PHYSIOLOGICAL REVIEWS   Vol. 79 No. 1 January 1999, pp. S175-S191
Copyright ©1999 by the American Physiological Society

Intracellular CFTR: Localization and Function

NEIL A. BRADBURY

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh Pennsylvania

I. INTRODUCTION
II. HISTORICAL PERSPECTIVE
III. SUBCELLULAR LOCALIZATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
    A. Immunologic Approaches to CFTR Localization
    B. Functional Approaches to CFTR Localization
    C. Recombinant Tagged CFTR Localization
IV. A ROLE FOR CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN VESICLE TRAFFICKING?
    A. CFTR and Exocytosis
    B. CFTR and Endocytosis
    C. Does CFTR Itself Undergo Regulated Trafficking?
    D. A Role for CFTR in Endosome Fusion
    E. How Does CFTR Regulate Membrane Vesicle Trafficking?
    F. Significance of CFTR and cAMP-Dependent Regulation of Membrane Traffic
V. DOES CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR FUNCTION AS AN INTRACELLULAR CHLORIDE CHANNEL?
    A. Determination of Vacuolar pH
    B. Mechanisms of Acidification
    C. Endosomal Chloride Channels and Their Regulation
    D. Defective Acidification in Cystic Fibrosis Cells?
    E. Consequences of Altered Trans-Golgi Network pH on Membrane Traffic
    F. Posttranslational Modifications, Trans-Golgi Network pH, and Cystic Fibrosis
    G. A Potential Pseudomonas Receptor
VI. DIRECTIONS FOR FURTHER STUDY
REFERENCES

	ABSTRACT

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Bradbury, Neil A. Intracellular CFTR: Localization and Function. Physiol. Rev. 79, Suppl.: S175-S191, 1999.  There is considerable evidence that CFTR can function as a chloride-selective anion channel. Moreover, this function has been localized to the apical membrane of chloride secretory epithelial cells. However, because cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein, it will also be present, to some degree, in a variety of other membrane compartments (including endoplasmic reticulum, Golgi stacks, endosomes, and lysosomes). An incomplete understanding of the molecular mechanisms by which alterations in an apical membrane chloride conductance could give rise to the various clinical manifestations of cystic fibrosis has prompted the suggestion that CFTR may also play a role in the normal function of certain intracellular compartments. A variety of intracellular functions have been attributed to CFTR, including regulation of membrane vesicle trafficking and fusion, acidification of organelles, and transport of small anions. This paper aims to review the evidence for localization of CFTR in intracellular organelles and the potential physiological consequences of that localization.

	I. INTRODUCTION

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The cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) in 1989 (116) heralded a new era in our ability to study the molecular pathophysiology of cystic fibrosis (CF), and an abundance of information on the genetics, tissue distribution of CFTR, and function of CFTR has been acquired subsequently. Studies of CFTR expression in a variety of cell types (51), including mammalian cells (4, 117), insect cells (77), and Xenopus oocytes (54) as well as functional reconstitution of CFTR protein in planar lipid bilayers (12) have provided clear evidence that CFTR can function as a cAMP-regulatable chloride-selective anion channel. Moreover, the tissue distribution of CFTR is consistent with its involvement in transepithelial fluid and electrolyte transport. However, the level of CFTR expression in various tissues has not necessarily correlated well with disease pathology. A clear correlation between CFTR genotype and clinical manifestations of the disease has remained elusive, and it is still not known how mutations in CFTR give rise to the variety of cellular abnormalities that have been associated with this disease, including changes in protein secretion (12, 92, 93), changes in the glycoproteins secreted (20, 36, 58), changes in bacterial adhesion to CF respiratory tissues (153), and a defective chloride conductance in CF cells (85, 112, 131).

Although it is conceivable that defective apical membrane chloride permeability could account for all of these observations, it is difficult to reconcile these many abnormalities with a single apical membrane chloride transport defect. Because of this enigma, roles for CFTR in addition to its function as an apical membrane chloride channel have been proposed. Thus it has been suggested that CFTR may function also as a chloride channel within intracellular organelles of the endosomal and biosynthetic pathways, potentially modifying their acidification (7, 9) as well as regulating the trafficking properties of intracellular organelles (26). Since the cloning of the gene for CFTR, many studies have been directed toward an understanding of the localization and possible function of intracellular CFTR. For example, roles for CFTR in modulating vesicle trafficking, organelle function, and Pseudomonas binding have been proposed (Fig. 1). This paper aims to review the evidence for localization of CFTR in intracellular organelles and the potential physiological consequences of that localization.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Proposed intracellular functions for cystic fibrosis transmembrane conductance regulator (CFTR). In addition to its role as a cAMP-regulated apical membrane chloride channel, CFTR may also function as an intracellular chloride channel regulating vacuolar acidification. Absence of CFTR in organelles of the biosynthetic pathway may cause alkalinization of these compartments, resulting in changes in sialylation and glycosylation of cell surface proteins. Under-sialylated GM1 may act as a receptor for Pseudomonas. CFTR may also serve to regulate vesicle trafficking of CFTR-containing vesicles, as well as mucin- and amylase-containing vesicles, in a cAMP-dependent manner.

	II. HISTORICAL PERSPECTIVE

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Reports of intracellular defects in CF cells appear throughout the CF literature, with many accounts being contradictory. This confusion may in part have arisen because of the pleiotropic nature of CF and because much past research overlooked the characterization of CF as a disease of epithelial tissues. Thus many contradictory reports of intracellular abnormalities in CF have focused on fibroblasts, platelets, lymphocytes, and erythrocytes. In addition, before the cloning of the gene, the differentiation between phenomenology and epiphenomenology is difficult to discern.

For example, altered mitochondrial calcium homeostasis was observed in fibroblasts from CF patients (57), but the intracellular calcium concentration and turnover was not altered in lymphocytes (148). Alterations in intermediary metabolism have also been reported in cells from CF patients (10, 79), including increased glycolytic activity. The observation that similar increases in glycolytic enzyme activities were also found in rats that were killed in a state of metabolic acidosis led to the suggestion that altered intracellular pH might play an important part in the pathophysiology of the disease (although such leads were never fully evaluated).

Alterations in cAMP-dependent processes have been recognized in CF for many years. Abnormal levels of cAMP in isoproterenol-stimulated fibroblasts (28), leukocytes (45), lymphocytes (44), and neutrophils (61) from CF patients were reported, with cAMP accumulation in isoproterenol-treated CF cells being two- to fivefold less than matched controls. Interestingly, cAMP accumulation in response to physiological agonists, PGE₁ or histamine, was normal. However, other workers reported no difference in isoproterenol-stimulated cAMP accumulation in CF fibroblasts compared with normal cells (84), and even reports of increased cAMP accumulation in isoproterenol-treated CF fibroblasts were made (120). A consensus regarding cAMP levels in CF tissue arose from studies on epithelial tissue. Reports from McPherson et al. (92), monitoring secretion from human submandibular cells, demonstrated that although the cAMP-dependent secretory response was diminished in CF tissue, cAMP accumulation was normal (92). Similarly, studies of -adrenergic responses in airway epithelial cells (22, 150, 152), sweat glands (128), and exocrine pancreas duct cells (130) showed that although cAMP-dependent changes in chloride transport were absent in CF tissues, cAMP accumulation was normal.

