The anti-protozoal drug pentamidine blocks K_IR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel

Background and purpose: Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U-wave has been linked to the cardiac inward rectifier current (I-K1), we examined the action and mechanism of pentamidine-mediated I-K1 block. Experimental approach: Patch clamp measurements of I-K1 were made on cultured adult canine ventricular cardiomyocytes, K(IR)2.1-HEK293 cells and K(IR)2.x inside-out patches. Pentamidine binding to cytoplasmic amino acid residues of K(IR)2.1 channels was studied by molecular modelling. Key results: Pentamidine application (24 h) decreased I-K1 in cultured canine cardiomyocytes and K(IR)2.1-HEK293 cells under whole cell clamp conditions. Pentamidine inhibited I-K1 in K(IR)2.1-HEK293 cells 10 min after application. When applied to the cytoplasmic side under inside-out patch clamp conditions, pentamidine block of I-K1 was acute (IC50 = 0.17 mu M). Molecular modelling predicted pentamidine-channel interactions in the cytoplasmic pore region of K(IR)2.1 at amino acids E224, D259 and E299. Mutation of these conserved residues to alanine reduced pentamidine block of I-K1. Block was independent of the presence of spermine. K(IR)2.2, and K(IR)2.3 based I-K1 was also sensitive to pentamidine blockade. Conclusions and implications: Pentamidine inhibits cardiac I-K1 by interacting with three negatively charged amino acids in the cytoplasmic pore region. Our findings may provide new insights for development of specific I-K1 blocking compounds.