-pore mutation (F315D) promotes intracellular Mg²⁺ block and inward rectification of BK channels

Large-conductance Ca²⁺- and voltage-activated potassium (BK) channels display a near-linear current-voltage relationship in excised membrane patches but exhibit inward rectification in intact cells due to block by intracellular cations. This rectification arises from electrostatic interactions between endogenous cationic blockers and negatively charged residues lining the inner pore. Here, we examined how the introduction of an additional negative charge within the inner vestibule affects BK channel permeation. Phenylalanine 315 in the S6 segment of the BK α-subunit was substituted with aspartate (F315D), and channel properties were examined using single-channel patch-clamp recordings in HEK293 cells. F315D channels exhibited reduced single-channel conductance, with a greater reduction in outward than inward current, resulting in strong inward rectification in the cell-attached configuration. Patch excision into Mg²⁺-free solutions substantially increased both inward and outward current amplitudes, indicating strong modulation by intracellular cations. Reintroduction of Mg²⁺ restored current suppression in a concentration-dependent manner, with mutant channels showing greatly enhanced Mg²⁺ sensitivity compared with wild-type BK channels. In addition, F315D channels exhibited reduced voltage dependence of activation and high open probability even at strongly negative potentials under low Ca²⁺ conditions. These data are consistent with the introduction of an additional electrostatic determinant within the inner vestibule that strengthens intracellular cation block and converts BK channels into strong inward rectifiers. Because F315 is also part of the hydrophobic deep-pore region that controls BK gating, we interpret the F315D phenotype as the combined result of altered pore electrostatics, local hydration/permeation, and modified energetic coupling between the pore and activation sensors.