	III. SUBCELLULAR LOCALIZATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

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Because CFTR is an integral membrane protein, it will, of necessity, traffic through vesicular membrane compartments. Therefore, one should expect, at some level, to detect CFTR within compartments such as the endoplasmic reticulum, Golgi stacks, and membranes of the endosomal/lysosomal pathways. Three approaches have been utilized to answer the question of subcellular localization of CFTR: immunocytochemical studies (including immunoelectron microscopy), subcellular fractionation (followed by reconstitution of channel activity in planar lipid bilayers or immunoblot of membrane fractions), and recombinant strategies employing "tagged" CFTR.

Most CFTR localization studies so far performed have relied primarily on immunodetection of CFTR by cell or tissue staining. To this end, several laboratories have generated antibodies capable of recognizing CFTR with varying degrees of specificity and sensitivity (for a critical review of CFTR antibodies, see Ref. 39). Since the initial studies by Cheng et al. (37), on COS-7 cells overexpressing wild-type CFTR, showing a diffuse staining for CFTR along the plasma membrane, numerous studies have focused on immunocytochemical and immunogold localization of CFTR in both native and overexpression systems. Confocal microscopy data derived from human epithelial cell lines natively expressing CFTR also showed a diffuse staining of CFTR predominantly associated with the apical membrane (47). In these studies, no CFTR was seen at the basolateral membrane. Similarly, studies employing SF9, COS-1, and Vero cell overexpression systems also demonstrated immunofluorescent staining associated with the plasma membrane (42, 43, 77). A question that must be raised concerning the localization of any protein in an overexpression system is the extent to which that localization reflects localization in a natively expressing tissue. This is a critical issue, especially in light of the data surrounding potential intracellular functional consequences of CFTR overexpression, for example, the role of CFTR in vesicular acidification. When the relevance of CFTR localization in overexpression systems is addressed, it may not be sufficient to document solely the presence of CFTR in the organelle of interest, but also its presence or absence in organelles that do not normally contain CFTR. Of interest is the observation that HeLa and 3T3 cells (cells that do not normally express CFTR), which have been engineered to overexpress CFTR, exhibited a staining that was predominantly intracellular (47). This difference in CFTR localization between natively expressing and overexpressing cells is a potential cause for concern when deciding precisely where subcellular populations of CFTR may reside, since overexpression is frequently used to increase the signal of an otherwise low-abundance protein.

Immunofluorescence microscopy has been of importance in determining which cell types express CFTR and in which plasma membrane domain (apical vs. basolateral) CFTR resides. Several studies have also suggested subapical localization of CFTR (presumably in subapical vesicles) in epithelial cells and the perinuclear area of CFTR-transfected L cells (43). However, the resolution required to definitively demonstrate subcellular localization of CFTR necessitates the use of immunoelectron microscopy. Early studies in CFTR-expressing SF9 cells showed the presence of CFTR in the plasma membrane, although other intracellular membranes, mainly endoplasmic reticulum, also bound CFTR antibodies (77). Several studies have now documented the presence of CFTR within subapical membrane vesicles (43, 110, 149). For example, Puchelle et al. (110), employing immunogold labeling in conjunction with quick-freeze techniques, have located CFTR in vesicles present beneath the plasma membrane of human airway epithelial cells, as well as vesicles in the process of fusion/scission from the plasma membrane. Gold particles were also identified along microtubules near the ciliary basal bodies. Immunogold labeling of CFTR-expressing L cells has also demonstrated the presence of CFTR beneath the plasma membrane as well as the membranes of the rough endoplasmic reticulum. Studies by Webster et al. (149) on rat submandibular duct cells showed gold labeling along the apical plasma membrane as well as many of the subapical membrane vesicles. Indeed, estimations of gold particle density suggested that intracellular CFTR expression may exceed that of the plasma membrane under resting conditions. Although the precise nature of these subapical vesicles is not entirely understood, at least a portion of these vesicles is likely to be part of the endosomal/recycling pathway. Some of the CFTR-labeled vesicles also labeled with antibodies against the transferrin receptor and rab4, two markers of early endosomes and recycling vesicles. Localization of CFTR by immunoperoxidase staining of CFTR-transfected CFPAC cells revealed staining for CFTR along the plasma membrane as well as in multivesicular bodies and occasionally in the rough endoplasmic reticulum, nuclear envelope, and Golgi complex (156). Finally, in preliminary immunoconfocal studies by Lehrich et al. (84a), DFTR, the dogfish homolog of CFTR, was detected in small round vesicles beneath the apical surface of intact rectal gland cells. There was also some diffuse staining within the apical membrane.

In addition to light and electron microscopic immunolocalization, immunologic strategies to localize CFTR have also been performed by coimmunoprecipitation of CFTR with proteins whose subcellular localization has been previously defined. For example, this approach has been used by Yang et al. (156) to localize CFTR to the endoplasmic reticulum. In these studies, wild-type and F508 CFTR were expressed in CFPAC cells using a recombinant adenovirus. Immunoprecipitation of cell lysates using antibodies against the cytosolic molecular chaperone 70-kDa heat shock protein (HSP70) precipitated both HSP70 and band B of CFTR. Pulse-chase experiments revealed that although wild-type CFTR was transiently associated with HSP70, interactions of HSP70 with F508 (the most common mutation in the CFTR gene giving rise to severe disease symptoms) were quite stable. These observations led to the speculation that the failure of F508 CFTR to exit the endoplasmic reticulum was related to its retention in the endoplasmic reticulum by HSP70. In contrast to the cytosolic chaperone HSP70, the endoplasmic reticulum luminal chaperone Bip showed no interaction with CFTR. We have employed a similar approach to localize CFTR to endocytic clathrin-coated vesicles. In these studies, clathrin-coated vesicles from T84 colonic epithelial cells were gently solubilized, and immunoprecipitations were performed using anti-CFTR antibodies (25). Analysis of proteins coprecipitated with CFTR revealed the presence of -adaptin, a coat protein component of endocytic clathrin-coated vesicles.

