The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR.
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.
Hug
M J
MJ
Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Thiele
I E
IE
Greger
R
R
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*
D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015056:1-Methyl-3-isobutylxanthine