This chapter summarizes the role of epithelial ion (K + , Na + , and Cl − ) channels in the gastrointestinal (GI) tract. Both cAMP- and Ca 2 + -activated K + channels are localized on both apical and basolateral membranes of the entire GI tract. Apical K + channels regulate H + ,K + -ATPase-mediated acid (i.e., H + ) secretion is stomach, while it regulates active K + secretion in colon. Basolateral K + channels regulate active Cl − secretion by maintaining cell membrane potential. Epithelial Na + channels (ENaC), which are present in the apical membrane of colon, mediates active Na + absorption. Cystic fibrosis transmembrane regulator (CFTR) Cl − channels that regulate electrogenic Cl − secretion are present on the apical membranes of the entire intestine, while Slc26a9 Cl − channels are strongly expressed in the upper GI tract. TMEM16 Ca 2 + -activated Cl − channels are important for the increase in intracellular Ca 2 + concentration during purinergic and cholinergic signaling.
KeywordsPotassium channels, Chloride channels, Epithelial sodium channel, Water absorption, Water secretion, Diarrhea, Inflammatory bowel disease, Glucocorticoids, Aldosterone
Electrolyte absorption and secretion mediated by ion channels of the epithelial cells play an important role in determining basal and active fluid movement (i.e., absorption or secretion) in the GI tract. Ion channels that are involved in regulating fluid movement include Na + , K + , and anion (Cl − and HCO 3 − ) channels. Active Na + absorption and active Cl − secretion associate with fluid absorption and secretion, respectively, while K + channels maintain the negative membrane potential that is necessary for active anion secretion. Both cAMP- and Ca 2 + -activated K + channels are localized on both apical and basolateral membranes. The cAMP-activated K + channels named KCNQ1/KCNE2 complex that regulates H + (i.e., acid) secretion is localized on the apical membranes of the gastric parietal cells, while KCNQ1/KCNE1 complex is localized in the basolateral membranes of the entire GI tract. The Ca 2 + -activated K + channels named large conductance K + (BK) channels that regulate active K + secretion and intermediate conductance K + (IK) channels that maintain the negative membrane potential during active Cl − secretion are localized on the apical and basolateral membranes, respectively. Epithelial Na + channels (ENaC), which mediate active Na + absorption, are restrictively present in the apical membranes of colon. The cystic fibrosis transmembrane regulator (CFTR) Cl − channel is the only currently identified apical anion channel in all intestinal segments and mediates cAMP (i.e., cholera toxin)-, cGMP (i.e., heat-stable Escherichia coli enterotoxin, guanylin)- and Ca 2 + -activated Cl − /HCO 3 − secretion. Slc26a9 and CLIC6 are apical/apical recycling anion channels in the stomach, but their functional role for acid secretion is still under debate. The TMEM16 family members expressed in the GI tract are involved in the Ca 2 + signaling itself, and therefore, essential for stimulation of anion secretion by any agonist. The CLC family members regulate organellar function; a role for the membrane-localized CLC2 has been demonstrated in colonic Cl − absorption and it may also be involved in GI barrier function. The role and regulation of these epithelial K + , Na + , and anion channels during physiological and pathophysiological conditions are discussed in this chapter.
Role of Ion Channels in GI Tract
In the 1960s, several research groups exploited the new “Ussing chamber” technique, in which an isolated epithelium was placed between two lucite half chambers, and the potential difference (PD) between the serosal and mucosal side of the epithelium was recorded, which was generated by an active transport of ions through the mucosa. If continuously clamped to zero voltage by a current passed through the mucosa, this “short circuit current” (Isc) was recognized to represent net transport of charge across the epithelium in real time. A set of agonists, namely, vasopressin, theophylline, and cyclic-AMP analogues, increased the Isc in a variety of epithelia such as frog skin, toad urinary bladder, and the rabbit ileum. While the agonist-mediated Isc increase in skin and bladder was generated by the influx of Na + , Field et al. realized that in the ileum, the agonist-induced Isc was mediated by the secretion of Cl − . At the same time, investigators had found that crude supernatant of Vibrio cholera induced hypersecretion in isolated intestinal loops of rabbits and dogs and increased cAMP in enterocytes. The Ussing technique has been proved to be a powerful tool for bilateral isotopic flux studies. A number of investigators interested in the pathophysiology of diarrhea soon realized the importance of the stimulation of electrogenic Cl − secretion in the pathophysiology of enterotoxin-mediated diarrhea.
A period of very active research in a number of laboratories resulted in the establishment of a model for intestinal salt transport, based exclusively on electrophysiological, isotope flux, and in vivo fluid movement measurements, which already envisioned many of the key features that were later proved correct by immunohistochemical studies and knockout mouse models, although additional ions such as HCO 3 − and pathways such as the Na + -HCO 3 − cotransporters (NBCs) have more recently been recognized to also play a major role in intestinal electrolyte transport. This included, for example, the fact that the NaCl absorptive pathway resides in the villi and the anion efflux pathway in the crypts, that cAMP acts in two ways, by inhibiting the coupled NaCl influx pathway and by stimulating an electrogenic Cl − efflux pathway, and that stimulating Na + -solute cotransport by glucose or amino acids reduced enterotoxin-mediated intestinal fluid loss in vivo ( Fig. 58.1 ).
During these years, it also became clear that the intestine expresses yet another system for active Na + absorption in the distal part of the colon, which was electrogenic and sensitive to low concentrations of the diuretic amiloride. Edmonds and colleagues studied the amiloride-sensitive PD in humans and already realized that the transport process, which caused this PD was almost completely abolished in ulcerative colitis, and that the antiulcer drug carbenoxolone caused an increase in the PD, possibly a reason for the frequent edema formation observed with this drug.
A decade later, the energy requirements for active intestinal Cl − secretion had also been clarified. Suitable models such as the shark rectal gland or other amphibian intestinal epithelia, the advancement of microelectrode studies, as well as the development of brush border and basolateral membrane vesicle preparations resulted in the understanding that a Na + -K + -2Cl − cotransport (NKCC) system in the basolateral membrane of enterocytes allowed the uphill transport of Cl − and K + by utilizing the Na + gradient established by the basolaterally located Na + ,K + -ATPase, and that Cl − leaves the enterocyte via a cAMP-activated apical conductance along its electrochemical gradient ( Fig. 58.2 ).
In the early 1980s, Dharmsathaphorn and colleagues described the use of a colon cancer cell line named T84 on permeable supports that could be placed in Ussing chamber systems, which appeared an ideally suited model for the cellular study of intestinal Cl − secretion. A large number of publications appeared in the following years that not only recapitulated the apical anion conductance and basolateral NKCC as important players in the secretory process, but also established the importance of activation of basolateral K + conductances by cAMP and Ca 2 + in maintaining the electrochemical driving force for electrogenic Cl − secretion. In addition, work in intestinal cell lines provided insight into the second messenger regulation of intestinal Cl − secretion, and it was recognized that cAMP- and Ca 2 + -elevating agonists had a synergistic action, suggested to occur via a cooperative action of Ca 2 + – and cAMP-activated basolateral K + conductance with an apical cAMP-activated Cl − conductance. Despite the fact that we now know that the intracellular regulation of secretion occurs through trafficking of transporters, cytoskeleton-anchored formation of multiprotein complexes, phosphorylation at multiple sites and by many kinases, and that the transmembrane transport of CO 2 and HCO 3 − , not represented in these models, also plays a major role in intestinal electrolyte transport regulation, the above model is what many of us still teach students today.
Epithelial K + channels that mediate cellular K + exit across plasma membranes help regulate several important physiological functions. The K + channels with distinct function are localized on the apical and basolateral membranes of the GI epithelial cells. Epithelial cells of the different part of the GI tract such as mouth (salivary secretion), stomach (acid secretion), intestine (nutrient absorption and electrolyte/water absorption/secretion), and colon (primarily electrolyte/water absorption/secretion) perform different functions. Thus, the K + channels present in different epithelial cells regulate different functions. Under basal condition , the basolateral K + channels regulate the Na + ,K + -ATPase (i.e., Na-pump) activity, which is the primary active transporter that catalyzes 3 Na + out of the cells in exchange for 2 K + into the cells. By regulating Na + ,K + -ATPase, the basolateral K + channels help maintaining intracellular low Na + and high K + concentration and a negative (~− 50 mV) membrane potential (i.e., electrochemical gradient). The electrochemical gradient provides the driving force for the secondary active transporters such as Na + -nutrient (e.g., Na + -glucose and Na + -amino acid) cotransporters, and electroneutral (e.g., Na-H exchange) and electrogenic (e.g., epithelial Na + channel, ENaC) Na + absorptive processes that are present in the apical membranes of intestinal and colonic epithelial cells ( Fig. 58.3 A ). Along with maintaining membrane potential, K + channels also regulate basolateral Na + -K + -2Cl − cotransporter (Cl − loader), which brings in Cl − during active Cl − secretion that is mediated through apical Cl − channels in salivary gland, intestine, and colon ( Fig. 58.3 B). In addition, the basolateral K + channels in coordination with apical H + ,K + -ATPase mediate active K + absorption in rat distal colon ( Fig. 58.3 C). In contrary to the basolateral K + channels, the apical K + channels regulate H + secretion (i.e., acid secretion), which is catalyzed by gastric H + ,K + -ATPase of the parietal cells in stomach ( Fig. 58.4 A ). Potassium secretion mediated by apical K + channels in part provides the driving force for secretion in salivary gland, while apical K + channels-mediated K + secretion regulates body K + homeostasis in colon ( Fig. 58.4 B).