In addition to light and electron microscopy methodologies to determine subcellular localization of CFTR in cells, several researchers have performed subcellular fractionation studies to characterize the location(s) of CFTR. Our own studies have involved the isolation and purification of clathrin-coated vesicles from the human epithelial cell line T84 (25). Immunoblotting and tryptic phosphopeptide mapping confirmed the presence of CFTR within these vesicles. Although our clathrin-coated vesicle preparation appears, by electron microscopic examination, to be >95% pure, it is possible that CFTR is present in a contaminating vesicle population. However, our ability to coimmunoprecipitate CFTR and -adaptin, after solubilization of clathrin-coated vesicle, suggests that CFTR is indeed targeted to endocytic clathrin-coated vesicles. Moreover, treatment of clathrin-coated vesicles with purified bovine brain uncoatase, followed by fusion of the resultant vesicles with planar lipid bilayers, results in the incorporation of a chloride-selective anion channel that is indistinguishable from CFTR identified in excised patch-clamp experiments. The observation that removal of clathrin is required before incorporation of CFTR chloride channels into the planar lipid bilayer can take place provides fairly strong evidence that CFTR is indeed a component of endocytic clathrin-coated vesicles. Thus we have been able to demonstrate both immunologically and functionally that CFTR is present within endocytic vesicles. The fact that we can detect fully functional CFTR within endocytic vesicles raises the question of whether CFTR does indeed play a functional role as a chloride channel within endosomes, a question addressed in section V.

Attempts to identify functionally CFTR within other intracellular membrane domains such as the endoplasmic reticulum have been performed by patch-clamp analysis. Pasyk and Foskett (103) addressed the question of CFTR localization and function in the endoplasmic reticulum by isolating a nuclear fraction from CFTR-expressing Chinese hamster ovary (CHO) cells (on the basis that the endoplasmic reticulum is contiguous with the nuclear membrane). Results from such studies suggested that CFTR-like channel activity could be detected in these membranes, indicating potential functional activity of CFTR along the biosynthetic pathway. A clear biophysical fingerprint of CFTR has emerged from the combined studies of several laboratories (see Ref. 69); however, beyond observing chloride-selective ion channel activity in the presence of protein kinase A and ATP, the studies by Pasyk and Foskett (103) did not provide definitive data that the ion channel they observed in the endoplasmic reticulum membranes was indeed CFTR.

Functional approaches to CFTR localization in endocytic vesicles have relied on the ability of chloride channels (for example, CFTR) to facilitate electroneutral proton transport across the limiting membranes of these vesicles (see sect. VB). Lukacs et al. (86) labeled endosomes of CHO cells transfected with wild-type CFTR, or mock transfected with viral vector alone, with the pH-sensitive fluorescent dye FITC-dextran. The CFTR-mediated endosomal chloride conductance was then measured as a function of the rate of dissipation of endosomal pH gradients after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In intact cells studied in suspension, activation of cAMP-dependent protein kinase [either by forskolin or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)] caused a twofold increase in the rate of pH gradient dissipation; in contrast, there was no such effect in mock-transfected cells. Similar results were obtained in vitro with a microsomal preparation obtained from CFTR- and mock-transfected CHO cells. Such results are consistent with the introduction of CFTR into an endosomal population. However, in these studies, there was no immunocytochemical evidence for the localization of CFTR to an endosomal population, nor were there any data to establish that the cAMP-dependent protein kinase alteration in pH gradient dissipation was indeed due to CFTR.

Similar studies were performed by Biwersi and Verkman (17) to determine whether functional CFTR was present in endosomes by using CFTR- and mock-transfected Swiss 3T3, as well as natively expressing T84 cells. Again, CFTR-mediated endosomal chloride conductance was measured as a function of the rate of dissipation of endosomal pH gradients following the addition of the protonophore CCCP. Pretreatment with forskolin resulted in a 1.5- to 2-fold increase in the rate of pH gradient dissipation in T84 and CFTR-transfected fibroblasts but had no significant effect on mock-transfected or F508 CFTR-transfected fibroblasts. Again, the data are consistent with the presence of functional cAMP-dependent CFTR chloride conductances within the endosomal membrane. However, no independent verification that cAMP was directly acting on CFTR chloride channels within the endosomal membrane was presented. Although the studies of Lukacs and co-workers (17, 86) described above clearly suggest that CFTR is present within endosomal membranes, immunologic evidence for the localization of CFTR within these structures would greatly enhance the significance of these findings.

In addition to localization of wild-type CFTR, a few studies have employed epitope tagging of CFTR as a means of detection. Preliminary studies by Moyer et al. (98) employing a CFTR-green fluorescent protein construct, expressed in the IB3-1 human airway epithelial cell line, localized CFTR to the endoplasmic reticulum, the Golgi apparatus, and the plasmalemmal region. Similarly, the M2 epitope tag has been inserted into the fourth extracellular loop of CFTR and expressed in HeLa and Madin-Darby canine kidney cells (72). Cell surface labeling of CFTR was detected using indirect immunofluorescence, and although intracellular localization of CFTR was not performed, the system may be amenable to confocal microscopic localization of subcellular CFTR.

CFTR is expressed in low abundance in many tissues, making localization of CFTR at the tissue level somewhat difficult, a difficulty which is only magnified when one attempts subcellular localization of CFTR in these same tissues. Although the specificity of some CFTR antibodies has been extensively characterized (40, 88), immunocytochemical studies on CFTR localization have frequently involved minimal characterization of antibodies. It is therefore prudent to examine individual antibody-dependent CFTR localization experiments in the context of other corroborating data. For example, our own studies have identified the presence of CFTR in clathrin-coated vesicles both immunologically (immunoblot, immunoprecipitation, and phosphopeptide map analysis) and functionally (incorporation of coated vesicle CFTR into planar lipid bilayers) (25). In retrospect, the observation that CFTR is present in rough endoplasmic reticulum, Golgi, and endosomal/recycling membranes is not unexpected. As mentioned before, CFTR is an integral membrane protein and therefore must traffic within a membrane environment. The questions that now need to be addressed include the rates at which CFTR traverses these various compartment and to what extent these rates are affected by the presence of secretagogues that impinge on the cAMP-mediated second messenger cascade.

	IV. A ROLE FOR CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN VESICLE TRAFFICKING?

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Studies addressing the role of CFTR in regulating vesicle trafficking have generated both positive and negative results. Such confusion in the literature may in part arise from differences in the model systems used and the markers employed to monitor regulated trafficking in these systems. Interestingly, the suggestion of a relation between the CF gene product and vesicle trafficking is not a new one. Over a decade before the cloning of the gene and the identification of CFTR, a connection between vesicle movement and the CF gene product was made. The basis for this association was the proposal that abnormalities in mucin secretion in CF airways may be the mechanistic basis for the chronic obstructive airway disease (18, 56, 58, 137). Because cAMP is known to stimulate both exocytosis (35, 46, 71, 90) and chloride secretion (52, 67) and because both of these processes are defective in CF tissue (26, 85, 92, 112, 131), the hypothesis was raised that in addition to direct activation of apical membrane resident chloride channels, regulation of chloride permeability could also occur through a cAMP-dependent recruitment of chloride channels in the apical membrane (Fig. 2). A growing number of transport proteins are now known to undergo regulated exocytic insertion into the plasma membrane upon stimulation of the cell, with subsequent endocytic retrieval following agonist removal (for review, see Ref. 24). Some membrane proteins (e.g., GLUT4, the insulin-responsive sodium/glucose cotransporter of adipocytes and myocytes) continuously cycle in the absence or presence of stimulus, but at different rates. Thus the observed movement of GLUT4 simply reflects a change in the steady-state distribution. Thus it is possible that CFTR may not only modulate the traffic of membrane vesicles on the exocytic and endocytic pathways, but may also regulate its own trafficking.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   CFTR regulates vesicle trafficking in a cAMP-dependent manner. In presence of agonists that activate cAMP-mediated second messenger cascade, CFTR effects an inhibition of endocytosis and an increase in exocytosis. Such cAMP-dependent regulation is missing in absence of CFTR.

