RECTIFICATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHLORIDE CHANNEL MEDIATED BY EXTRACELLULAR DIVALENT-CATIONS

We report here distinct rectification of the cystic fibrosis transmemb rane conductance regulator (CFTR) chloride channel reconstituted in li pid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extrac ellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEP ES), the inward currents (intracellular --> extracellular chloride mov ement) through a single CFTR channel were similar to 20% larger than t he outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-vol tage relationship of the channel could be restored through the additio n of millimolar concentrations of MgCl2 or CaCl2 to the trans solution . The dose responses for [Mg](0) and [Ca](0) had half-dissociation con stants of 152 +/- 72 mu M and 172 +/- 40 mu M, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid did not alter rectification of the CFTR channel. The non linear conductance property of the CFTR channel seemed to be due to ne gative surface charges on the CFTR protein, because in pure neutral ph ospholipid bilayers, clear rectification of the channel was also obser ved when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids o n several extracellular loops joining the transmembrane segments, whic h could constitute the putative binding sites for Ca and Mg.