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Mechanisms of Anion Secretion in Epithelial Cells

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Mechanisms of Anion Secretion in Epithelial Cells Scott M. O’Grady ANSC/PHSL 5702/PHSL 4702 Cell Physiology Lecture 19 Mechanisms of anion secretion across epithelial cells Objectives 1. Understand the polar distribution of critical ion transport mechanisms that contribute to Cl- - and HCO3 secretion. 2. Know the properties of the CFTR anion channel and it’s regulation by PKA and ATP 3. Understand the structural basis for CFTR protein-protein interactions at the apical membrane - and its significance relative to HCO3 secretion in pancreatic duct cells. 4. Understand the coordinated regulation of CFTR and NKCC1 activity through PKA-dependent phosphorylation. 5. Understand the role of KCNQ1/KCNE3 K channels in cAMP-activated anion secretion. 6. Understand the concept of charge compensation as it applies to sustaining the electrical driving force for anion secretion. 7. Understand the mechanism of cholera toxin and its effects on intestinal anion secretion and NaCl ansorption. 8. Know the function of TMEM16 and bestrophin in Ca2+-dependent Cl- secretion. Readings 1. Kunzelmann, K. et al., Bestrophin and TMEM16: Ca2+ activated Cl channels with different functions. Cell Calcium 46:233–241, 2009. 2. Kim, D. and M.C. Steward, The role of CFTR in bicarbonate secretion by pancreatic duct epithelia. J. Med. Invest. 56:336-342, 2009 3. Rao, M.C., Oral rehydration therapy: New explanations for an old remedy, Annu. Rev. Physiol., 66:385–417 2004. 1 Scott M. O’Grady ANSC/PHSL 5702/PHSL 4702 Cell Physiology Lecture 19 A. General mechanisms for anion secretion 1. Mechanism of Cl- secretion Chloride secretion in vertebrate epithelial tissues occurs by secondary active transport that depends on the coordinated function of transport proteins localized in the apical and basolateral membranes. The primary basolateral transporters include Na-K-ATPases, Na-K- 2Cl cotransporters and K channels. Cl channels constitute the rate-determining transport pathways in the apical membrane, which are typically regulated by intracellular second messengers such as cAMP and Ca2+. Transepithelial Cl- secretion from the blood into the lumen of epithelial structures such as glands within the GI tract and airways involves + - electroneutral uptake of Na , Cl Na+ Cl- and K+, followed by active Na+ efflux mediated by the Na-K - ATPase and passive K+ movement down its electrochemical gradient through channels present in the basolateral membrane. Intracellular [Cl-] accumulates above V electrochemical equilibrium under these conditions and exits across the 2K+ K+ apical membrane when the Cl- channels are activated. The active - transport of Cl establishes a + 3Na+ transepithelial potential difference + K+ Na that drives the paracellular 2Cl- movement of Na+ between the cells and into the lumen. Figure 1: Transepithelial Cl- secretion - 2. Mechanisms for HCO3 secretion Figure 2: Transepithelial HCO3 secretion + - Bicarbonate secretion can occur by Na HCO3 - different combinations of transport HCO3 - pathways that transport both Cl- and CFTR - HCO3 ions depending on epithelial P P cell type. Figure 2 is a model that - CA Cl illustrates the spectrum of + - CO2 → H + HCO3 transporters known to participate in + - Na HCO3 secretion. Uptake of HCO3 V into the cell can occur by several + mechanisms involving specific 2K - + transporters (e.g. Na-HCO3 H cotransport) or by diffusion of CO2 across the membrane and + + + 3Na subsequent conversion into H and Na+ HCO - - 3 HCO3 by carbonic anhydrase (CA). - CO2 HCO3 generation by CA must be supported by acid extrusion pathways such as Na-H exchange or H-ATPase activity in order 2 Scott M. O’Grady ANSC/PHSL 5702/PHSL 4702 Cell Physiology Lecture 19 - to regulate intracellular pH as HCO3 exits the cell across the apical membrane. Uptake of - - HCO3 by Na-HCO3 cotransport can be mediated by NBCn1, an electroneutral transporter + - - that relies on the [Na ] gradient to drive HCO3 into the cell. Efflux of HCO3 across the apical - membrane can be directly mediated by ion channels like CFTR that are permeable to HCO3 , but at a level that is 4 fold less than their permeability to Cl-. Another configuration that has been identified in pancreatic duct cells involves a cooperative interaction between apical Cl - channels and Cl-HCO3 exchangers, where Cl channels function to recycle Cl in the vicinity of the exchanger back into the apical unstirred fluid layer associated with the outer leaflet of the membrane. Cl- recycling reduces accumulation of intracellular Cl-, thus sustaining the chemical - driving force for HCO3 efflux and ensures that the extracellular [Cl-] within the unstirred layer is never rate limiting for the exchange process. Another - mechanism for HCO3 efflux can include - electrogenic Na-HCO3 exchange where 3 HCO3 ions are transported out of the cell with one Na+ ion. This stoichiometry is necessary to provide enough electrical driving force to offset the chemical gradient for Na+ in most epithelial cells. Figure 3: Expression of DF508 CFTR - - B. Cyclic AMP-activated Cl and HCO3 