In experiments by McPherson and co-workers (92, 93), trafficking of mucin and amylase vesicles along the exocytic pathway was monitored using freshly isolated tissue from control and CF submandibular salivary glands (24). Secretion of amylase was monitored enzymatically, and the release of glycoconjugates was monitored after the incorporation of a [¹⁴C]glucosamine precursor to label the carbohydrate moities. These studies indicated that although -adrenergic agonists were able to stimulate the release of mucin and amylase from control tissue, the secretory responses in CF tissues were attenuated to ~40% of that observed in control tissue. Control experiments showed that this was not because of an inability of CF tissue to synthesize mucins, since the total mucin pool of control and CF tissues was similar. Moreover, generation of cAMP was not impaired in these tissues, implicating an abnormality in the protein machinery of the exocytic pathway. Interestingly, the xanthine derivative IBMX (100 mM) was able to partially restore the secretory response in CF tissue. This did not appear to be because of an increase in the level of cAMP, since IBMX was unable to increase significantly cAMP levels above that observed with maximal isoproterenol stimulation.

More recently, similar observations were made using CFTR "knockout" mice (97). In these studies, release of [¹⁴C]glucosamine-labeled mucins from mouse submandibular glands was monitored in response to activation of the cAMP-mediated second messenger cascade. Again, although a large sustained release of mucin was observed in control mouse tissue, the stimulated release of mucin from CFTR knockout mouse tissue was reduced significantly. Defective cAMP-dependent regulation of exocytosis in CF tissue has been also observed in airway epithelial cells. Mergey et al. (95) studied the release of mucin after incorporation of [¹⁴C]glucosamine into complex carbohydrates. Stimulation of immortalized normal human airway epithelial cells, with either isoproterenol or forskolin, led to a 40% increase in mucin release. In contrast, stimulation of immortalized CF airway cells resulted only in a 1-3% increase in mucin release. The defective cAMP-mediated regulation of mucin secretion was restored after adenovirus-mediated gene transfer of wild-type CFTR to these cells. Stimulation of secretion by activation of protein kinase C with phorbol esters was similar in CF and normal cells.

Our own studies have shown a similar role for CFTR in mediating cAMP-dependent regulation of membrane vesicle recycling and exocytosis (26) in a pancreatic adenocarcinoma cell line (CFPAC) from a CF patient (130). This cell line is homozygous for the common F508 mutation and, consequently, produces very little plasma membrane CFTR. For these studies, CFPAC cells were stably transduced with wild-type CFTR (CFPAC-PLJ-CFTR) (53). This manipulation results in the expression of functional wild-type CFTR, as monitored by a variety of techniques including halide efflux. The plasma membrane of CFPAC and CFPAC-PLJ-CFTR cells were labeled with biotinylated wheat germ agglutinin (WGA), and following a period to allow the cells to internalize the marker and load recycling compartments, remaining cell surface biotin-WGA was blocked with excess unlabeled avidin. Binding of fluorescently labeled streptavidin to the plasma membrane monitored recycling back to the cell surface from intracellular compartments. Cells expressing wild-type CFTR showed increased recycling and exocytosis of internalized marker upon exposure to forskolin, with a 2.5- to 3-fold increase in cell surface recruitment. Forskolin had no effect on WGA-biotin recycling and exocytosis in parental CFPAC cells or mock transfected CFPAC-PLJ cells.

The regulation of recycling/exocytic vesicular trafficking has also been assessed in airway epithelial cells (132), in which recycling was monitored by release of previously internalized fluoresceinated dextran (FITC-dextran, a fluid-phase endocytosis marker) and by increases in membrane capacitance (monitored using whole cell patch clamp). Treatment of non-CF airway cells with CPT-cAMP resulted in a significant increase in membrane capacitance, consistent with net incorporation of membrane into the cell surface. Simultaneous release of FITC-dextran in response to CPT-cAMP suggested that the cAMP-dependent increase in plasma membrane capacitance was, at least in part, due to fusion and incorporation of exocytic vesicles into the plasma membrane. Under identical experimental conditions, CPT-cAMP had no effect on either plasma membrane capacitance or exocytosis of internalized marker in airway cells derived from a CF patient. Studies by Howard et al. (72) using an extracellular epitope-tagged CFTR demonstrated that cAMP increased the level of CFTR expression at the cell surface of transfected polarized MDCK cells, such that steady-state labeling of plasma membrane CFTR was increased two- to threefold upon stimulation by forskolin.

In contrast to the above studies showing a close correlation between wild-type CFTR expression and regulation of exocytosis in epithelial cells, studies on CFTR-dependent regulation of exocytosis in nonepithelial expression systems have yielded both positive and negative results. Peters et al. (105) injected mRNA for an extracellular epitope-tagged CFTR into Xenopus oocytes. Although little or no staining was observed at the cell surface under basal conditions, a combination of forskolin and IBMX caused a marked increase in the amount of tagged CFTR at the cell surface, consistent with an exocytic insertion of CFTR into the plasma membrane upon activation of the cAMP-mediated second messenger cascade. Studies by Takahashi et al. (136) using the same system were able to demonstrate a parallel increase in chloride conductance and membrane surface area (monitored by both morphometry and membrane capacitance) when oocytes expressing CFTR were stimulated with forskolin and IBMX. Studies aimed at investigating the role of CFTR on exocytosis in nonepithelial mammalian cells have not been able to demonstrate a correlation between CFTR expression and cAMP-dependent exocytosis. For example, Dho et al. (48) labeled the recycling compartment of parental CHO fibroblast cells, and CHO cells stably expressing wild-type CFTR, with biotinylated WGA. They observed no regulation of membrane recycling by cAMP in the presence or absence of CFTR. Using the same CHO cell lines, Hug et al. (74) monitored changes in membrane surface area in response to raised cAMP levels by alteration in membrane capacitance. Although overexpression of wild-type CFTR but not F508 CFTR in CHO cells induced a cAMP-activatable chloride conductance, this activation was not accompanied by an increase in membrane capacitance. Such negative data concerning the role of CFTR in exocytic activity in CHO cells may reflect a difference between polarized epithelial cells and nonpolarized fibroblasts. Indeed, in the various epithelial cell types that have so far been evaluated, all studies have reported a close correlation between the expression of wild-type CFTR and the regulation of exocytosis. The possible reasons for the difference between fibroblasts and epithelial cells are discussed in section IVE.

