The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.
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 Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ 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.
Chen
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P
Department of Electrical Engineering, University of Missouri-Columbia, Columbia, MO 65211, USA.
Hwang
T C
TC
Gillis
K D
KD
D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism* / Q000502:physiology; D004553:Electric Conductivity; D005089:Exocytosis / Q000502:physiology; D005456:Fluorescent Dyes; D006801:Humans; D018408:Patch-Clamp Techniques; D010775:Photic Stimulation; D010782:Photolysis; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
C089981:(2-nitrophenyl)ethyl-cyclic adenosine monophosphate; C505032:CFTR protein, human; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D002118:Calcium