Molecular Identities of K + Channels
Epithelial K + channels are classified into two major classes—Ca 2 + -activated (K Ca ) and cAMP-activated K + channels. There are five different K Ca channels in mammals: one large conductance K + (BK, also known as Slo, K Ca1.1 and KCNMA1), one intermediate conductance K + (IK; also known as SK4, K Ca3.1 and KCNN4), and three small conductance K + (SK1–3) channels. Only BK and IK, but not SK channels are expressed on the apical and/or basolateral membranes of the intestinal epithelial cells. Thus, this chapter will discuss the molecular identities and functional role of IK and BK channels. The BK channels exhibit K + conductance of ~ 150–230 pS, while IK channels exhibits K + conductance of ~ 12–39 pS. Furthermore, cAMP-activated K + channels with conductance of ~ 1–3 pS have also been characterized on both apical and basolateral membranes of intestinal epithelial cells.
Ca 2 + -Activated K + Channels
Ca 2 + -activated K + channels are present in both apical and basolateral membranes of epithelial cells. Ion flux studies performed under voltage clamp condition have shown that K + channels mediate K + efflux across the apical membranes, while K + channels localized on the basolateral membranes provide the driving force for active Cl − secretion in trachea and colon. Subsequent patch clamp studies have characterized K + channels with conductance of approximately 39 and 200 pS that are designated intermediate K + (IK) and large conductance K + (BK) channels, respectively. BK channels are activated by Ca 2 + and cAMP and inhibited by Ba 2 + , tetraethyl ammonium (TEA), and quinidine. The IK channel is activated by both Ca 2 + and cAMP and inhibited by Ba 2 + , diphenylamine-2-carboxylic acid (DPC), and quinidine, but not by TEA. Patch clamp studies have also characterized BK and IK channels on the basolateral membranes of rat and guinea pig small intestine, and rat, guinea pig, rabbit, and turtle colon. Immunological studies have established the localization of both IK and BK channel-like proteins on both apical and basolateral membranes of epithelial cells of the intestinal tract.
Large conductance K + (BK) channels : The BK channels composed of pore forming α (BKα)- and Ca 2 + /voltage sensing β (BK β )-subunits. Two BKα splice variants (STREX and ZERO) that are transcribed from a single gene, and four BKβ-subunits (β 1–4 , known as KCNMB 1–4 ) that display tissue- specific distribution have been identified. Expression of BKα alone results in large-conductance (~ 150–200 pS) K + channel function, while coexpression with different β-subunits exhibit different sensitivity to Ca 2 + and voltage. The BKα splice variants STREX and ZERO have distinct sensitivities to cAMP (STREX being inhibited and ZERO being activated by cAMP). BK exon 18 is the splice target defining STREX (with exon 18) and ZERO (without exon 18). Only ZERO transcripts have been shown expressed in guinea pig and human colon. However, STREX and ZERO transcripts have been shown expressed in mouse colon. In addition, BKα (ZERO) has also been shown to exhibit three splice variants of COOH terminal in human brain and guinea pig colon. The BKα with exon-27 termination resulted in an amino acid sequence ending of QEERL, whereas the termination in exon-28 resulting in an amino acid sequence of EMVYR. The splicing of exon-28 has been shown to occur with two variants that differed by an omission of three base pair at the beginning of exon-28. The BKα variants that end with amino acid sequences QEERL and EMVYR are identified as BKα RL and BKα YR , respectively. Both BKα RL and BKα YR have been identified to express in guinea pig and rat colon (Rajendran, V. M., unpublished observation). The COOH-terminal variants of BKα RL and BKα YR contribute to the membrane (apical vs. basolateral membrane)-specific delivery of BK channels. BKα YR variant that has been localized on both apical and basolateral membranes of immature cells (i.e., bottom of the crypt) has been shown to be expressed only on the apical membranes of mature surface cells in guinea pig colon. Although the molecular identities of BKα splice variants of rat and human colon have not yet been established, the demonstration that cAMP-activated BK channel activity and forskolin (an adenylate cyclase activator that increases intracellular cAMP levels)-activated K + secretion suggest that the BK channel encoded by ZERO transcripts mediate the K + secretion in rat and human colonic epithelial cells. Aldosterone (dietary-Na + depletion) and high-K + diet enhance BKα-specific mRNA abundance and protein expression and stimulate iberiotoxin (BK channel specific blocker)-sensitive K + secretion in rat distal colon. It has been shown that aldosterone also stimulated K + secretion through ZERO isoform, as forskolin (cAMP) did not inhibit the aldosterone stimulated K + secretion in rat distal colon.
The BKα forms complexes with the auxiliary β-subunit, which has the ability to modify the activation kinetics (i.e., Ca 2 + and voltage sensitivity) and inhibitor sensitivity of BK channel function. Four isoforms of BKβ-subunits have been identified, each of which may associate with the BKα-subunit to modulate BK channel activities in a unique way. Earlier studies have identified the expression of all four β-subunits, while a recent study has identified only β2-subunit expression in mouse colon. Similarly, although different studies have shown all four β-subunits expressed, only β1- and β3-subunits have been identified as the predominant β-subunits expressed in the human colon. Only β1 and β4 are the only β-subunits detected in guinea pig colon. It is likely that BKα splice variants (BKα RL and BKα YR ) may coexpress with different β-subunits in mouse, guinea pig, rat, and human colon. Further studies are required to establish whether BKα RL and BKα YR isoforms coexpress with same or different β-subunits, and whether BKα RL and BKα YR express in same or in different cell types (villus vs. crypt cells) and different membrane (apical vs. basolateral membranes) in colonic epithelial cells.
Intermediate conductance K + (IK) channels : The IK channel (known as hSK4) that encodes 427 amino acids was originally cloned from human placenta. Human and rat colonic IK ortholog that encodes 424 amino acids has been cloned from T84 cells and rat colonic cDNA library, respectively. The rat colonic IK channel expressed in vitro exhibited Ca 2 + -activated K + current with a single channel conductance of 36 pS and was inhibited by clotrimazole (CLT, an antifungal inhibitor). Immunofluorescence studies have localized IK channel-like proteins on both apical and basolateral membranes of epithelial cells in esophagus, glandular stomach, duodenum, jejunum, ileum, and proximal and distal colon of rat, while immunogold labeling studies have localized IK-like proteins on both apical and basolateral membranes of rat and human colon. Further extensive cloning studies have isolated two additional IK splice variants that encode 425 and 395 amino acid proteins. The IK channel with 425 amino acid protein is identical to that of mouse IK channel (mIK1) ortholog that was cloned from smooth muscle. The IK channel isoform mRNAs that encode 425, 424, and 395 amino acid proteins were designated as KCNN4a, KCNN4b, and KCNN4c, respectively. It has been concluded that Kcnn4a encodes smooth muscle IK channels, while KCNN4b and KCNN4c encode basolateral (40 kD) and apical (37 kD) IK channels in intestinal epithelial cells, respectively.
cAMP-Activated K + Channels
cAMP-activated K + channels have been identified on the apical and basolateral membranes of gastric parietal cell and basolateral membranes of rat and human colon. This cAMP-activated K + channel is inhibited by chromanol-293B, a slow delayed rectifier K + current blocker. Molecular studies have established that this cAMP-activated K + channel is encoded by voltage-gated K v LQT1 channel, which is mutated in hereditary Long QT syndrome type-1. K v LQT1 composed of α-(KCNQ1) and β-(KCNE) subunits. Five different β-subunits (KCNE1–KCNE5) have been isolated. KCNQ1 expressed in different cells and membrane domain that exhibit various functions have been shown to coassemble with different KCNE isoforms. KCNQ1 coassembles with KCNE1 are localized on the basolateral membranes of trachea, pancreas, jejunum, and colon, while KCNQ1 coassembled with KCNE2 (i.e., KCNQ1/KCNE2 complex) forms apical membrane K + channels in gastric parietal cells. KCNQ1/KCNE3 complex have also localized on the basolateral membranes of mouse tracheal and intestinal epithelial cells. Thus, it is unequivocally established that KCNQ1/KCNE2 complex mediated K + exit is critical for apical H + ,K + -ATPase-regulated acid (i.e., H + ) secretion in gastric parietal cells. Although KCNQ1 is expressed in the basolateral membranes of colon, conflicting observations have been shown about its contribution to Cl − secretion. The chromanol 293 sensitive KCNQ1 K + channel has been shown to provide the cAMP active Cl − secretion in mouse colon, but it has been shown to be not essential for cAMP-dependent activated Cl − secretion in guinea pig colon. Since both cAMP and Ca 2 + have been shown to activate basolateral IK channels in human colon, it is likely that the basolateral IK, but not KCNQ1 channels play a central role in regulating infectious and other types of secretory diarrhea.
