title: The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR.
abstract: The aim of this study was to examine the question of whether activation of wt-CFTR (wild-type cystic fibrosis transmembrane conductance regulator) by cAMP and the opening of a Cl- conductance is paralleled by exocytosis and corresponding increases in membrane capacitance. To this end three types of Chinese hamster ovary (CHO) cells were examined: a control group of CHO cells; a group of CHO cells stably expressing wt-CFTR at high levels (also called BQ2-CHO); and a group of CHO cells stably expressing the frequent mutation DeltaF508-CFTR. Whole-cell patch-clamp studies were performed to measure the membrane voltage (Vm), the membrane conductance (Gm) and the membrane capacitance (Cm). Cm was assessed by a two-frequency lock-in amplifier method. Forskolin (Fsk, 0.1 micromol/l) and isobutylmethylxanthine (IBMX, 0.1 mmol/l) were used to increase cytosolic cAMP. It is shown that Fsk and IBMX had no effect on Vm and Gm in control CHO and DeltaF508-CFTR-CHO cells. Fsk and IBMX depolarized wt-CFTR-expressing CHO cells significantly (from -40 +/- 1.5 to -32 +/- 1.6 mV, n = 41) and enhanced Gm strongly from 5.0 +/- 0.9 to 36 +/- 3.9 nS (n = 65). The conductance increase was mostly for Cl-, because under stimulated conditions a reduction in bath Cl- concentration depolarized these cells further and significantly from -26 +/- 1.8 to -10 +/- 1.2 mV (n = 16). This conductance had the characteristic wt-CFTR selectivity of Br- > Cl- > I- (n = 16). Despite this large increase in the Fsk- and IBMX-induced conductance Cm was not altered significantly (15.5 versus 15.7 pF, n = 50). These data indicate that stable overexpression of wt-CFTR but not of DeltaF508-CFTR in CHO cells induces a cAMP-activated Cl- conductance. The activation of this large conductance obviously proceeds with little if any exocytosis.
authors.affiliation: Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
authors.forename: M J
authors.initials: MJ
authors.lastname: Hug
authors.forename: I E
authors.initials: IE
authors.lastname: Thiele
authors.forename: R
authors.initials: R
authors.lastname: Greger
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D018118:Chloride Channels / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004553:Electric Conductivity; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D009154:Mutation; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015056:1-Methyl-3-isobutylxanthine