Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle.
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 Ca(2+) 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 microM. 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 microM 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.
Salvail
Dany
D
Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4.
Cloutier
Martin
M
Rousseau
Eric
E
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation; D018118:Chloride Channels / Q000302:isolation & purification / Q000378:metabolism*; D004553:Electric Conductivity; D004594:Electrophysiology; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism; D014132:Trachea; D014665:Vasodilator Agents / Q000494:pharmacology
D018118:Chloride Channels; D008051:Lipid Bilayers; D008081:Liposomes; D014665:Vasodilator Agents; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium