title: Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.
authors.affiliation: Department of Physiology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: T
authors.initials: T
authors.lastname: Tao
authors.forename: J
authors.initials: J
authors.lastname: Xie
authors.forename: M L
authors.initials: ML
authors.lastname: Drumm
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: P B
authors.initials: PB
authors.lastname: Davis
authors.forename: J
authors.initials: J
authors.lastname: Ma
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004553:Electric Conductivity; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008051:Lipid Bilayers; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D010472:Perchlorates / Q000494:pharmacology; D011487:Protein Conformation; D017670:Sodium Compounds / Q000494:pharmacology
chemical_list: C505032:CFTR protein, human; D008051:Lipid Bilayers; D010472:Perchlorates; D017670:Sodium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; C031068:sodium perchlorate; D008274:Magnesium