The precise molecular mechanisms by which CFTR may regulate exocytosis are not known. However, studies by Schweibert et al. (132) have investigated the role of heterotrimeric G proteins in chloride secretion. Stimulation of the heterotrimeric G protein G_i-2 inhibited both cAMP-activated chloride secretion and cAMP-stimulated exocytosis in cells endogenously expressing CFTR. In contrast, inhibition of G_i-2 stimulated exocytosis and allowed cAMP to increase chloride secretion. Although no description of CFTR movement was made, the results are consistent with a model describing CFTR insertion into the apical membrane to permit chloride exit. Because CFTR may undergo continual recycling between the plasma membrane and subapical vesicles, it is not possible to determine whether the observed effect of cAMP is on inhibiting CFTR retrieval from the plasma membrane or increased exocytic insertion of CFTR into the plasma membrane. Indeed, as is the case for GLUT4, it is possible that both events occur simultaneously.

We have investigated the regulation of endocytosis by the cAMP-mediated second messenger cascade in CFTR expressing epithelial cells using both fluid- and adsorptive-phase markers, in combination with enzymatic and optical detection systems (27). Using these approaches, we observed that forskolin-stimulated increases in cAMP resulted in a significant inhibition (~60%) of endocytic activity in the human colonic epithelial cell line T84. Dideoxyforskolin, a forskolin analog which exhibits many of the non-cAMP-mediated effects of forskolin but does not stimulate adenylate cyclase, was without effect in inhibiting endocytosis (confirming that the inhibitory effect of forskolin on endocytosis was a cAMP-mediated effect). Studies from other laboratories on T84 cells and the human tracheal cell line 9HTEo have also demonstrated a cAMP dependence to inhibition of endocytosis (108, 127). Interestingly, activation of protein kinase C using phorbol esters was unable to inhibit endocytosis, nor did it modulate forskolin-inhibited endocytosis (23).

The CFPAC/CFPAC-PLJ-CFTR expression system has also been used to monitor the role of CFTR in endocytosis. Similar to our studies with T84 cells, we monitored endocytosis enzymatically by uptake of horseradish peroxidase (HRP) and optically by the uptake of FITC-dextran. In either parental CFPAC cells or the mock-transfected CFPAC-PLJ cells, cAMP had no effect on endocytosis (26). However, in cells expressing wild-type CFTR (CFPAC-PLJ-CFTR), cAMP was able to inhibit endocytosis to a similar extent to that observed in T84 cells. Using this same system, Dunn et al. (55) investigated the role of CFTR in regulating internalization of the transferrin receptor (a marker for basolateral endocytosis, Refs. 59, 106). Forskolin had no effect on transferrin receptor internalization in either CFPAC or CFPAC-PLJ-CFTR cells. Although colocalization of CFTR and transferrin receptors was not performed in these studies, immunogold electron micrographs of ductal epithelia from rat submandibular glands have suggested the CFTR and transferrin receptors can colocalize in vesicular compartments within the cell (149). However, since transferrin receptors are localized to the basolateral membrane, and CFTR to the apical membrane (but see also Refs. 113, 114), it is hard to envisage how CFTR could directly modulate the rate of transferrin receptor endocytosis in the opposite membrane, even though they may colocalize postendocytically in a sorting compartment which may contain material from both apical and basal membranes.

Although very little CFTR is predicted to be extracellular, CFTR is glycosylated on its fourth predicted extracellular loop, allowing the traffic of CFTR to be monitored through chemical modification of its carbohydrate residues. Using a two-step biotinylation procedure to label cell surface glycoconjugates, Prince et al. (108) determined the ratio of plasma membrane to intracellular CFTR in T84 cells. Immunoprecipitation, in vitro phosphorylation, and autoradiography subsequently quantitated biotinylated and nonbiotinylated CFTR. Assuming 100% efficiency in biotinylating cell surface CFTR, these authors concluded that ~50% of mature CFTR was on the cell surface and 50% was intracellular, inaccessible to biotinylation. Moreover, CFTR is rapidly internalized from the cell surface at 37°C (87, 108). This internalization rate is very similar to that of the low-density lipoprotein and transferrin receptor, both of which are internalized through clathrin-coated vesicles. Indeed, it is known that CFTR is internalized by entering clathrin-coated vesicles (25, 87). Prince et al. (109) also used the cell surface labeling approach to investigate the role of cAMP in modulating endocytic activity. Elevation of cAMP led to a decrease in the rate of CFTR internalization from the cell surface. Moreover, the degree to which CFTR internalization was inhibited in the presence of cAMP was similar to the cAMP-dependent inhibition of fluid-phase endocytosis described above. The G551D mutant of CFTR, unlike F508 CFTR, escapes the endoplasmic reticulum to become fully glycosylated and enter the plasma membrane, but has little functional activity (although it is not a "dead" channel, Refs. 13, 60, 155). Internalization of G551D CFTR appears to proceed at a rate similar to that of wild-type CFTR in unstimulated cells. However, cAMP did not decrease the rate of G551D CFTR internalization, suggesting that chloride channel activity may in some way be involved in CFTR-dependent regulation of membrane trafficking.

Attempts to directly correlate CFTR's function as a chloride channel with its ability to stimulate exocytic insertion and/or inhibit endocytic retrieval of CFTR containing vesicles from the plasma membrane have been performed by several groups. The shark rectal gland has been used as a model of chloride secretion across epithelial tissues, utilizing DFTR (the shark homolog of CFTR) (89). Qi et al. (111) performed transepithelial impedance and capacitance measurements in conjunction with transepithelial short-circuit measurements on primary cultures of shark rectal gland. In the presence of IBMX, both membrane capacitance and chloride current increased in parallel in a dose-dependent manner, consistent with a model of exocytic insertion of CFTR upon activation of the cAMP-mediated second messenger cascade. Correlation of membrane surface area and chloride conductance has been monitored in Xenopus oocytes injected with CFTR mRNA (136). Chloride conductance (monitored by 2-electrode voltage clamp) and membrane surface area (as monitored by membrane capacitance) increased in parallel when Xenopus oocytes expressing CFTR were stimulated with forskolin and IBMX. The cAMP-induced increases in membrane surface area were confirmed by morphometric analysis of transmission electron micrographs. Measurement of cell surface CFTR under identical conditions (using an extracellular epitope-tagged CFTR construct) showed an increase in cell surface CFTR levels upon increasing cellular cAMP (105). It must also be kept in mind that these studies were undertaken in a heterologous expression system, and therefore, care must be taken in extrapolating data to the function of CFTR in natively expressing tissues. However, other studies have provided evidence of the usefulness of the oocyte system in studying acutely regulated insertion of membrane transport proteins. For example, the GABA transporter GAT-1 relocates to the surface membrane of oocytes after activation of protein kinase C (41). Hug et al. (74), monitoring membrane capacitance and whole cell chloride conductance in parental and CFTR overexpressing CHO cells, were, however, unable to document increases in membrane capacitance, although CFTR expressing CHO cells did acquire a cAMP-activatable chloride conductance.