Apical Membrane K + Channels
Role of Apical Membrane BK Channels in Colonic K + Secretion
Slow marker perfusion studies have shown that the human colon, but not small intestine, secretes 4.7 mEq K + per day. Both passive K + permeation and active K + secretion contribute to luminal and stool K + concentration. Para cellular passive K + permeation occurs as a result of transepithelial lumen-negative (~ 20–30 mV) electrical PD, which may exist through the entire GI tract. In contrast, the transcellular active electrogenic K + secretion has only been localized in salivary gland, gastric mucosa, and large intestine of the digestive tract. Active K + secretion contributes to increased stool K + content during secretory diarrhea. Every day, normal humans excrete 10 mEq K + in stool, while patients with severe diarrhea such as cholera secrete 119 mEq/day. Active K + secretion requires K + uptake across basolateral membrane and K + exit across the apical membranes ( Fig. 58.5 ). In addition, electrogenic K + secretion also requires exit mechanisms for Na + and Cl − across basolateral membranes. Potassium uptake across the basolateral membrane is mediated by Na + ,K + -ATPase, and Na + -K + -2Cl − cotransport, while K + exit across the apical membrane is mediated by K + channels. The Na + brought in by Na + -K + -2Cl − cotransport is pumped out by Na + ,K + -ATPase, while Cl − brought in by Na + -K + -2Cl − cotransport exits via chloride channel-2 (CLC2) that has been shown to be localized on the basolateral membranes of mouse and guinea pig. Inhibition by serosal ouabain (Na + ,K + -ATPase inhibitor) and bumetanide (loop diuretic, Na + -K + -2Cl − inhibitor) has established that active K + secretion is regulated by basolateral Na + ,K + -ATPase and Na + -K + -2Cl − cotransport. Na + ,K + -ATPase regulates the active K + secretion under basal condition, while Na + -K + -2Cl − cotransport is the predominant regulator of the stimulated active K + secretion. In stoichiometric point of view K + uptake across the basolateral membrane far exceeds its transepithelial movement, thus suggesting the presence of K + recycling across the basolateral membrane. The observation of an increased K + secretory rate when Ba 2 + (nonspecific K + channel blocker) is added to the serosal bath suggested that K + recycling across the basolateral membranes of turtle and rabbit colon contributes to enterocyte K + homeostasis. Since Ba 2 + -sensitive K + channels have not been observed, it was suggested that K + -Cl − cotransport might be responsible for K + recycling across the basolateral membranes in rat distal colon. Since active K + secretion is enhanced in IK channel knockout mouse, and BK and IK channel-like proteins have been localized, it is likely that either BK and/or IK channel might regulate the K + recycling across basolateral membranes in guinea pig, mouse, and rat distal colon. The colonic K + secretion is activated by a cellular second messenger (e.g., cAMP and Ca 2 + ) and is stimulated by dietary-K + loading, dietary-Na + depletion (aldosterone) and dextran sulfate sodium (DSS)-induced colitis. Active colonic K + secretion is increased in patients with end-stage renal disease (ESRD), colonic pseudo-obstruction, and adenomas.
Ion flux studies performed under voltage clamp condition have shown that iberiotoxin or paxilline (BK channel-specific blockers) sensitive K + channels present on the apical membranes mediate both basal and stimulated K + secretion in guinea pig, mouse, and rat distal colon, while patch clamp studies have characterized K + channels with large conductance in rat and human colon. The absence of K + secretion in BKα knockout mouse has established that BK channel mediates both resting and activated K + secretion in mouse colon. It is current dogma that the absorptive (e.g., ENaC) and secretory (e.g., CFTR) transport processes are distributed differentially along the surface-crypt axis, as the absorptive process being localized on the surface cells, while the secretory processes are localized on the crypt cells. In contrast, conflicting observations exist regarding BKα protein localization in normal guinea pig, mouse, and human colon. In normal human colon, BKα-like proteins have been localized only on the apical membranes of surface cells in normal human colon, while in mouse it has been localized only to crypt cells. Absence of BKα proteins in BK channel knockout mouse justified the presence of BK channels in the crypt cell of mouse colon, while surface cell expression of BK channel has been established by extensive patch clamp characterization of large conductance K + channel in normal human colon. In contrary to both mouse and human, BKα proteins have been localized on both surface and crypt cells in guinea pig distal colon. BKα proteins have been localized on both surface and crypt (top 2/3rd of crypt) cells, while the bottom third of crypt cells are completely devoid of BKα protein, in human colonic biopsy specimens (Rottgen T, Nickerson A and Rajendran VM; unpublished observations). Despite the existing conflict on cell specific localization, it is well established that BK channels localized on the apical membrane mediate basal and activated K + secretion in mammals and human colon.
Role of Apical Membrane BK Channels in K + Secretion in Salivary Glands
The K + concentration of protein-rich saliva is from 5 to 10-fold higher than that of plasma K + concentration. Micropuncture studies have shown that acinar cells are capable of K + secretion and that the salivary K + concentration is inwardly proportional to the perfusion rate in rat and mouse salivary glands. Although K + channels were originally described in the basolateral membranes, recent functional studies have shown TRAM-34 (IK channel blocker) and paxilline sensitive K + secretion, as evidence for the presence of IK and BK channels on the apical membranes of mouse parotid acinar cells. Molecular studies have established the expression of IK and BKα channels in mouse parotid acinar cells. BKα variant of salivary gland parSlo has been cloned and shown to express in both mouse and human parotid glands. As discussed, BKα coexpresses with different BK β subunit (BK β1–4 ) that expresses distinct Ca 2 + and voltage-sensitive K + current. Molecular studies have identified that both β1 and β4 expression in parotid glands. However, the biophysical characteristics of BK channels of parotid acinar cells have not been altered in β1/β4 double knockout mouse. As a result, further studies have identified leucine-rich repeat containing proteins 26 (LRRC26, known as BKγ1-subunit), which is highly expressed in salivary glands, as an accessory protein for parSlo. The parSlo coexpressed with LRRC26 has been shown to exhibit biophysical properties comparable to native BK channels in CHO cells. Thus, in contrast to other BKα, which coexpresses with β1–4, parSlo coexpresses with LRRC26 in salivary glands.
Although it has been hypothesized that K + channels are involved in the regulation of fluid secretion, the salivary gland fluid secretion was not affected either in IK or BKα channel gene knockout mice. However, the fluid secretion has been shown to be severely impaired in IK and BKα double gene knockout mice parotid gland. Based on these observations, it was suggested in the absence of one channel, the other channel compensates to regulate fluid secretion in salivary gland. However, although IK channels have been shown to provide the driving force for Cl − (i.e., fluid) secretion, it is not known whether BK channel can also provide similar driving force for Cl − secretion. Although fluid secretion was not affected, K + secretion was reduced by > 75% in submandibular salivary glands of BKα knockout mouse. Thus, the BK channel significantly contributes to the K + secretion in salivary glands.
Role of Apical Membrane IK Channels in K + and Cl − Secretion
Patch clamp studies have characterized IK channels only in the basolateral membranes of crypts from rat and human colon. Immunofluorescence studies have localized IK channel proteins on the apical membranes of esophagus, glandular stomach, ileum (both villus and crypt cells), cecum, proximal colon and distal colon of rat, and human colon, while functional studies have identified IK channels in mouse parotid acinar cells. The apical IK channel has been shown to be encoded by KCNN4c transcripts (IK splice variant) in rat distal colon. Although the role of apical IK channels in small intestine is unknown, the role for apical IK channels has been shown in rat proximal and distal colon. Inhibition of carbachol stimulated K + secretion by clotrimazole (IK channel blocker) has been shown as evidence for the apical membrane IK channels in normal rat proximal colon. Mucosal DC-EBIO (IK channel opener) has been shown to stimulate TRAM-34 (IK channel specific blocker)-sensitive K + secretion, as evidence for apical membrane IK channels in normal rat distal colon ( Fig. 58.6 ). Further, the complete absence of mucosal DC-EBIO-induced K + secretion in dietary-K + depleted rat distal colon established the presence of IK channels in apical membranes. In addition to stimulating IK channel, mucosal DC-EBIO has also stimulated Inh- 172 (CFTR specific blocker)-sensitive Cl − secretion (measured as short circuit current). The TRAM-34, which blocked IK channel mediated K + secretion, also blocked the Cl − secretion. The inhibition of Cl − secretion by TRAM-34 indicates that apical membrane IK channel also provides the driving force for CFTR-mediated Cl − secretion. It is likely that in addition to basolateral membrane IK channels, the apical membrane IK channels also provide the driving force for active Cl − secretion in distal colon. The role of apical membrane IK channels need to be identified in the small intestine.
