Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle

Abstract

We describe the biochemical properties of an eicosanoid-modulated Cl−channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability ( P o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl− channel is a 55-kDa glycosylated monomer with voltage- and Ca2+concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 μM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 μM but failed to alter the P o of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET− anions with the channel pore, whereas the alteration of P o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.