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Original Article

^a Department of Electrical Engineering, University of Missouri-Columbia, Columbia, MO 65211
^b Department of Physiology, University of Missouri-Columbia, Columbia, MO 65211
^c Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211
Dalton Cardiovascular Research Center, University of Missouri-Columbia, Research Park, Columbia, MO 65211.(573) 884-4232

gillisk{at}missouri.edu

The mechanism whereby cAMP stimulates Cl^– flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1–43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl^– current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1–43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca²+ in Calu-3 cells. However, no increase in Cl^– current accompanies Ca²+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca²+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.

Key Words: exocytosis • endocytosis • membrane capacitance • FM1-43 • caged calcium

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