Role of Apical Membrane KCNQ1/KCNE2 Channels on Gastric H + ,K + -ATPase Regulation
The human stomach secretes about 1–2 L of hydrochloric acid (~ 100 mmol/L) per day. This extraordinary function is catalyzed by an electroneutral apical H + ,K + -ATPase, which pumps out intracellular H + into the lumen in exchange for extracellular K + in gastric parietal cells, requires a continuous luminal K + supply. More than three decades ago, experiments with vesicles isolated from parietal cells have suggested that K + replenishment and accompanied Cl − secretion are accomplished through apical membrane localized K + and Cl − channels, respectively. Several other studies, however, have suggested that K + -Cl − cotransport, instead of K + and Cl − conductive pathways, as a possible mechanism for K + exit across apical membrane. Although K + conductance has not been observed in vesicles isolated from resting parietal cells, K + conductance that is augmented by phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown in vesicles isolated from preactivated parietal cells. This apical K + channel has unexpectedly been identified as KCNQ1, although Kir Channels are also localized in the tubulovesicular membrane in resting state and secretory membrane in secreting. KCNQ1 knockout mouse exhibited a stomach phenotype (i.e., enlarged stomach as a result of hyperplasia of the antrum and fundus mucosa), decreased number of parietal cells, hypochlorhydria with increased stomach pH of 6–7 (wild-type mouse stomach pH is 1–2) and elevated plasma gastrin level. Immunofluorescence studies have localized KCNQ1 on gastric luminal compartment and have suggested a KCNQ1 recycling mechanism in mouse and human. Further a functional study has demonstrated that chromanol 293B (KCNQ1 inhibitor) almost completely inhibited the acid secretion in mouse, rat, and dog, but a later study revealed nonspecific effects of chromanol 293B and an incomplete inhibition of acid secretion by a more specific KCNQ1 inhibitor. In addition, Kir channels 4.1 and 5.1 × 100% cotrafficked with the H + /K + -ATPase, while KCNQ1 expression only partially overlapped with H + /K + -ATPase, raising the question of a complementary role for the two K + channels. However, the newborn KCNQ1-deficient but not Kir 4.1-deficient gastric mucosa is unable to secrete acid but its secretory rates are restored to normal levels when a high K + concentration was applied to the luminal bath, demonstrating an absolute dependence of acid secretion on the presence of KCNQ1. High K + rescue was not possible any more in adult KCNQ1-deficient stomach, which displays severely abnormal parietal cell ultrastructure and an enormous foveolar hyperplasia. Kir 4.1 channels, on the other hand, appear to play a role in apical membrane recycling. As discussed, KCNQ1 complexed with a different regulatory subunit (i.e., KCNE1–5) have been shown to exhibit various functions in different tissues. Of the five regulatory subunits, only KCNE2 and KCNE3 have been shown abundant in human stomach. This study has also shown that KCNE2 coexpressed with KCNQ1 exhibited voltage insensitive, luminal acid pH: 5.5 (compared to alkaline pH: 7.5) activated, chromanol 293B-sensitive whole cell current in COS cells. KCNE2 has been identified to colocalize with H + -K + -ATPase in stimulated, but not in nonstimulated rat gastric parietal cells. Studies with gene knockout animal have shown that similar to KCNQ1 knockout, KCNE2 knockout mice also exhibited abnormal parietal cell, hypergastrinemia, glandular hyperplasia and impaired gastric acid secretion, and have established that the regulatory subunit KCNE2 is critical for the proper function of KCNQ1 in gastric parietal cells. Thus, the KCNQ1/KCNE2 complex expressed on the apical membranes mediates the electrogenic K + secretion that is required for the continuous operation of H + ,K + -ATPase in gastric parietal cells. Intracellular second messenger (cAMP, Ca 2 + , and PIP 2 ) and extracellular acid pH that activated the luminal K + conductance of parietal cells have all been shown to activate the in vitro expressed KCNQ1/KCNE2 activity. The H + ,K + -ATPase location has been shown to have dramatic difference in resting and stimulated conditions in the parietal cells of the gastric mucosa. Most of the H + ,K + -ATPase has been shown localized in cytoplasmic tubulovesicles with no access to the canaliculi system in parietal cells. Upon stimulation, the H + ,K + -ATPase-containing tubulovesicles have been shown to exocytotically fuse into canaliculi that involve complex vesicular trafficking. Under a resting state (i.e., nonstimulated condition), H + ,K + -ATPase has been shown to most of the evenly distributed in cytoplasmic tubular vesicles, which is devoid of K + conductance, throughout the parietal cells. In contrast, in stimulated parietal cells, H + ,K + -ATPase has been shown localized in canalicular space, where KCNQ1 has also shown colocalized. Based on these observations, the cellular models have been proposed for both acid and K + secretion in parietal cells. In that the agonists (e.g., acetyl choline, gastrin and histamine) stimulate acid secretion by actively recruiting the H + ,K + -ATPase-containing tubulovesicles into canaliculi compartment. In contrast, KCNQ1/KCNE2 K + and CFTR Cl − channels, which are readily present on the canaliculi, are activated by second messenger (cAMP, Ca 2 + , and PIP 2 ) regulated phosphorylation. In addition to second messengers, luminal H + secreted through H + ,K + -ATPase also stimulates KCNQ1/KCNE2-mediated K + secretion. The K + secreted through KCNQ1/KCNE2 channels utilized by H + ,K + -ATPase for active H + secretion. Immunofluorescence studies have observed that H + ,K + -ATPase and KCNQ1 are unevenly expressed along the gastric gland, as H + ,K + -ATPase and KCNQ1 are predominantly localized on the parietal cells of the upper and lower part of the gastric gland, respectively. Thus, acid secretion (i.e., H + ,K + -ATPase) and K + and Cl − channels have been illustrated to localize in different cell types (i.e., top and lower cells, respectively) of gastric glands.
Basolateral Membrane K + Channels
Potassium channels that exit K + across basolateral membranes into the systemic circulation regulate both the Na + ,K + -ATPase and the Na + -K + -2Cl − cotransporter. Under basal condition, constitutively open basolateral K + channels maintain a negative membrane potential, which is the driving force for Na + -dependent nutrient absorption and Na + /H + -exchanger mediated electroneutral Na + absorption in the small intestine. The constant K + loss is counterbalanced by the regulation of the Na + ,K + -ATPase, which maintains intracellular high K + levels. The electrochemical gradient also provides the driving force for electrogenic Na + absorption mediated through epithelial Na + channel (ENaC) in the distal colon. In contrast, under stimulated conditions, basolateral membrane Na + -K + -2Cl − cotransporter brings in Cl − , which exits through apical membrane Cl − channels and depolarizes the membrane potential. Potassium exits through agonist-activated basolateral membrane K + channels and maintains the negative membrane potential that provides the driving force for sustained electrogenic Cl − secretion. Patch clamp studies have identified characteristically different population of K + channels in the basolateral membranes of various epithelia of digestive tract (i.e., salivary acinar cells, gastric parietal cells, and intestinal and colonic crypt cells) of different species ( Table 58.1 ). Although it is difficult to generalize the properties and physiological roles of the basolateral K + channels between different species, and between different tissues, these K + channels can be divided into three categories: (1) small (< 6 pS), (2) intermediate (19–35 pS), and (3) large (67–240 pS) conductance K + channels. Molecular studies have identified that the small, intermediate, and large conductance K + channels are encoded by KCNQ1/KCNE3, IK (Kcnn4), and BK (KCNMA1) K + channels, respectively. In addition to these three categories, nonselective cation channels with 27–30 pS that conduct Na + and K + have also been identified in turtle and rat colon. However, the molecular identities of the nonselective cation channels are not known. Small conductance (KCNQ1/KCNE3) and BK K + channels are activated by cAMP and Ca 2 + , respectively, while IK channels are activated by both Ca 2 + and cAMP. Despite the electrophysiological and immunofluorescence localization, physiological functions of basolateral BK channels have not been identified. Since the contribution of KCNQ1/KCNE3-mediated K + conductance has been shown small in the basolateral of human colonic crypts, it is likely that the highly abundant IK channel that is activated by both Ca 2 + and cAMP plays critical role in activated Cl − secretion that drives fluid secretion.
