Carbonic-anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch-clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+-activated-K+ (BK) channel of K+-deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10(-6)M)>BFT(DE50=5.93x10(-5)M)>ETX(DE50=1.17x10(-4)M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra-molecular hydrogen bond with calculated inter-atomic distances ranging between 1.82 A degrees and 3.01 A degrees and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.