News Physiol Sci 15: 57-61, 2000;
1548-9213/00 $5.00
News in Physiological Sciences, Vol. 15, No. 2, 57-61,
April 2000
© 2000 Int. Union Physiol. Sci./Am. Physiol. Soc.
CFTR Chloride Channels: Binding Partners and Regulatory Networks
Anjaparavanda P. Naren and
Kevin L. Kirk
A. P. Naren and K. L. Kirk are in the Gregory Fleming James Cystic Fibrosis Research Center and the Department of Physiology and Biophysics at the University of Alabama at Birmingham, Birmingham, Alabama 35294. K. L. Kirk is also in the Department of Neurobiology.
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Abstract
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The cystic fibrosis gene encodes a chloride channel (CFTR) that regulates transepithelial salt and water transport. Two classes of CFTR-binding proteins appear to link the opposing cytoplasmic tails of this channel to distinct regulatory networks. Such interactions may constitute new paradigms for modulating CFTR activity in health and disease.
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Introduction
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is encoded by the gene that is defective in cystic fibrosis. CFTR is predominately localized to the lumen-facing (apical) membranes of epithelial cells, in which it participates in the regulated transport of salt and water across epithelial tissues. The biophysical properties of this ion channel, and the effects of the many disease-associated mutations on these properties, have been studied extensively by many laboratories (see Ref. 4 for an excellent review). Much less is known about the identities of those proteins that physically and functionally interact with CFTR to modulate the transport activity of this chloride channel in epithelial cells. The purposes of this article are to briefly review recent data that indicate (perhaps not surprisingly) that such CFTR-binding partners do exist and to speculate about the functional relevance of these interactions.
The CFTR channel is a symmetrical, polytopic protein with two membrane-spanning domains (MSDs), two cytoplasmically oriented tails, two cytosolic nucleotide-binding domains (NBDs), and a central regulatory domain (R domain) with multiple phosphorylation sites (Fig. 1
). Phosphorylation of the R domain (principally by protein kinase A) is a prerequisite for channel activation. The gating of the phosphorylated channel appears to be additionally controlled by a cycle of ATP binding and hydrolysis at the NBDs (4). To what extent the cytoplasmic tails contribute to CFTR function is poorly understood, although recent evidence indicates that these tails may possess signals that mediate channel internalization from the cell surface (11). The subjects of this review are two classes of proteins that physically interact with the opposing tails of this chloride channel: 1) syntaxin 1A, a membrane protein that inhibits CFTR-mediated currents at least in part by binding to the amino-terminal tail and 2) PDZ (for PSD-95, discs large, ZO-1) domain-containing proteins that bind to the carboxy-terminal tail of this ion channel. These interactions imply that the two cytoplasmic tails of CFTR couple this ion channel to distinct regulatory networks.
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FIGURE 1. Schematic illustration of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with syntaxin 1A binding at amino-terminal tail and PDZ (for PSD-95, discs large, ZO-1) domain-mediated interactions at carboxy-terminal tail. NBD, nucleotide-binding domain; H1, H2, and H3, helical domains 1, 2, and 3; R, regulatory domain.
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Syntaxin interactions at the amino-terminal tail of CFTR
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Syntaxin 1A was originally identified in brain, in which it is an essential component of the protein complex that mediates the fusion of the synaptic vesicle with the presynaptic plasma membrane (reviewed in Ref. 1). This integral membrane protein is also expressed in gut epithelial cells, albeit at much lower levels than in brain (9). The coexpression of syntaxin 1A with CFTR in heterologous expression systems such as Xenopus oocytes leads to an inhibition of CFTR-mediated currents (Fig. 2). Moreover, CFTR can be rescued from this inhibition by multiple reagents, including: 1) Munc-18a, a high-affinity syntaxin-binding protein (6) that is also expressed in gut epithelial cells (9); 2) soluble syntaxin 1A peptides (Fig. 2C); and 3) botulinum neurotoxin C1, an endoprotease that degrades membrane-anchored syntaxin 1A. Importantly, each of these reagents also markedly stimulates CFTR-mediated chloride currents in various colonic epithelial cell lines that normally express CFTR and syntaxin 1A (e.g., see Fig. 3). Thus syntaxin 1A tonically inhibits CFTR activity in tissue culture models of gut epithelial cells.
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FIGURE 3. CFTR-mediated currents in T84 colonic epithelial cells are potentiated by the inclusion of recombinant syntaxin 1A cytoplasmic domain in whole cell patch pipette. Representative whole cell current traces at varying holding potentials and current-voltage curves for 2 T84 cells are shown, one stimulated at t = 0 with 0.5 mM 8-(4-chlorophenylthio) cAMP (CPT-cAMP) alone (A) and one stimulated with CPT-cAMP plus syntaxin 1A (Syn 1A) peptide in pipette (B). Reprinted by permission from Nature 390: 302–305, copyright 1997, MacMillan Magazines, Ltd.
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The negative modulation of CFTR currents by syntaxin 1A is at least in part mediated by a direct physical interaction between these membrane proteins (10). It would seem plausible that syntaxin 1A could remotely modulate CFTR function by regulating membrane fusion events that subsequently influence the numbers of CFTR channels at the cell surface, given its participation in vesicle fusion reactions at the synapse (1). However, the interaction between these proteins is more direct. In particular, syntaxin 1A (but no other syntaxin isoform that was tested) binds to the amino-terminal tail of CFTR with a 1:1 stoichiometry (10). This binding interaction is inhibited by Munc-18a, which itself is unable to bind to CFTR. Furthermore, the functional interaction between CFTR and syntaxin 1A is eliminated either by deleting the amino-terminal tail from CFTR or by blocking this interaction with recombinant amino-terminal tail peptide. The direct interaction between CFTR and syntaxin 1A raises the interesting possibility that syntaxin 1A could modulate the channel-gating properties of CFTR, perhaps in addition to any effects on protein traffic that it may have.
What is the physiological or pathophysiological relevance of this protein-protein interaction in epithelial cells? This interaction could fine tune CFTR activity in response to signals that up- or downregulate its binding to syntaxin 1A (e.g., Munc-18 or kinases that modulate the CFTR-syntaxin 1A interaction). In addition, if CFTR activity in epithelial cells is normally limited by its interaction with syntaxin 1A, then reagents that block this interaction may be useful for augmenting the activities of partial-loss-of-function CFTR mutants that cause cystic fibrosis. Indeed, the functional activity of the most common cystic fibrosis-associated mutant (
F508) could be stimulated severalfold by soluble syntaxin 1A peptides in an epithelial cell culture model (10). Whether or not such peptides can also potentiate the activities of wild-type CFTR or disease-associated mutants in vivo remains to be determined. If so, reagents that block the CFTR-syntaxin 1A interaction may serve as starting points for developing new strategies for treating certain forms of cystic fibrosis.
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Syntaxin-ion channel interactions as a general theme
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In broader terms, the interaction between the CFTR chloride channel and syntaxin 1A may serve to coordinate the regulation of ion transport with the regulation of membrane traffic in epithelial cells. In this regard, it is interesting to note that syntaxin 1A also physically interacts with voltage-gated calcium channels in neurons (14) and in neuroendocrine cells (15), i.e., cells that engage in regulated membrane traffic (Fig. 4). Syntaxin 1A modulates the gating of N-type (2) and L-type calcium channels (15) in heterologous expression systems. In vivo, these interactions appear to be important for optimizing the efficiency of calcium-evoked secretion by physically tethering the secretory vesicles to sites of calcium influx (13). Thus the interactions between syntaxin 1A and calcium channels serve to spatially and temporally couple the regulation of membrane traffic to the regulation of ion transport in neurons and neuroendocrine cells.
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FIGURE 4. Minimal model of protein complex that couples synaptic calcium channel to vesicle-fusion apparatus. VAMP, vesicle-associated membrane polypeptide. SNAP 25, synapse-associated membrane protein of 25 kDa.
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The regulation of fluid and electrolyte transport is also coupled (at least temporally) to the regulation of membrane traffic in CFTR-expressing epithelial cells. For example, the same factors that stimulate CFTR chloride channel activity and thereby fluid secretion in gut epithelial cells (e.g., cholera toxin via cAMP) also inhibit apical membrane endocytosis (3) and stimulate the secretion of proteins that are important for mucosal homeostasis (7). Indeed, there is some evidence to indicate that CFTR itself may contribute to the regulation of membrane traffic in these cells (3). It may be relevant that the CFTR-binding site on syntaxin 1A maps to that domain that is most critical for the participation of syntaxin 1A in membrane fusion reactions (i.e., the third helical domain, or H3 domain; see Ref. 10). This observation implies that CFTR may regulate the availability of syntaxin 1A for membrane fusion reactions (i.e., this functional interaction may be bidirectional), which could contribute to the coordination of fluid secretion and membrane traffic in epithelial tissues. Such interactions between ion channels and syntaxins may be a more general theme in biology, as evidenced by the recent identification of a plant syntaxin that regulates chloride channels and potassium channels in stomatal guard cells (8). Physical and functional interactions between ion channels and syntaxins would provide a direct means to couple the regulation of ion transport to the regulation of membrane traffic in those tissues for which coordinating these processes is physiologically advantageous.
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PDZ domain-mediated protein interactions at the carboxy-terminal tail of CFTR
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Whereas the amino-terminal tail couples CFTR to components of the membrane traffic machinery, the carboxy-terminal tail binds to proteins that possess binding modules referred to as PDZ domains. Proteins that possess PDZ domains are often multivalent (i.e., they contain multiple PDZ domains) and can promote homotypic and heterotypic protein-protein interactions in a variety of tissues (reviewed in Ref. 12). Such interactions can facilitate the clustering of ion channels within microdomains at the cell surface and the assembly of signaling complexes at the plasma membrane. Several PDZ domain-containing proteins have been shown to bind with high affinity both in vitro and in vivo to a consensus sequence (TRL) at the extreme carboxy terminus of CFTR (e.g., NHERF or EBP-50; see Ref. 5). The relevance of these interactions to the intracellular traffic or the functional activity of CFTR is unknown. However, on the basis of studies from other systems, it seems reasonable to propose that PDZ domain-mediated interactions at the carboxy-terminal tail of CFTR could link this ion channel to a variety of proteins, including signaling molecules (i.e., kinases and phosphatases), cytoskeletal elements, and other transport proteins. This latter point is particularly intriguing, given the accumulating evidence that CFTR can modulate the functional activities of other ion channels, such as epithelial sodium channels. Conceivably, PDZ domain-mediated interactions permit cross talk between CFTR chloride channels and parallel ion transport pathways. The presence of a PDZ domain-binding motif at the carboxy-terminal tail of CFTR raises the possibility that this chloride channel engages in combinatorial interactions with multiple proteins, especially since there are large numbers of proteins that either possess PDZ domains or that can bind to these domains in various tissues.
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Conclusions
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In summary, the opposing cytoplasmic tails of CFTR link this ion channel to distinct sets of proteins with presumably different functional consequences. Outstanding issues include: 1) the mechanism by which the binding of syntaxin 1A to the amino-terminal tail influences CFTR activity (altered channel gating, altered channel traffic, or both?); 2) the physiological importance of this interaction in coordinating the regulation of ion transport and membrane traffic in epithelial tissues; and 3) the functional relevance of the interactions of PDZ domain-containing proteins with the carboxy-terminal tail of CFTR. Resolving these issues will improve our understanding of the various roles that are played by this chloride channel in epithelial physiology and may provide new strategies for manipulating CFTR activity in diseases such as cystic fibrosis and secretory diarrhea.
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Acknowledgments
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We thank Dr. Erik Schwiebert for his helpful comments. We also thank our collaborators, in particular Drs. Deborah Nelson and Michael Quick. We apologize to the many investigators in the CFTR field whose work we could not cite due to length restrictions.
Our work is supported primarily by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51868 and DK-50830.
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References
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Introduction
Syntaxin interactions at the...