|Mouth||Sheep, rat and human||Ca 2 +||30||TEA,|
|Stomach||Necturus||Ca 2 +||< 6||Not known|
|Ca 2 +||67||Not known|
|Duodenum||Rat||cAMP and Ca 2 +||19–28||Ba 2 + and TEA|
|Colon||Rat||cAMP||< 3||Chromanol 293B|
|Ca 2 +||12||Ba 2 + and TEA|
|Ca 2 +||187||Ba 2 +|
|Rabbit||Ca 2 + and cAMP||90–220||Ba 2 + and TEA|
|Turtle||Nonselective||30||Quinidine and DPC|
|Ca 2 +||35||Ba 2 + , quinidine and DPC|
|188||Ba 2 + and quinidine|
|cAMP and Ca 2 +||23||Ba 2 + , quinidine and DPC|
|Ca 2 +||138||Ba 2 + , quinidine and TEA|
|Cell line||T84||cAMP and Ca 2 +||28||CLT|
|Ca 2 +||161||Ba 2 +|
Role of basolateral IK channels in active Cl − secretion : Electrogenic Cl − secretion, which is the critical event underlying in secretory diarrhea, occurs predominantly from crypts of small intestine and colon. Secretory diarrhea may be caused by a variety of infective and neurohumoral factors that stimulate intestinal Cl − secretion and water secretion by activating intracellular protein kinase A (PKA, Ca 2 + -dependent) and protein kinase C (PKC, cAMP-dependent) signaling pathways. The current model for electrogenic Cl − secretion requires activation of basolateral K + channels to promote hyperpolarization, and recycling of K + taken into the cell via basolateral Na + -K + -2Cl − cotransporter and Na + ,K + -ATPase. Ion flux studies have shown that basolateral K + conductance is important for cAMP-stimulated Cl − secretion in rat and human colon. Activated K + channels that exit K + across basolateral membranes generate the membrane potential gradient required to drive the sustained Cl − secretion via cAMP-stimulated CFTR Cl − channels localized on the apical membranes. Two pharmacologically distinct K + channels have been shown, one that is activated by Ca 2 + -dependent, while the other is activated by cAMP-dependent Cl − secretory agonists, in the basolateral membranes of T84 cells. Blockage of these basolateral K + channels has been shown to inhibit active Cl − secretion in T84 cells. Electrophysiological studies have characterized low-conductance basolateral K + channels that are activated during cAMP-stimulated Cl − secretion and inhibited by chromanol 293B in rat colonic crypts, while it has been identified as cAMP-stimulated K + channel encoded by KCNQ1/KCNE3 complex in T84 cells. In contrast, however, intermediate conductance K + (IK) channels have been shown abundant and activated by Cl − secretagogues carbachol (a Ca 2 + -mediated muscarinic agonist) and dibutyryl-cAMP (a membrane permeant cAMP analogue) in basolateral membranes of T84 cells rat and human colonic crypts. Activation of basolateral IK channels stimulates active Cl − secretion, while cAMP-stimulated Cl − secretion is completely inhibited by TRAM-34 (an IK channel inhibitor) in rat distal colon. Thus, the membrane potential maintained by basolateral IK channels provides the driving force for both Ca 2 + -stimulated and cAMP-stimulated Cl − secretion in human and mammalian colon.
Selective blockade of basolateral IK channels as a means of controlling epithelial Cl − secretion is being explored. Basolateral IK channels can be inhibited by Ba 2 + in human colonic crypts, and thus it has been shown to block both Ca 2 + and cAMP-stimulated Cl − secretion when added to the basolateral surface of T84 monolayer. The IK channel-specific inhibitors CLT and TRAM-34 have also been shown to inhibit both Ca 2 + and cAMP-stimulated Cl − secretion in mouse and rabbit intestine and rat colon. Somatostatin, a tetradecapeptide normally present in intestinal mucosa, has long been recognized as a potent antisecretory peptide that inhibits all forms of Ca 2 + and cAMP-stimulated Cl − secretion. Somatostatin has been shown to exert its effects via G-protein coupled receptors. Although somatostatin has been shown to reduce intracellular cAMP levels via a G-protein dependent inhibition of adenylate cyclase, it has also been shown to have additional antisecretory effects distal to the intracellular second messenger production cascades. Further studies have shown that somatostatin markedly inhibited basolateral IK, but not BK channels in human colonic crypts. Since multiple somatostatin receptors exist, identification of specific receptor subtypes linked to basolateral IK channels in intestinal and colonic epithelia may provide a basis for the development of new antidiarrheal drugs, which might act on a specific receptor.
Corticosteroid Regulation of K + Channels
Corticosteroids that include glucocorticoids and mineralocorticoids are secreted from adrenal gland and are required for basal intestinal and colonic functions. Glucocorticoids regulate electrolyte transport in both small intestine and colon, while mineralocorticoids primarily regulate the colonic electrolyte transports. Glucocorticoids and mineralocorticoids exert different aspects of electrolyte transport through specific receptors present in the epithelial cells of small intestine and colon. Although K + channels are present in the entire intestinal tract, only colonic K + channels are regulated by corticosteroids. Mammalian distal colon is a major target for the mineralocorticoids, as the mineralocorticoid receptor expression is high in distal colon. In vitro studies that measured unidirectional ion fluxes under voltage clamp conditions and intracellular microelectrode techniques that monitored ion movement have shown that glucocorticoids produce qualitatively different K + transport in proximal and distal segments of rat colon. These segmental variations have been suggested to occur as a result of different types of corticosteroid receptors in the mucosa. Receptor-binding studies have shown the presence of both glucocorticoid and mineralocorticoid receptors through the entire colon; however, the number of glucocorticoid receptors in the proximal colon are greater than that of mineralocorticoid receptors. Initial studies that examined the effect corticosteroids on electrolyte transport have shown that both glucocorticoids and mineralocorticoids stimulate electrogenic K + secretion in colon. However, studies that utilized RU-28362 (a glucocorticoid receptor specific agonist) have shown that a mineralocorticoid (i.e., aldosterone), but not glucocorticoid stimulated the electrogenic K + secretion in rat proximal and distal colon. The glucocorticoid agonist RU-28362 has been shown to bind more avidly to glucocorticoid receptor than dexamethasone or corticosterone and does not compete for radiolabeled aldosterone binding to the mineralocorticoid receptor. Thus, mineralocorticoid (aldosterone), but not a glucocorticoid (RU-28362) receptor-specific agonist stimulates K + secretion in mammalian colon.
Ion flux studies have shown that hyperaldosteronism, produced by exogenous aldosterone administration or secondary to chronic dietary-Na + depletion, stimulates electrogenic K + secretion in mouse and rat distal colon. Electrophysiological studies performed using patch clamp techniques and site-directed intracellular microelectrode techniques have shown enhanced K + conductance on the apical membranes of surface epithelial cells in aldosterone rat distal colon. Active K + absorption present in normal rat distal colon is reversed to active K + secretion in hyperaldosteronism rat distal colon ( Fig. 58.7 ). Both IK (i.e., KCNN4c) and BK (KCNMA1) channels contribute to aldosterone induce K + secretion, while inhibitor studies have shown that BK and IK channels mediated 64% and 29%, respectively, of aldosterone-induced K + secretion in rat distal colon. Western and quantitative PCR analyses have shown that aldosterone enhanced both KCNN4c and KCNMA1α-specific protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone has also enhanced KCNN4c and KCNMA1α mRNA levels that was prevented by exposure to actinomycin D (DNA-dependent RNA polymerase inhibitor). These observations indicate that aldosterone induced active K + secretion by enhancing mucosal KCNN4c and KCNMA1α expression at the transcriptional level.
Hyperaldosteronism is accompanied by an increased KCNN4 expression, suggesting that the expression of KCNN4 gene is controlled via the action of aldosterone on its cognate receptor (i.e., mineralocorticoid receptor; MR). Chromatin immunoprecipitation (ChIP) assay has identified the MR response elements (MREs) in a region that spanned 20 kb upstream and 10 kb downstream of the presumed transcription start site (TSS) in the chromatin of colonic epithelial cells of normal and aldosterone-treated rats. MREs have immunoprecipitated in an approximately 5 kb region that spanned the first and second introns in the aldosterone rats. When co-expressed with MR, these clones have exhibited aldosterone-activated enhancer activity in HEK293T and CaCo2 cells. Bioinformatics analyses have identified two MRE regions. These clones lost their enhancer activity after mutation of the presumptive MREs, and thus established the functionality of the MREs in KCNN4 genes. Bioinformatics analyses have also identified single MRE in KCNMA1α genes (Rajendran VM, et al.; unpublished observations). These observations have established that hyperaldosteronism regulate both IK and BK channels in the colon.
Pathophysiology of Colonic K + Channels
Role of K + Channels in Ulcerative Colitis
The healthy human colon absorbs 1.5–2 L of water a day, which is driven by the net absorption of large amounts of Na + and Cl − . These net absorptive fluxes of Na + and Cl − reflect several different transport mechanisms operating in different segments of the colon, which have been described in detail elsewhere. Healthy colon is also capable of water secretion, but in normal individuals fluid absorption far outweighs fluid secretion. However, in patients with active ulcerative colitis (UC), where the mucosa of the rectum and (to a variable extent) the colon is inflamed, diarrhea is the main and most debilitating symptom. Based on the results of a variety of studies, it is now clear that impaired Na + and Cl − absorption, rather than increased Cl − secretion, is the dominant ion transport abnormality underlying the pathogenesis of diarrhea in this disease. This creates ineffective fluid retention and increased fluid excretion.
