Raymond A. Frizzell

Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer

We have used retrovirus-mediated gene transfer to demonstrate complementation of the cystic fibrosis (CF) defect in vitro. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC-1, a pancreatic adenocarcinoma cell line derived from a patient with CF that stably expresses the chloride transport abnormalities characteristic of CF. CFPAC-1 cells were exposed to control virus (PLJ) and CFTR-expressing virus (PLJ-CFTR); viral-transduced clones were isolated and subjected to molecular and physiologic analysis. RNA analysis detected a viral-derived CFTR transcript in all of the PLJ-CFTR clones that contained unrearranged proviral sequences. Agents that increase intracellular cAMP stimulated 125I efflux in PLJ-CFTR clones but not PLJ clones. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses were due to cAMP stimulation of Cl conductance. Our findings indicate that expression of the normal CFTR gene confers cAMP-dependent Cl channel regulation on CF epithelial cells.

Cystic Fibrosis: a disease of ion channels?

Abstract Cystic fibrosis is of interest to neuroscientists because it appears to be a disease of ion channels. It is not the conduction properties of ion channels that are affected, but rather their gating by chemical agonists. These conductance pathways appear to be unique to epithelial cells in which salt and water transport rates are governed by cAMP- and Ca 2+ -dependent regulatory processes.

Single chloride channel currents from canine tracheal epithelial cells

Patch-clamp techniques were used to characterize the properties of anion-selective channels in canine tracheal epithelial cells that had been maintained in primary culture. Gigaohm seals (10-30 G omega) were obtained in single isolated cells or cells at the edge of a confluent sheet, and channels were studied in the cell attached or the inside-out, excised patch configuration. Pretreatment with isotonic KCl caused the cells to round-up and allowed us to have better success in obtaining good seals. Based on conductance, anion-cation selectivity and voltage-dependent kinetic properties, four anion channel types could be detected in symmetrical solutions of 0.15 M NaCl: (i) a 30-50 pS Cl- channel of high selectivity, active at negative potentials and inactivated by large positive potentials; (ii) an approx. 20 pS Cl- channel of high selectivity, active at positive potentials and inactivated at negative potentials; (iii) an approx. 250 pS channel of moderate selectivity (PCl/PNa = 4) that was not voltage-dependent, and (iv) an approx. 10 pS Cl- channel with characteristics similar to (iii) above, but remaining somewhat active at large negative voltages. All excised patches were exposed to relatively high calcium concentrations on the intracellular side. Channel activity was increased in tracheal cells treated with 1 mM cAMP, suggesting that at least one of these channels plays a role in the increase of the apical membrane Cl- conductance that is mediated by cAMP and elicited by agonists of active Cl- secretion.

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl‐ conductance defect in cystic fibrosis lymphocytes by transfection with wild‐type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non‐CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector‐specific sequences. Both fluorescence and patch‐clamp assays showed that transfectants expressing wild‐type CFTR acquired properties previously associated with Cl‐ conductance (GCl) regulation in non‐CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist‐induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild‐type CFTR corrects the defects in Cl‐ conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR‐mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

Biophysical Properties of a Chloride Channel in the Apical Membrane of a Secretory Epithelial Cell

1. Patch clamp studies on colonic tumor cell line T84 show the presence of chloride channels. 2. The channels are activated by forskolin, PGE2, or 8-Br-cAMP. 3. Single channel conductance was ca 40 pS at the reversal potential, increasing to 70 pS at +80 mV and decreasing to 25 pS at -80 mV. 4. Relative permeabilities were I greater than Br greater than Cl greater than F.

Conductance Pathways Involved in Chloride Secretion and Their Regulation

Cystic fibrosis impairs the secretory activities of a variety of exocrine glands and other secretory epithelia in the intestines and airways. The secretion of salt and water across epithelia of this type is driven by a secondary active Cl transport mechanism (Frizzell et al., 1979). Chloride enters secretory cells due to the combined activities of three basolateral membrane transport events: Na/K/Cl co-transporters, Na/K pumps, and K channels. Chloride leaves secretory cells across the apical membranes by diffusion, and alterations in apical Cl conductance represent a pivotal control point that determines Cl secretion rate. A variety of hormones and neurotransmitters stimulate salt secretion via their intracellular mediators, cAMP and Ca. The overall Cl secretory process is electrogenic so that the co-ion, Na, accompanies Cl to the lumen via paracellular pathways, driven by the lumen-negative voltage arising from Cl secretion.

CFTR targeting in epithelial cells.

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.

Protein Targeting and the Control Of C1− Secretion in Colonic Epithelial Cells

Human colonic epithelial cells can secrete chloride in response to cAMP-dependent stimulation following cellular polarization. This is correlated with the presence of 8 pS cAMP-activated Cl- channels within the apical membranes of these cells and their absence from the plasma membranes of unpolarized cells (Morris et al., 1992). The protein responsible for this cAMP-activated Cl-conductance is now recognized to be the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, Rommens et al., 1989; Bear et al., 1992). We have, therefore, postulated that the apical membrane expression of CFTR, and the ability of these cells to secrete Cl- in response to cAMP, is manifest when this protein targets to the apical membrane during epithelial cell differentiation (Morris et al., 1992).