DiscoveryofanovelBKCachannelactivatorwith isoxazoleringandcharacterization ofitsactivationmechanism

Abstract

The BKCa channel (large-conductance calcium-activated potassium channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle (UBSM) cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, I screened a chemical library containing 8,364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazole ring (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure–activity relationship study to obtain 4-(4-(4-chlorophenyl)-3 (trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance–voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. Also, CTIBD relaxes the urinary bladder muscle strip of rats and alleviates the symptoms of overactive bladder in a rat model induced with overactive bladder. To identify and characterize the mechanism of CTIBD, I tested whether Ca2+ and voltage sensing affect the activation effect of CTIBD. As a result, CTIBD can activate the BKCa channel without the Ca2+ sensing domain and at highly negative membrane voltages. These results suggest that the Ca2+ and voltage sensing are not essential for the channel activation of CTIBD, and suggest that the intrinsic pore gating may be affected. I tried to find binding sites of CTIBD. Based on the mutant study, i possible binding sites are located on the turret region of the BKCa channel, which is close to the ion conduction pore. I mutated three residues (P262, W263, and F266) located in the binding site to alanine. When those residues were changed to alanines, the channel activation effects of CTIBD decreased significantly. The triple mutant channel (P262A/W263A/F266A) showed the smallest V1/2 shift by 10 µM CTIBD. The triple mutant did not show any differences in activation and deactivation kinetics by CTIBD treatment. The increase in open probability by CTIBD was also smaller in the triple mutant BKCa channel compared to the wild-type channel. In this study, I identified a novel activation mechanism of BKCa channel by a small chemical, CTIBD, stabilizing its open conformation by binding near ion-conduction pore of BKCa channel.