Studies have recently been initiated toward an understanding of the role of CFTR in endosome fusion. Using a pulse-chase approach, Biwersi et al. (16) labeled endosomes from CFTR expressing and control (nonexpressing) Swiss 3T3 fibroblasts, first with Bodipy-avidin, and then following a wash/chase period, with biotin-albumin or biotin transferrin. Endosome fusion was then monitored by an increase in Bodipy fluorescence upon binding of avidin to biotin. Fusion of endosomes in unstimulated CFTR-expressing cells was not different from that in control cells. However, in CFTR-expressing cells exposed to forskolin, fusion rates were increased. Endosome fusion was not stimulated by forskolin in chloride-depleted CFTR-expressing cells. The authors thus hypothesized that CFTR provides a cAMP-dependent chloride conductive pathway in endosomal vesicles. However, no evidence was presented that chloride transport across the endosomal membrane was involved in increasing endosome fusion, only that cellular chloride was required. It is also possible that CFTR may modulate the activity of an endosomal resident protein in a cAMP-dependent manner, effecting cAMP-dependent regulation of fusion. This is an interesting possibility given the suggestion that CFTR may directly regulate epithelial sodium channels (83). Activation of plasma membrane CFTR may lead to alterations in cytosolic chloride and/or sodium concentrations, which may also result in changes in endosome fusion kinetics. This also is an intriguing possibility, since it has been shown that cytosolic chloride is important for the activation of G proteins that may be involved in endosome fusion (100).

The choice of an appropriate model system is a critical one when investigating the role of cAMP-dependent regulation of membrane trafficking and, indeed, the cAMP-dependent trafficking of CFTR itself. Thus it is quite possible to observe trafficking of CFTR in one model system and not in another. Such model-dependent trafficking has been observed for other integral membrane transport proteins. For example, GLUT4, normally expressed in adipocytes and myocytes, traffics between intracellular stores and the plasma membrane in response to insulin. However, although heterologous expression of GLUT4 in 3T3 fibroblasts and Hep G2 hepatoma cells results in intracellular GLUT4 localization similar to that seen in resting adipocytes (68, 73), no movement to the plasma membrane upon stimulation is detected (68). These observations supported the notion that GLUT4 could target to an appropriate intracellular location in a variety of cell types but that the machinery that allows GLUT4 to traffic was only expressed in tissues that natively express GLUT4.

Although it is evident that further study into the functional role of CFTR in controlling vesicular movement is required, a consensus does appear to have been reached. In general, the majority of studies performed using epithelial cell model systems have supported the notion that CFTR can regulate endocytosis, exocytosis, and membrane recycling in a cAMP-dependent manner. Moreover, there is accumulating evidence that CFTR continually recycles between the plasma membrane and intracellular vesicles in epithelial cells. In contrast, studies that have addressed these same issues in nonepithelial cells have not been able to assign a role for CFTR in these processes (Xenopus oocytes being an exception).

Given the accumulation of thick viscous mucus in the airway of CF patients, it is perhaps surprising to find that CF tissues actually have a decreased rate of stimulated mucin release compared with non-CF tissue (92, 93). However, these two observations are easily reconciled by the finding that there is extensive hyperplasia of submucosal glands (14). The accumulation and stagnation of mucus in the airways of CF patients may also reflect an inability of the airways to adequately flush the secretions from the underlying secretory glands, due to impaired fluid movement. Airway epithelial cells also secrete a host of other proteins (including defensins, superoxide dismutase, ₁-antitypsin, and surfactant protein A) that act as anti-inflammatory or antibacterial agents. Regulated secretion of these molecules could play a vital role in the protective response of the airway milieu to environmental assault from particulate and microbial agents. Clearly, a defect in the ability of airways to respond by upregulating traffic of these molecules could contribute to or exacerbate any inflammatory state of the airway, making an individual susceptible to bacterial colonization.

	V. DOES CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR FUNCTION AS AN INTRACELLULAR CHLORIDE CHANNEL?

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The role of CFTR in regulating and maintaining an acidic pH within various intracellular organelles (the various compartments of the Golgi apparatus and endosomes for example) has been somewhat controversial. This may, in part, reflect our poor understanding of the role of intracellular chloride channels. Although the function of chloride channels at the plasma membrane has been extensively studied, such attention has not been directed toward the transport of chloride across intracellular membranes. Before discussing the specific role of CFTR in mediating the transport of chloride across intracellular membranes, it is perhaps worthwhile to discuss the general role of chloride fluxes across these membranes and, specifically, the role of chloride ions in modulating the pH of intracellular organelles. It has long been recognized that living cells have developed remarkably sophisticated mechanisms for regulating the pH within the cytoplasm and inside organelles. The acidification of intracellular compartments, and possible abnormalities of this process in CF, is currently an active area of research. However, it is interesting to note that insightful studies on acidification of intracellular organelles were performed over 100 years ago by Metchnikoff (96). In a series of elegant experiments on phagocytic cells, Metchnikoff (96) observed that in the process of intracellular digestion, protozoa "secrete around the object they have englobed an amount of acid sufficient to convert the colour of litmus from blue to red." For a more detailed discussion of the proteins and mechanisms involved in organelle acidification, the reader is directed to several excellent review articles (2, 94, 124, 143).

Several strategies to identify acidic compartments and to measure acidification in vitro and in vivo have been employed, including biochemical, functional, and morphological approaches. The organelles that are the best understood are those for which more than one line of evidence is available and include lysosomes, endosomes, certain secretory granules, and some compartments of the Golgi.

Several methodological approaches have been employed to study vacuolar acidification, including lipophilic weak bases [such as acridine orange and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP)] (5, 94, 118, 122) and fluoresceinated dextrans (91, 102, 141). Using such techniques, investigators from several laboratories have estimated the internal pH of intracellular vesicles. Thus, in intact cells, the intralysosomal environment is maintained at a pH of 4.6-5.0 (65, 102, 141). Endosomes maintain an internal pH of between 5.0 and 6.0 (64, 141), although the precise pH monitored by an internalized probe (e.g., FITC-dextran) may vary as probes appear to be transferred from less acidic to progressively more acidic endosomes as a function of time after internalization. It should also be noted that endosomes from different cell types can also display different internal acidities. Thus single-cell fluorescence measurements of endosomal pH in CHO cells failed to detect endosomal compartments of pH <6.5 (154).

