Subunit-specific inhibition of BK channels by piperine

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca²⁺-activated K⁺ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαβ_1,4, BKαγ_1,3) expressed in HEK293T cells. Piperine in zero Ca²⁺ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 4.8 μM. Elevating the internal Ca²⁺ concentration from 0 to 100 μM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKβ₁ or β₄ regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ₁ or BKγ₃ subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαβ_1,4 subunits but not BKαγ_1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.