SLOW CONVERSIONS AMONG SUBCONDUCTANCE STATES OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHLORIDE CHANNEL

The cystic fibrosis transmembrane conductance regulator (CFTR) chlorid e channel exhibits multiple subconductance states. To study the regula tion of conductance states of the CFTR channel, we expressed the wild- type CFTR protein in HEK 293 cells, and isolated microsomal membrane v esicles for reconstitution studies in lipid bilayer membranes. A singl e CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub- open states with conductances of similar to 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl 2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on bo th sides of the channel. In 200 mM KCl, both H and L states could be m easured in stable single-channel recordings, whereas M could not. Spon taneous 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, be came stable and seemed to gate independently from each other. The exis tence of multiple stable conductance states associated with the CFTR c hannel suggests two possibilities: either a single CFTR molecule can e xist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, an d different conductance states are due to different aggregation states of the CFTR protein.