947-113 Aprotinin Produces Ion Channels in lipid Bilayers

Abstract

Aprotinin, a 58 Amino Acid peptide protease inhibitor, is used clinically to prevent postoperative blood loss and reduce transfusion requirements in those procedures which employ extracorporeal circulation. Its mechanism of action is unknown. We have observed that Aprotinin has produced channel-like artifacts in other experimental systems. We tested the hypothesis that Aprotinin could form ion channels in lipid bilayers. Lipid bilayers were created on the tips of patch clamp pipets using phosphatidylethanolamine, phosphatidylserine and cholesterol in a 5:3:2 ratio respectively. Single channel currents were recorded in symmetric solutions of KCI + Tris/HEPES, pH = 7.2, as well as asymmetric solutions. Aprotinin was added to the bath solution. Data were digitized and recorded on video tape for off-line analysis using custom software. 8 JLM Aprotinin produced typical ion channel currents in symmetric solutions of KCI at concentrations of 50 mM. 100 mM, 150 mM, and 250 mM, with resultant peak currents of 7.33 pA. The conductance of the Aprotinin channel was 31.8 pS in 250 mM KCI. Aprotinin exhibited multiple conductance levels and complex kinetics. In 250 mM potassium, the channel had a steady state Popen of 0.5. Popen was not voltage dependent. The channels were saturable, selective, and highly specific for potassium ions over sodium ions. No definite blockers of the channel have yet been identified. Our experiments show that Aprotinin produces ion channels in lipid bilayers, and this in turn may lead to better understanding this drug's mechanism of action.