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The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.

Chen P, Hwang TC, Gillis KD - J. Gen. Physiol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of Missouri-Columbia, Columbia, MO 65211, USA.

ABSTRACT
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.

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Flash photolysis of caged cAMP rapidly activates CFTR current but results in no change in Cm. All experiments included 100 μM NPE-cAMP in the [Cl−]i = 125 mM pipette solution. The [Cl−]o = 30 mM bath solution was used, and the holding potential was +15 mV. (A) Sample response to flash with Cm depicted in the top trace and current given in the bottom trace. The I-V response at peak current activation is shown in the inset. (B) Averaged response of 15 cells. The dotted line indicates the exponential fit to the averaged response, which had a time constant of 6.2 s.
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Figure 5: Flash photolysis of caged cAMP rapidly activates CFTR current but results in no change in Cm. All experiments included 100 μM NPE-cAMP in the [Cl−]i = 125 mM pipette solution. The [Cl−]o = 30 mM bath solution was used, and the holding potential was +15 mV. (A) Sample response to flash with Cm depicted in the top trace and current given in the bottom trace. The I-V response at peak current activation is shown in the inset. (B) Averaged response of 15 cells. The dotted line indicates the exponential fit to the averaged response, which had a time constant of 6.2 s.

Mentions: Since membrane capacitance often slowly drifts slightly during the course of an experiment, small changes in capacitance can be more accurately resolved in response to a rapid perturbation. Therefore, we used flash photolysis of caged cAMP to fully activate the CFTR current within seconds. Fig. 5 A depicts a sample response of a Calu-3 cell to photorelease of 100 μM NPE-cAMP and Fig. 5 B presents the average response from 15 cells that have activated currents of 100 pA or greater. The average peak current response of these cells is −521 pA (±80 pA, SEM) and the time constant of the exponential fitted to the average response is 6.2 s (Fig. 5 B, dotted line), which is consistent with a previous report (Nakashima and Ono 1994). To measure Cm accurately with higher time resolution than the sine + square technique, we used a technique where a stimulus containing two frequencies is applied (Rohlicek and Schmid 1994). Note that there is no apparent increase in Cm for either the sample or averaged traces despite a robust activation of the CFTR current. We performed a parallel set of experiments in NIH3T3 cells stably transfected with CFTR. In four cells, the average maximal current evoked by photorelease of caged cAMP was −581 pA (±157 pA, SEM), yet there was no increase in Cm (data not shown).


The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.

Chen P, Hwang TC, Gillis KD - J. Gen. Physiol. (2001)

Flash photolysis of caged cAMP rapidly activates CFTR current but results in no change in Cm. All experiments included 100 μM NPE-cAMP in the [Cl−]i = 125 mM pipette solution. The [Cl−]o = 30 mM bath solution was used, and the holding potential was +15 mV. (A) Sample response to flash with Cm depicted in the top trace and current given in the bottom trace. The I-V response at peak current activation is shown in the inset. (B) Averaged response of 15 cells. The dotted line indicates the exponential fit to the averaged response, which had a time constant of 6.2 s.
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Figure 5: Flash photolysis of caged cAMP rapidly activates CFTR current but results in no change in Cm. All experiments included 100 μM NPE-cAMP in the [Cl−]i = 125 mM pipette solution. The [Cl−]o = 30 mM bath solution was used, and the holding potential was +15 mV. (A) Sample response to flash with Cm depicted in the top trace and current given in the bottom trace. The I-V response at peak current activation is shown in the inset. (B) Averaged response of 15 cells. The dotted line indicates the exponential fit to the averaged response, which had a time constant of 6.2 s.
Mentions: Since membrane capacitance often slowly drifts slightly during the course of an experiment, small changes in capacitance can be more accurately resolved in response to a rapid perturbation. Therefore, we used flash photolysis of caged cAMP to fully activate the CFTR current within seconds. Fig. 5 A depicts a sample response of a Calu-3 cell to photorelease of 100 μM NPE-cAMP and Fig. 5 B presents the average response from 15 cells that have activated currents of 100 pA or greater. The average peak current response of these cells is −521 pA (±80 pA, SEM) and the time constant of the exponential fitted to the average response is 6.2 s (Fig. 5 B, dotted line), which is consistent with a previous report (Nakashima and Ono 1994). To measure Cm accurately with higher time resolution than the sine + square technique, we used a technique where a stimulus containing two frequencies is applied (Rohlicek and Schmid 1994). Note that there is no apparent increase in Cm for either the sample or averaged traces despite a robust activation of the CFTR current. We performed a parallel set of experiments in NIH3T3 cells stably transfected with CFTR. In four cells, the average maximal current evoked by photorelease of caged cAMP was −581 pA (±157 pA, SEM), yet there was no increase in Cm (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of Missouri-Columbia, Columbia, MO 65211, USA.

ABSTRACT
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.

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