Role of basolateral IK (KCNN4) channels during ulcerative colitis : Most of the studies to compare electrolyte transport processes in normal patients with those in active UC have been done in the distal colon and rectum, where electrogenic Na + absorption is normally the main Na + absorptive process. This generates a substantial lumen-negative transmucosal electrical PD in noninflamed distal colon. By contrast, a marked decrease (depolarization) or loss of this PD is the bioelectric hallmark of active UC, which reflects defective apical Na + channel function with the virtual disappearance of electrogenic Na + absorption, as well as basolateral membrane depolarization. As described, during Na + absorption, the basolateral membrane (and thus the cell interior) is normally maintained in a hyperpolarized state by K + ions recycling across the membrane via K + channels. Indeed, the negative intracellular potential is a prerequisite for apical Na + entry. The first study of a K + channel in human colonic epithelium, and subsequent studies, identified intermediate conductance (∼ 25 pS) Ca 2 + -activated K + channels as the dominant basolateral K + channel in human colonic crypt cells. These channels, encoded by the KCNN4 gene, are often referred to as IK channels. Molecular studies and patch clamp recording have evaluated the IK channels in colon from normal and active UC patients. Immunolabeling revealed basolateral IK channels are distributed uniformly along the surface-crypt axis in normals, with greatly decreased immunolabeling in active UC colon. Patch clamp analysis showed cell conductance to be dominated by basolateral IK channels in normals, but channel abundance and overall activity were decreased by 53% and 61%, respectively, in active UC, equating to a 75% decrease in basolateral membrane K + conductance in patients with active disease. Thus, in addition to defective apical Na + channel function, substantial decreases in basolateral IK channel expression and activity occur in active UC. This loss of IK channel function most likely accounts for the epithelial cell depolarization that occurs in this disease, resulting in a decreased electrical driving force for electrogenic Na + absorption across the inflamed mucosa. Interestingly, “normal” levels of IK channel expression and activity were seen in UC patients in clinical remission, which fits well with results from in vivo rectal dialysis studies, where UC patients in clinical remission had lumen-negative transmucosal PDs and net Na + , Cl − , and water absorptive fluxes identical to those in normals. Thus, chronic inflammation damages the mechanisms, which control Na + and K + transporters.
Role of basolateral KCNQ1/KCNE3 channels during ulcerative colitis : While IK channels dominate the basolateral K + conductance in human colon, the basolateral membranes of mouse colonic crypt cells also contain small conductance K + (KCNQ1/KCNE3) channels. KCNQ1 is a K + channel protein with six transmembrane domains that interacts with the smaller β-subunit KCNE3 protein, which has a single transmembrane domain. The KCNQ1/KCNE3 complex functions as a constitutively open basolateral K + channel, and is activated by cAMP, inhibited by chromanol 293B, and has a critical role in cAMP-stimulated electrogenic Cl − secretion. The KCNQ1/KCNE3 or SK channels maintain the Cl − secretory response by recycling K + entering the cell via the basolateral Na + -K + -2Cl − cotransporter, thus hyperpolarizing the cell while Cl − entering basolaterally exits the cell via apical CFTR channels. Although the inflamed mucosa in active UC contains high levels of a number of inflammatory cytokines that increase intracellular cAMP, the low/absent transmucosal PD seen in active UC is inconsistent with electrogenic Cl − secretion, and Cl − secretion has not been observed in previous transport studies, raising the possibility that the expression/activity of putative SK channels in the inflamed colon might be decreased. In a recent study, however, despite similar levels of KCNQ1 and KCNE3 mRNA expression in colonic crypts from normal and active UC patients, single cAMP-activated 6.8 pS channels were seen in 36% of basolateral patches in normals and in 74% of patches in active UC, with two or more channels per patch. Furthermore, overall channel activity was 10-fold greater in active UC, with a 20-fold greater contribution to basolateral conductance than in normals. Thus, SK channels appear to make a relatively small contribution to basolateral K + conductance in normal colonic epithelial cells, and even though enhanced, SK channel activity in active UC is insufficient to prevent cell depolarization. This provides additional evidence that defective Na + absorption rather than enhanced Cl − secretion is the main pathophysiological mechanism of diarrhea in UC.
Role of apical BK channels during ulcerative colitis : Apical BK channels have been studied extensively in mouse colon, where they are present along the entire surface cell-crypt cell axis. By contrast, in rat colon, apical BK channels localize to surface cells and cells in the upper 20% of crypts, with relatively low levels of channel abundance (as judged by patch clamp recording) in both the proximal and distal segments, although BK channel expression and abundance are greatly enhanced in the distal colon during chronic dietary K + loading. The distribution of apical BK channels (214 pS) along the crypt axis in normal human colon is similar to that in normal rat colon, without any obvious proximal-distal variation in channel expression. However, in patients with UC, the pattern of BK channel distribution is altered, so that BK channel protein is expressed uniformly along the entire surface cell-crypt cell axis, a change that is present irrespective of whether the disease is active or quiescent. It is presently unclear whether the wider distribution of BK channel protein along the entire crypt in UC patients results in an increase in luminal (apical) K + permeability, but if that is indeed the case, it may explain the increased colonic K + secretion that occurs in some patients with active UC, leading to excessive fecal K + losses and hypokalemia. The fact that the wider cryptal distribution of BK channel expression persists in quiescent UC (where colonic K + secretion is likely to be normal) suggests that fecal K + losses in UC are also dependent upon overall BK channel activity, which is likely to be stimulated in active UC by cAMP and/or Ca 2 + -mediated inflammatory cytokines.
Recent studies using an experimental model of DSS-induced distal colitis in rats have shown changes in apical BK channel expression and upregulation of active K + secretion, as well as histological changes, that are remarkably similar to those seen in human active UC. Whereas there was zero net K + transport in control animals, there was active K + secretion in DSS-treated animals, which was inhibited by 98%, 76%, and 22% by Ba 2 + (a nonspecific K + channel blocker), iberiotoxin (IbTX; a specific BK channel blocker), and TRAM-34 (a specific IK channel blocker), respectively. Compared to controls, apical BK channel α-subunit mRNA abundance and protein expression were enhanced six- and threefold, respectively. Thus, in DSS-induced colitis, active K + secretion involved upregulation of apical BK channel expression. Since similar changes in K + transport occur in patients with UC, diarrhea in this disease may reflect water secretion driven by increased colonic K + secretion, in addition to defective Na + and Cl − absorption. It is worth noting that both Ca 2 + and cAMP-stimulated Cl − secretion are defective in DSS-colitis rat distal colon ( Fig. 58.8 ). This experimental model of chronic colitis is likely to be extremely useful for future studies into the intracellular mechanisms that determine the ion transport defects underlying diarrhea in human UC.
Role of K + Channels in End-Stage Renal Disease
An important feature of the natural progression of end-stage renal disease (ESRD) is that patients tend to remain normokalemic for long periods in the face of steadily deteriorating renal excretory function. Adaptive changes occur within the remaining functional renal tubules, namely, enhanced K + uptake across the basolateral (peritubular) membrane, which is mediated by increased cortical and outer medullary Na + ,K + -ATPase activity. However, this response alone cannot entirely explain the maintenance of K + homeostasis in the predialysis phase (during which there is usually no or only a relatively small restriction of dietary K + intake), because urinary K + losses are generally substantially lower than in healthy individuals. This raises the question of how the body continues to excrete K + when renal K + excretory capacity is so impaired. A possible clue came from metabolic studies performed nearly 50 years ago, which showed enhanced fecal K + losses in patients with ESRD, with a strong correlation between dietary K + intake and fecal K + output, raising the possibility that some part of the intestinal tract developed an accessory K + excretory role during progressively deteriorating renal function. Indeed, such an adaptive response may account for sustained normokalemia in many patients, before they eventually require additional intervention in the form of continuous ambulatory peritoneal dialysis (CAPD), hemodialysis, and ultimately, renal transplantation. Subsequent studies into intestinal K + transport in rats with normal renal function fed a high K + diet for 10–14 days indicated that the colon, but not the small intestine, was capable of increasing its capacity for K + secretion in response to dietary K + enrichment. Chronic dietary K + loading stimulated a pancolonic active K + secretory process, which involved increased Na + ,K + -ATPase-mediated K + uptake across an amplified basolateral membrane, a rise in intracellular K + concentration, and an increase in apical membrane K + conductance. Furthermore, in vivo dialysis studies in healthy individuals and patients with ESRD indicated that rectal K + secretion was substantially greater than normal in normokalemic patients with ESRD who were not yet established on dialysis, normokalemic patients maintained on CAPD, and patients undergoing hemodialysis. Enhanced rectal K + secretion in these groups was independent of the transmucosal electrical PD, as well as the rate of Na + absorption and the circulating level of plasma aldosterone, which suggested that altered K + transport reflected the stimulation of an active K + secretory process.