Proton pumps (H⁺-ATPases) have been shown to be important in vesicular acidification (Fig. 3); however, they not only generate a pH across the organelle membrane as they acidify the lumen, they also generate a membrane potential () due to the electrogenic nature of the proton transport. If the proton permeability of the subcellular membrane compartment is low, the ATPase will generate a membrane potential large enough to reach its own reversal potential and thereby inhibit any subsequent proton transport and any further generation of pH (1). The presence of a parallel chloride conductance within the membrane of many intracellular organelles allows for counterion transport, helping to collapse the membrane potential and facilitating further intraorganelle acidification. That such chloride conductances are important in vesicle acidification is exemplified by the essential role of chloride transport in the generation of a pH in chromaffin granules (76), endosomes (6, 115, 144, 145), lysosomes (70, 129), Golgi vesicles (66), clathrin-coated vesicles (99, 133), thyroid parafollicular cell granules (8), neurohypophysial granules (123), zymogen granules (63), and platelet granules (34).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Vacuolar proton pumps generate both a membrane potential () and a pH gradient. In absence of a counter conductance, interior-positive potential inhibits inward proton movement and hence acidification (left). An inward chloride conductance, such as CFTR, can dissipate gradient, allowing acidification (right).

Although the role of chloride conductances, especially the role of the CFTR chloride channel, in mediating electroneutral acidification has received attention, it should be kept in mind that there are likely mechanisms in addition to chloride conductances to permit electroneutral acidification. Some lysosomes and endosomes have potassium conductances that can support acidification by allowing potassium exit from the vesicle in a charge compensatory manner (62, 70). Studies in Swiss 3T3 cells (29, 94) but not in early rat liver endosomes (3) suggest a role for electrogenic pumps such as the Na⁺-K⁺-ATPase in modulating vesicle pH by generating a opposing that generated by the proton pump.

The observation that chloride conductances can dramatically affect vesicle acidification rates suggests that chloride channels potentially provide a means of regulating vesicle pH. Although second messengers and protein kinases have been shown to modulate chloride conductances and/or acidification in some intracellular organelles, there appears to be considerable tissue and organelle specificity to the response. Bae and Verkman (6) and Reenstra et al. (115) demonstrated that chloride conductive pathways in kidney endosomes (and hence endosomal acidification) could be controlled by treatment with protein kinase A. Exposure to phosphatases decreased the ability of endosomes to acidify. Moreover, studies using chloride channels from bovine brain clathrin-coated vesicles reconstituted into liposomes (99) showed enhanced ³⁶Cl uptake upon exposure to protein kinase A. In addition, phosphorylation of stripped clathrin-coated vesicles resulted in increased acridine orange accumulation, whereas phosphatase treatment resulted in a decrease in ATP-dependent acidification. In addition to changing chloride conductance across vesicle membranes, cAMP and/or protein kinase A may have other effects including modulating the proton pump and other cation conductances, or maturation of endosomes (144, 146). Finally, a nucleotide-activated chloride channel has been identified in rat liver lysosomal membranes, although it is not known if changes in gating of this channel alter lysosomal acidification (140).

The most common mutation in CFTR, giving rise to severe clinical symptoms, results from the loss of a phenylalanine residue in the first nucleotide binding fold (F508). In contrast to wild-type CFTR which displays a "mature" glycosylation pattern consistent with passage through the Golgi apparatus, F508 CFTR does not appear to acquire heavy glycosylation but rather accumulates within the endoplasmic reticulum where it is degraded (37). Indeed, there is now convincing evidence that lack of an apical CFTR-mediated chloride conductance is a hallmark of CF. If, indeed, CFTR chloride channels also contribute to the electroneutral acidification of intracellular organelles, then in CF the loss of such a conductance would affect and hence pH. This hypothesis was investigated by Barasch et al. (9), who suggested that indeed such a defect in acidification of intracellular organelles was present in CF cells expressing F508 CFTR. Freshly isolated nasal polyps from CF patients, as well as immortalized respiratory epithelial cells, were incubated with DAMP, fixed, and the DAMP visualized by electron immunocytochemistry. Accumulation of DAMP was then monitored as an assessment of vesicular pH. Results from such studies indicated that although DAMP accumulation was not significantly different between CF and non-CF cells in the nucleus, cis-Golgi, and lysosomes, significant differences were observed for the trans-Golgi and prelysosomal compartments. Semiquantitative correlation of DAMP accumulation with pH suggested that trans-Golgi compartments in CF cells were 0.5 pH units more alkaline than those in non-CF cells. In addition, Barasch et al. (9) monitored membrane potential (indirectly as a function of pH-dependent acridine orange accumulation) in a light microsomal fraction from CF and non-CF epithelial cells. When valinomycin, an ionophore that is expected to collapse any membrane potential () was added, the rate of acidification of CF light microsomes was significantly greater than that observed in non-CF microsomes. This indicated that acidification (proton pumping) was limited in CF cells by a high , which would form in the absence of a counterion conductance. Barasch and Al-Awqati (7) extended these studies to the CFTR-transfected and parental CFPAC pancreatic cell lines, again demonstrating lower intravesicular acidification in CFPAC cells expressing F508 CFTR compared with the wild-type CFTR transfected cells. Transfection of wild-type CFTR into cells that do not normally express CFTR does not alter acidification rates or steady-state intracellular pH of endosomes or lysosomes (17, 86, 119, 147).

Part of the confusion surrounding acidification defects in CF cells arises from the use of different cell lines and the assessment of pH in different organelles. Thus Barasch and Al-Awqati (7) have argued that they have been monitoring the role of CFTR in acidifying a biosynthetic compartment, since the defect was observed in sialyl transferase-positive vesicles (trans-Golgi network) and in mannose-6-phosphate-positive vesicles (Golgi and prelysosomes). Lysosomes from CF and non-CF tissues acidify equally well. In contrast, Lukacs et al. (86) studied the role of CFTR and cAMP on intravesicular pH in CFTR-transfected CHO cells. Monitoring intravesicular pH through internalization of FITC-dextran into acidic compartments, Lukacs et al. (86) were unable to document any sensitivity of acidification to cAMP. Fluorescein isothiocyanate-dextran is initially internalized into early endosomes and then directed to lysosomes compartments that do not mix with mannose-6-phosphate-positive vesicles (134); thus the defect in acidification in CF cells appears to be confined to specific biosynthetic compartments. It is also important to distinguish between the role of CFTR in its native environment, i.e., a polarized epithelial cell, and in an expression system. Clearly much information has been gleaned on the biophysics of CFTR using various expression systems, and indeed, it is likely that the kinetics of CFTR channels will be context insensitive. However, for functions of CFTR such as trafficking and endosomal or trans-Golgi network acidification, such functions are very likely to be context specific. Transfection and expression experiments only allow one to ask whether CFTR can perturb the normal intravesicular pH in these cells. This will only be true if a chloride conductance is rate limiting in the acidification of these organelles.

