Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations

Abstract

We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.

Voltage-dependent Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channel

“…Second, inward rectification has been observed using cells expressing either native or recombinant wild-type human CFTR (e.g., Quinton and Reddy, 2000 ; Lansdell et al, 2000 ; Linsdell and Gong, 2002 ; this study), indicating that inward rectification is independent of the expression system used to investigate CFTR. Third, inward rectification of CFTR Cl − channels reconstituted into planar lipid bilayers was unaffected by changes in the composition of the lipid bilayer ( Zhao et al, 1996 ), suggesting that the surface charge of the membrane lipid does not affect inward rectification. Fourth, the effects of voltage on the function of the murine CFTR Cl − channel (present study) indicate that inward rectification is a common characteristic of human and murine CFTR.…”

“…Using excised membrane patches from baby hamster kidney (BHK) cells expressing wild-type human CFTR, Linsdell and colleagues observed similar results in some studies ( Linsdell and Hanrahan, 1999 ; Linsdell and Gong, 2002 ), but not in others (e.g., Linsdell et al, 1998 ; Linsdell and Hanrahan, 1998b ). Moreover, Zhao et al (1996) reported inward rectification of CFTR Cl − channels reconstituted into planar lipid bilayers. In this study, we investigate the mechanism of inward rectification of the CFTR Cl − channel.…”

Function of the second nucleotide‐binding fold in the CFTR chloride channel

“…A human embryonic kidney cell line (293‐EBNA, Invitrogen) was used for transfection and expression of wt and mutant CFTR proteins. The culture conditions for maintaining the HEK 293 cells were as described previously [20,21]. The parental cell line was grown to confluence in a 37°C incubator with 5% CO 2 and passaged 1:5, 2 days before gene transfer.…”

“…Lipid bilayer membranes were formed across an aperture of ∼200 μm diameter with a lipid mixture of PS:PE:cholesterol of 6:6:1. The lipids are dissolved in decane to a concentration of 40 mg/ml [21]. The recording solutions are as follows: cis (intracellular): 200 mM CsCl, 2 mM Mg‐ATP, 10 mM HEPES‐Tris (pH 7.4) and trans (extracellular): 50 mM CsCl, 10 mM HEPES‐Tris (pH 7.4).…”