A critical component of the upregulated active K + secretory process in rat colon elicited by chronic dietary K + loading is the induction and/or activation of high-conductance (BK; 220 pS) apical K + channels in surface colonic epithelial cells. Similar if not identical apical BK channels occur in surface cells around the luminal openings of human colonic crypts. In a recent study, the apical K + permeabilities of the proximal rectum in ESRD patients and patients with normal renal function were compared using the rectal dialysis technique, and the expression of BK channels in the distal colon of these two groups of patients evaluated using a specific BK channel antibody. Rectal K + secretion was almost threefold greater in ESRD patients than in patients with normal renal function, and intraluminal barium ions (a general inhibitor of K + channels) decreased K + secretion in the ESRD patients by 45%, but had no effect on K + transport in normal patients. Immunostaining with a specific antibody to the BK channel α-subunit demonstrated significantly greater levels of BK channel protein expression in surface colonocytes and crypt cells in ESRD patients than in patients with normal renal function, in whom low levels of expression were mainly restricted to surface colonocytes. Taken together, these results suggest that upregulated colonic K + secretion in ESRD reflects enhancement of the apical K + permeability of the large intestinal epithelium, most likely the result of increased expression of apical BK channels. What drives this adaptive change in apical BK channel expression in ESRD is unclear, but one possibility is postprandial increases in plasma K + concentration. This hypothesis was tested by measuring plasma K + concentrations in the fasting state, and for 180 min after the oral administration of 30 mmol of K + to control subjects and normokalemic patients with ESRD who were “predialysis” or undergoing CAPD. Plasma K + concentrations were also monitored in fasting controls and ESRD patients who were not given the oral K + load. Oral K + loading caused plasma K + concentration to rise within the normal range in control subjects, while significantly higher concentrations were achieved in “predialysis” patients and sustained hyperkalemia developed in CAPD patients. Thus, by raising the “K + load” presented to the colonic epithelium, postprandial increases in plasma K + concentration may be an important signal in maintaining the colon in a state of K + hypersecretion.
Despite the rise in colonic K + secretory capacity in ESRD, interdialytic hyperkalemia remains a potentially life-threatening problem in hemodialysis patients. cAMP elicits a greater proximal colonic net K + secretory response in dietary K + -loaded rats than in normal animals. This raises the possibility that interdialytic hyperkalemia might be attenuated by further enhancing colonic K + secretion using a cAMP-mediated laxative, thus limiting postprandial increases in plasma K + concentration in hemodialysis patients. This was evaluated by measuring plasma K + concentrations in control subjects and hemodialysis patients before and during two weeks treatment with bisacodyl (a cAMP-mediated laxative), and in hemodialysis patients before and during two weeks treatment with lactulose (an osmotic laxative). Bisacodyl treatment had no effect on plasma K + concentrations in control subjects, but significantly decreased the mean interdialytic plasma K + concentration in hemodialysis patients, whereas there was no change during lactulose treatment. Since cAMP stimulates both colonic apical BK channels and net colonic K + secretion, these findings suggest that cAMP-mediated laxatives may be a novel approach to the reduction of severe interdialytic hyperkalemia in hemodialysis patients.
Epithelial Na + Channel (ENaC)
The amiloride-sensitive apically located Na + channel (ENaC) is the rate limiting component of electrogenic Na + absorption. It is present in a variety of different organs including kidney, lung, and large intestine/rectum. Under basal (unstimulated) conditions, it is only expressed at low levels and provides a reserve capacity for Na + uptake. Following acute stimulation of mineralocorticoid or glucocorticoid receptors (MR and GR; Fig. 58.9 ), it is dramatically upregulated and can be further activated during chronic stimulation. Under these conditions, it becomes the predominant Na + uptake mechanism in the distal colon/rectum. When stimulated, there is an axial ENaC expression gradient along the large intestine of being very active in the distal colon and rectum and much less in proximal colon segments.
Molecular Identification and Function of ENaC
ENaC was originally cloned from Xenopus and rats, and later in man. ENaC consists of three subunits (α, β, and γ), which assemble as 1:1:1 heterotrimers. Furthermore, the subunit combinations are able to form higher-order structures containing up to three individual trimers. The role of a δ-ENaC subunit, which can substitute for the ENaC α-subunit, is less well defined, and is more important in the brain, being dispensable in the intestine. Immunohistochemistry localized ENaC to the apical border of surface cells in the piglet distal colon. In enterocytes, Na + entry across the apical membrane takes place along the electrochemical gradient provided by a low intracellular Na + concentration and the negative intracellular electrical potential created by the basolateral membrane Na + pump. ENaC mutations can lead to disturbed function, as in Liddle’s syndrome (a severe form of hereditary hypertension), where ENaC mutations in kidney and colon contribute to Na + overload. In this disease, increased ENaC function reflects changes in subcellular distribution rather than changes in channel conductivity.
Commercially available ENaC-expressing cell lines do not exist. To study ENaC expression, intestinal cell lines need to be transfected with either the mineralocorticoid and/or the glucocorticoid receptor. Without this transfection, they do not exhibit significant electrogenic Na + absorption, even if stimulated with corticosteroids.
Regulation of ENaC Expression
ENaC stimulation in the colon is the result of transcriptional upregulation and insertion of the β- and γ-ENaC subunit, while the α-ENaC subunit is constitutively expressed. The γ-ENaC subunit mRNA has a particularly short half-life. This mode of regulation is seen in the colon, whereas in the kidney, ENaC activity is regulated via changes in the expression of the ENaC α-subunit. The main stimuli are mineralocorticoids and/or glucocorticoids, the latter at high concentrations exhibiting significant crossover binding to the mineralocorticoid receptor (MR). The intracellular glucocorticoid receptor (GR) and MR dimerize and form homodimers but there is also experimental evidence for heterodimeric ENaC activation.
While aldosterone is effective in concentrations as low as 1–3 nM, glucocorticoids are only effective at much higher concentrations in the upper nanomolar range. This is due to the presence of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) in the colon, which oxidizes the active glucocorticoid cortisol to the inactive metabolite cortisone, and protects the human colon (and other aldosterone-sensitive epithelia, e.g., distal nephron) against intense ENaC activation even by low physiological levels of circulating cortisol (normal range 0.2–0.8 μM). 11β-HSD2 is inhibited by glycyrrhizin , a bioactive component of liquorice, as a consequence of which physiological levels of serum glucocorticoid activate ENaC in kidney and colon, giving rise to pseudo-hyperaldosteronism.
In addition to upregulation of the ENaC subunits, their insertion into the apical membrane is another key step of the activation in all ENaC-expressing tissues. The E3 ubiquitinin ligase Nedd4.2 plays a central role, which acts together with SGK-1 and other proteins in a multiprotein complex, the ENaC-regulatory complex (ERC). It phosphorylates the ENaC subunits and renders removable from the apical membrane. Nedd4.2 can be inhibited by the serum- and glucocorticoid-regulated kinase-1 (SGK-1), which is expressed during steroid signaling. Thus, this regulation of membrane insertion is essential and especially evident during exposure to short-chain fatty acids, which strongly upregulate β- and γ-ENaC mRNA, but do not increase Na + absorption, since the synthesized β- and γ-ENaC subunits are not inserted into the apical membrane without concomitant steroid signaling.
While mineralocorticoids and glucocorticoids are the main regulators, other signaling influences are important as well. In the colon, propionate and butyrate are abundant and intensify steroid-induced ENaC-dependent Na + absorption by altering the responsiveness of the β- and γ-ENaC-subunit promoters (see below). Other regulatory inputs on ENaC activity are stimulation by insulin and vasopressin. In addition, ENaC is affected by CFTR, since ENaC activity is increased in the colon in cystic fibrosis.
There is evidence that Na + absorption via ENaC is seriously disturbed in intestinal inflammation. Electrogenic Na + absorptive capacity and β- and γ-ENaC expression have been shown to be impaired in ulcerative colitis , Crohn’s disease, and microscopic colitis, which can be detected when intestinal biopsy specimens are studied during aldosterone stimulation in vitro. In general, ENaC-dependent Na + absorption is relatively modest in healthy controls (without external stimulation). In diarrheal diseases, however, ENaC-dependent Na + absorption becomes activated, in order to compensate for the ion and water loss as demonstrated, for example, in the NHE3-knock out model. This activation is due to endogenous mineralocorticoid stimulation as the result of dehydration. Thus, the inflammatory impairment of ENaC function is an important cofactor for the onset of diarrhea in IBD. Impaired ENaC function has also been shown to contribute to diarrhea in infectious diarrheal diseases.
The mechanisms involved in this inflammatory defect of ENaC expression are only partially understood. Proinflammatory cytokines like interleukin-13, which are released in ulcerative colitis, cause ENaC inhibition by p38-dependent expression arrest of the β- and γ-ENaC subunits, as well as by arrest of Nedd4.2-dependent ENaC insertion into the apical membrane due to decreased SGK-1 expression. However, in Crohn’s disease and microscopic colitis, TNFα is the key cytokine that hampers the transcription of β- and γ-ENaC, leading to lowered mRNA levels and arrest of insertion of the synthesized subunits into the plasma membrane, as the result of which electrogenic Na + absorption is attenuated. That this impairment can be normalized by MEK/ERK inhibition with U0126 in Crohn’s disease and microscopic colitis biopsies, as well as in rat colon exposed to TNFα in vitro, is a strong evidence for the involvement of MAPK-dependent intracellular signaling in this cytokine-induced impairment of active Na + absorption.
Pharmacological doses glucocorticoids such as hydrocortisone and prednisolone are effective in decreasing diarrhea in IBD and have been shown to cause a rapid activation of electrogenic Na + absorption in normal and inflamed human colon. This has to be assumed to be mainly caused by transcriptional upregulation and membrane insertion of the β- and γ-ENaC subunits, which overcomes the inhibitory influence of the proinflammatory cytokine. Indeed, synergism between therapeutic glucocorticoids and TNFα occurs, which likely explains the strong and rapid proabsorptive effect of this type of corticosteroid, since TNFα elevates the glucocorticoid receptor pool in epithelial cells via p38 signaling.