Observations by Yilla et al. (157) indicated a close relation between vesicle acidification, sialylation of secreted proteins, and the rate of protein secretion. Addition of concanamycin (a specific inhibitor of the vacuolar H⁺-ATPase) to Hep G2 cells caused a reduction in ₁-antitrypsin sialylation and a reduction in the rate of constitutive secretion. Studies by Jilling and Kirk (75) using the human colonic cell lines T84 and HT-29 demonstrated that cAMP stimulated sialylation of ₁-antitrypsin and an increase in apical, but not basolateral, secretion of ₁-antitrypsin. These observations led these authors to conclude that the regulation of protein sialylation and apical secretion have a common origin, which they postulated was acidification of the trans-Golgi network. Support for such a postulate comes from the work of Zeuzem et al. (158), which demonstrated that the binding of ADP-ribosylation factor (ARF) to microsomal membranes was enhanced by vacuolar acidification. Moreover, such pH-dependent binding of ARF was also chloride dependent, since ARF binding was inhibited by removal of chloride or the addition of chloride channel blockers. If CFTR regulates trans-Golgi network acidification in a cAMP-dependent manner, it is conceivable that this could lead to a CFTR and cAMP-dependent association of coat proteins to the trans-Golgi network and the regulation of secretory vesicle formation from the trans-Golgi network. This could, in part, explain why cAMP fails to stimulate protein secretion from CF tissues.

Alterations in mucins from the gastrointestinal (38, 50, 151) and respiratory tracts (19, 20, 58, 80) of CF patients have been reported for many years. A phenotype found by many, though not all (101), investigators is an increase in sulfation and fucosylation with a decrease in sialylation of mucins and other glycoproteins secreted by CF cells (9, 121).

Enzymes involved in sialylation, sulfation, and glycosylation of glycoproteins and mucins have been identified in the trans-Golgi system, the only region of the organelle which is acidic (15, 30, 31, 135). Terminal glycosylation reactions appear to be competitive for substrate; thus desialylated thyroglobulin is a good substrate for sulfation, whereas sialylated thyroglobulin is a very poor substrate for sulfation (78). Similarly, sialylation and fucosylation also appear to be mutually exclusive. Thus desialylated transferrin can either be resialylated by -galactoside 2,6-sialyltransferase, or fucosylated by N-acetylglucosamine 1,6,3-fucosyltransferase, the reactions being mutually exclusive (104). Because fucosylation, sialylation, and sulfation are mutually exclusive events, perturbations in the functions of any of the enzymes responsible for posttranslational modifications could lead to alterations in the oligosaccharide conformation. Thus a decrease in the activity of -galactoside 2,6-sialyltransferase would be expected to lead to an undersialylation of oligosaccharide moieties and a concomitant increase in either sulfation or fucosylation, a condition reported for mucins from patients with CF (9, 121). Such differences in enzyme activity may arise as a result of the different pH optima of each of the transferase enzymes. Thus sialyltransferases have an acid pH optima (especially mucin sialyltransferase, pH 5.8) (11, 32). In contrast, sulfotransferases (pH optima 6.8-7) (33, 78) and fucosyltransferases (pH optima 7-8.5) (21, 107) generally have a pH optimum centered around 7.0. If a defect in Golgi acidification arises because of the absence of CFTR, then, as proposed by Barasch and co-workers (7, 9), this could be the mechanistic basis for decreased sialylation and increased fucosylation and sulfation of mucous glycoproteins in CF.

Part of the attractiveness of the defective acidification hypothesis is that it provides a unifying model for the way in which abnormalities in a chloride channel could give rise to the pathology observed in the CF airway. Chronic bacterial colonization of the conducting airway, particularly by Pseudomonas, is an almost universal condition in CF patients. Such extensive colonization appears to require adherence of the pathogen to a cell surface receptor. Thus there is a sevenfold increase in Pseudomonas (rough form) binding to buccal cells of CF patients compared with control and a twofold increased binding of mucoid forms compared with control (153). A mechanism for this binding was initially reported by Krivan et al. (81), who demonstrated that Pseudomonas isolated from CF patients bound avidly to the cell surface glycosphingolipids asialo-GM1 and asialo-GM2, but not to sialyated GM1 and GM2, suggesting that asialated GM1 and GM2 were receptors for Pseudomonas. Similarly, another common infectious agent observed in the airways of CF patients, Haemophilus influenzae, was also found to bind asialo-GM2 but not sialo-GM2 (82). An extensive amount of literature documents the importance of pili as a virulence factor for many gram-negative bacteria, including Pseudomonas (125). Indeed, asialo-GM1 but not sialated GM1 has been shown to be a receptor for Pseudomonas pilin (126, 138), a major adhesin in this organism. Moreover, exogenous asialo-GM1 was found to inhibit competitively Pseudomonas adherence to epithelial cells.

However, it should be pointed out that other organisms that also bind to asialo-GM1, including Streptococcus pneumoniae and Klebsiella pneumonia, are not clinically important in CF (82). In addition, the precise extent to which cell surface glycosphingolipids are undersialated in CF cells compared with control cells is still under investigation. Flow cytometric studies on primary airway epithelial cells from two patients with CF and three normal controls revealed the presence of superficial asialo-GM1 on 12% of primary CF cells, compared with 2.9% on normal cells (126). Interestingly, neuraminidases secreted by Pseudomonas were found to be able to increase the level of asialo-GM1 on CF cells, but not normal cells. Recently, it has been shown that once bound to the cell surface (by whatever mechanism) Pseudomonas elicits an immune response, with CF cells secreting higher levels of interleukin-8 and other cytokines than control cells (49, 138), resulting in an abnormal inflammatory response. In addition, interleukin-2, an important T-cell growth activator, is inactivated by elastase and alkaline protease (139), which are both secretory products of Pseudomonas aeroginosa. It is clear that further studies are warranted to define the precise difference in asialo-GM1 expression between normal and CF airway epithelial cells, as well as to determine what contributions these differences make to airway colonization.

	VI. DIRECTIONS FOR FURTHER STUDY

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Although many of the observations that link CFTR to vesicle trafficking and vaculoar acidification are intriguing, the molecular mechanisms underlying the causal relations between CFTR genotype and cellular dysfunction remain elusive. Clearly, more studies are required to provide definitive evidence for any abnormalities in these processes being associated with clinical symptoms. It is important to ascertain which cell models should be used for these studies since, as described above, there can be great variation in measured parameters between different cell lines. Clearly, expression systems are much more amenable to experimental manipulation, but without a deeper understanding of the molecular mechanisms involved in vesicle trafficking and vacuolar acidification, it is possible that epithelial cell specific components of these processes may be absent in other cells. Moreover, it is important to place the role of intracellular CFTR function within the context not only of an individual cell, but also within an intact epithelium. Thus it will be necessary to use an integrative approach to study the role of intracellular CFTR at a tissue and organ level, and not rely entirely on the reductionist approaches the have dominated the study of CF in recent years. Furthermore, it is important to understand precisely how CFTR functions in the intracellular environment, since it may not be necessary to correct all of the cellular defects associated with CFTR to intervene therapeutically in individuals affected by CF.

	FOOTNOTES

   I express my appreciation to Drs. C. Widnell, R. A. Frizzell, and R. J. Bridges for their critical review of this manuscript.

   Work in the author's lab is funded by the National Institutes of Health and the Cystic Fibrosis Foundation.

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