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Activation of a CFTR-mediated chloride current in a rabbit corneal epithelial cell line
1Department of Physiology, School of Medicine, University of Missouri-Columbia, 65212, USA. al-nakkashl@missouri.edu
Purpose:
To determine whether there is gene expression and functional activity of cystic fibrosis transmembrane conductance regulator protein (CFTR) in an SV40-immortalized rabbit corneal epithelial cell line, tRCE.
Methods:
Both whole-cell and cell-attached patch-clamp techniques were used to examine the biophysical characteristics of the cAMP-dependent chloride current. The molecular identity of this conductance was evaluated using RT-PCR analysis.
Results:
In whole-cell patch-clamp studies, a cAMP-dependent chloride conductance was further facilitated by the known CFTR activator genistein (20 microM). Kinetic analysis of cell-attached patches containing few channels ascertained that genistein increased the chloride channel activity by increasing channel open probability (via an increased channel open time and a decreased channel closed time). In addition, in the presence of a reduced forskolin concentration (i.e., 100 nM), the chloride conductance generated could be augmented by the nonspecific phosphodiesterase enzyme inhibitor, IBMX (100 microM), implicating the importance of intracellular cAMP in the regulation of this conductance. Furthermore, this conductance exhibited voltage-dependent inhibition in the presence of the CFTR chloride channel blocker glibenclamide (250 microM), but was DIDS insensitive (500 microM). Consistent with the presence of a CFTR-mediated chloride conductance, the expression of CFTR-mRNA was detected using RT-PCR. Sequence analysis of the product revealed 99.4% homology to that described for rabbit CFTR.
Conclusions:
In tRCE cells, there is gene expression and functional CFTR activity. Its presence may have important therapeutic implications in corneal epithelial diseases resulting from declines in transepithelial secretory and fluid transport activity.
Insights
Cystic fibrosis transmembrane conductance regulator protein (CFTR) is expressed and functionally active in rabbit corneal cells (tRCE). This finding suggests potential therapeutic applications for corneal diseases involving fluid transport.
Area of Science:
- Ocular surface science
- Ion channel physiology
- Molecular biology
Background:
- Corneal epithelial cells play a crucial role in maintaining ocular surface homeostasis.
- Dysregulation of ion transport can lead to various corneal pathologies.
Purpose of the Study:
- To investigate the presence and functional activity of the cystic fibrosis transmembrane conductance regulator protein (CFTR) in an immortalized rabbit corneal epithelial cell line (tRCE).
Main Methods:
- Patch-clamp electrophysiology (whole-cell and cell-attached) was employed to characterize cAMP-dependent chloride currents.
- Reverse transcription polymerase chain reaction (RT-PCR) was utilized to detect CFTR gene expression.
Main Results:
- Functional, cAMP-dependent chloride conductance was observed in tRCE cells, modulated by CFTR activators (genistein) and inhibitors (glibenclamide).
- Genistein increased channel open probability, while glibenclamide inhibited the conductance.
- RT-PCR confirmed the expression of CFTR mRNA with high homology to rabbit CFTR.
Conclusions:
- Rabbit corneal epithelial (tRCE) cells express functional CFTR.
- The presence of CFTR in tRCE cells offers potential therapeutic avenues for corneal diseases linked to impaired transepithelial transport.