efflux 1. Cyclic AMP-activated Cl secretion and the role of CFTR The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein belonging to the superfamily of ATP-binding cassette (ABC) transporters In mammalian tumor cells, certain ABC transporters function as ATP-hydrolyzing pumps that transport antineoplastic drugs out of the cell across the plasma membrane (e.g. the multidrug resistance P-glycoprotein). CFTR is the only member of the ABC superfamily to exhibit Cl- channel activity. This channel is expressed in epithelial tissues as well as non- epithelial tissues including cardiac myocytes smooth muscle cells, endothelial cells and red blood cells. In epithelial tissues, CFTR participates in transepithelial electrolyte and fluid transport and mutations that inhibit CFTR activity cause cystic fibrosis. About 1500 distinct disease-associated mutations have been identified, but one single codon deletion at position 508 (F508), is the most common and is present in nearly 70% of patients with severe disease (Figure 3). Figure 4: CFTR channel structure 3 Scott M. O’Grady ANSC/PHSL 5702/PHSL 4702 Cell Physiology Lecture 19 2. CFTR structure and gating Figure 5: NBD binding of ATP CFTR is comprised of two membrane- spanning regions which contain six transmembrane helixes, two nucleotide- binding domains (NBDs), each possessing amino acid sequences that bind ATP and a regulatory domain that has several consensus phosphorylation sites. The membrane spanning regions form an anion-selective channel with a low single channel conductance (<10 pS). PKA-dependent phosphorylation of the regulatory domain is necessary for activating the channel. NBDs interact in a head-to-tail manner with two ATP-binding sites present at the interface. ATP-binding site 1 is formed by the Walker A and B motifs of NBD1 and the LSGGQ motif of NBD2), however ATP is not hydrolysed at site 1. At site 2 ATP binding is associated with the Walker A and B motifs of NBD2 and the LSGGQ sequence of NBD1. ATP hydrolysis does occur at site 2 and is necessary for channel closure. Dimerization of the NBDs occurs as a consequence of phosphorylation and ATP binding, producing channel activation as shown in figure 6. Hydrolysis of ATP at site 2 promotes channel closure and mutation of a critical residue that prevents ATP hydrolysis causes channel closure to be 1,000 fold slower than normal following ATP with-drawl. Figure 6: CFTR activation by phosphorylation and ATP At this time no X-ray crystal structures of eukaryotic ABC transporters have been determined, but crystal structures of prokaryotic ABC transporters suggest that when ATP is bound the extracellular gate located within in the transmembrane domains is open while the cytoplasmic-side gate is closed. Approximately 107 Cl- ions/second flows through the CFTR channel when ATP is bound. This indicates that the extracellular gate is open, and that the cytoplasmic gate of CFTR is either atrophied or uncoupled from the extracellular gate compared to other ABC transporters. Hence, CFTR appears to represent an example of an ABC transporter where a single extracellular gate controls anion movement through the pore. Figure 7: ATP hydrolysis and channel closure 4 Scott M. O’Grady ANSC/PHSL 5702/PHSL 4702 Cell Physiology Lecture 19 3. CFTR interactions with other membrane proteins CFTR assembles with other membrane proteins to from dynamic macromolecular signaling complexes that contain receptors, other ion channels, transporters, PDZ domain-containing scaffolding proteins and regulatory enzymes. Protein–protein interactions that affect expression and/or functional activity of CFTR have physiological significance since this channel not only transports Cl- and - HCO3 , but also regulates the function of other ion channels and transporters. Certain physical interactions between CFTR and other proteins have been shown to be dependent on the presence of a protein binding motif located in the C-terminus known as a PDZ domain. The name comes from the first three proteins for which these domains were initially identified: postsynaptic density protein PSD-95, the Drosophila junctional protein Disclarge DLG, and the epithelial zonula occludens (ZO)-1 protein. PDZ domain-containing proteins often possess multiple PDZ domains allowing them to facilitate both homotypic and heterotypic protein– protein interactions. As a result, apical PDZ proteins typically participate in the formation of multiprotein complexes that modulate protein trafficking and signaling in polarized epithelial cells. Presently, several distinct PDZ domain containing proteins have been shown to bind to the C-terminal region of CFTR and include: NHERF1&2, PDZK1&2, CAL (CFTR-associated ligand), and Shank 2. The last four amino acids (DTRL) located at the C-terminus of CFTR constitute the PDZ domain that is recognized by these adaptor proteins. Figure 8: PDZ proteins that bind CFTR Figure 9:-adrenergic receptor coupling to CFTR An example of a macromolecular complex that contains CFTR is shown in figure 9. CFTR, NHERF1, and 2 adrenergic receptor (2AR) interact to form a signaling complex at the apical membrane of airway epithelial cells.
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