Furthermore, short-chain fatty acids therapeutically improve β- and γ-ENaC expression, and butyrate enemas have been used successfully to treat active ulcerative colitis. One of the mechanisms of this short-chain fatty acid effect has already been described above in this chapter. Butyrate can inhibit through histone deacetylase inhibition the binding of nuclear binding factors (such as SP3) to the promoter of the γ-ENaC subunit (that is, SP3 binding is increased), as the result of which promoter activity is increased.
Recently, in a search for plant glucocorticoids, endiandrin-A was identified as an activator of ENaC. This is caused by β- and γ-ENaC upregulation and activation of the serum- and glucocorticoid-induced kinase (SGK)-1 through an increase in glucocorticoid receptor (GR) expression via c-Jun N-terminal kinase (JNK) and p38 signaling.
Anion channel research was an underrepresented area of research within the ion channel field until the 1990s. This was different in the field of GI research, where it was recognized early that Cl − was the counterion secreted with protons for gastric acid secretion and, as mentioned in the introduction, that electrogenic Cl − secretion was the key event for intestinal fluid secretion. For both purposes, Cl − was intracellularly accumulated above the electrochemical gradient by basolateral Cl − /HCO 3 − exchangers and Na + K + 2Cl − cotransporters. Besides classical Ussing-chamber electrophysiology, the sequentially developed techniques for ion channel research, such as isotope fluxes in isolated epithelia, cells and membrane vesicles, ion and voltage sensitive microelectrodes, patch-clamp techniques and anion-selective dyes were all employed early in GI epithelial cells, and a variety of Cl − channels were characterized based on biophysical principles.
It is now clear that in most cells, Cl − ions are actively transported; that the intracellular Cl − concentration is not in electrochemical equilibrium, and that therefore Cl − channels or transporters are capable of volume, pH i and membrane voltage regulation both of the cell and of intracellular organelles. Cl − channels are involved in cellular events such as proliferation, apoptosis, protein synthesis, trafficking and degradation, and signaling. Members of all recently identified anion channel families are expressed in intestinal cells, likely involved in the above named “homeostatic” properties of Cl − channels, and the interested reader can find more information on Cl − channels in general in excellent recent reviews. This chapter will concentrate on intestinal anion channels that are directly involved in electrolyte absorption and secretion in GI epithelia.
The CFTR Channel
CFTR Is of Major Importance in Intestinal Fluid Balance—Lessons Learned From Knockout Mice
Soon after the description of a Cl − channel defect as the likely genetic basis for the disease called “cystic fibrosis” (CF) or “mucoviscidosis,” it became clear that the isolated small intestinal epithelium of patients with cystic fibrosis lacked a short circuit current response to cAMP-dependent secretagogues. The first description of apical Cl − channels in the intestine was in the late 1980s by Giraldez and colleagues, who used conventional and Cl − selective microelectrodes in Necturus intestine, and Frizzell and colleagues, who used the newly developed patch-clamp technique in T84 cells. Halm et al. demonstrated the existence of apical cAMP-activated Cl − channels in enterocytes. Such apical Cl − channels were unresponsive to cAMP in CF airway cells, and in isolated jejunal mucosal resectates or biopsies of CF patients. A year later, the CFTR gene was cloned by isolating overlapping complementary DNA clones from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus on chromosome 7. It was called “cystic fibrosis transmembrane regulator,” because of the predicted nonchannel structure of its gene product. Its expression in CHO cells (Chinese Hamster Ovary cells), however, resulted in the appearance of Cl − conductance. Despite some initial controversies whether the cftr gene encoded a Cl − channel or a regulator of Cl − channels, the former hypothesis was proven correct by the insertion of mutations into the CFTR gene and the demonstration of altered anion conductance properties when the gene was expressed. Soon after the cloning of the gene, the first CFTR-deficient mouse strains became available, which could recapitulate many although not all aspects of the human disease.
CFTR-deficient mice display an absent small intestinal cAMP-induced Isc, defective colonic Isc stimulation, as well as absent second-messenger induced small intestinal HCO 3 − secretion. All CFTR-deficient or CFTR-mutant animal models develop an intestinal obstruction soon after birth or during the weaning phase as the leading cause for death, unless rigorous preventive measures are taken. Transfer of CFTR gene into the epithelium of these mice could rescue the phenotype. In contrast, the lungs, the pancreas, and the liver of CFTR-deficient rodents are not severely affected, probably because of a relatively higher expression of non-CFTR apical anion channels in those organs compared to man. CFTR-deficient pigs invariably die of intestinal obstruction at an early age unless a intestine-specific CFTR knockin prevents obstruction, in which case the pigs can grow up to display pulmonary and pancreatic disease. While these findings highlighted the enormous importance of the CFTR anion channel in intestinal physiology of rodents and pigs, it also called for caution when extrapolating the results obtained in gene-deficient mice to humans with cystic fibrosis, which have defective, but rarely absent, CFTR expression. The presence of “residual CFTR activity,” but also of “non-CFTR anion conductance activity” assessed by placing rectal biopsies of CF patients into mini-Ussing chambers and measuring the short circuit during sequential addition of substances that were known to activate CFTR and active or inhibit non-CFTR anion channels, were correlated with overall disease outcome, but it is still not clear what the “non-CFTR anion conductances” may be. Such “alternative” agonist-activated apical anion channels (proposed candidates are discussed at the end of this chapter) could easily be identified in cultured intestinal cell lines, but so far escaped molecular identification in human or rodent intestine.
Regulation of CFTR Activity
Anion efflux via CFTR is regulated at several levels, described in brief below. In addition to its role as a Cl − channel, it is discussed as a “hub” protein, interacting with a large variety of proteins and influencing a plethora of cellular and organ functions. This function of CFTR as an “interactome” platform is reviewed in detail elsewhere, and will only be discussed in its importance in intestinal transport regulation. In addition, a huge body of literature exists on the inhibition of CFTR activity by endogenous and exogenous substances and the potential detrimental effects on normal, or beneficial effects on dysregulated CFTR function. Substance-induced CFTR inhibitory effects will not be a major part of this review.
CFTR channel gating : The CFTR channel bears no similarity to any other Cl − channel family, but is a member of the ABC transporter family, which hydrolyzes ATP. In contrast to all other members of this family, which use the energy from ATP hydrolysis to transport large hydrophobic ions against a concentration gradient, CFTR utilizes ATP hydrolysis for channel opening and closing. The CFTR channel consists of two transmembrane domains, two nucleotide-binding domains (NBD), and a large cytoplasmic regulatory domain. ATP binding to the NBDs triggers a dimerization of the NBDs, resulting in a conformational change of the transmembrane regions with an accessibility of the pore from the cytoplasmic side. Recent data suggest, however, that the gate of CFTR is located toward the extracellular part of the pore. More detailed information on CFTR gating by ATP binding can be found in several excellent recent reviews. ATP-independent CFTR gating has been found to occur through the insertion of constitutive point mutations of the ATP-binding mutant G551D-CFTR, and the NBD2 deletion mutant, which resulted in channel opening when triggered by PKA phosphorylation.
While ATP is necessary, the regulated step of CFTR channel opening is the phosphorylation of the regulatory (R) domain. The protein kinase A (PKA) is best studied in this respect, but other kinases such as the cGMP-dependent kinase II, tyrosine-, or Ca 2 + -regulated kinases may have similar effects. The phosphorylation results in dynamic changes of the R domain that allosterically modulate intramolecular interactions in the NBD1:NBD2 interface and also within the R domain and in membrane domains, making the CFTR molecule more compact. A disturbance of phosphorylation is the underlying defect of many disease-causing mutations in cystic fibrosis, and to counter this effect is one of the strategies employed by the CFTR potentiators. CFTR gating is also influenced by binding of the R domain to the STAS domain of SLC26A transporters and to the regulatory protein IRBIT (IP3-receptor binding protein released with IP3). These interacting proteins will be discussed below in more detail.
Regulation of CFTR synthesis, trafficking, and membrane stability : McDonald and colleagues demonstrated the existence of a cAMP-responsive element in the promotor region of the CFTR gene and proposed that PKA may increase the basal expression of CFTR by increasing transcriptional activity and possibly by stabilizing the mRNA. Once synthetized, CFTR possesses multiple PKA consensus sites that display a low level of constitutive phosphorylation, which is necessary for CFTR trafficking in the early secretory pathway. Since the majority of disease-causing mutations are trafficking mutants, very intense research by many laboratories delineated the biogenesis of CFTR in cytosolic ribosomes, and the insertion to the endoplasmic reticulum (ER), where core glycosylation and chaperone-assisted folding takes place, and where the protein meets its first-quality control processes. This leads to retention of misfolded CFTR mutants such as F508del CFTR in the ER and from there to degradation via the ubiquitin-proteasome pathway. Transport of the core-glycosylated CFTR (band B) to the Golgi via the coat protein II (COPII)-coated vesicles is another quality control checkpoint through which mutated CFTR may not pass because of exposed ER retention motifs. Mature, fully glycosylated CFTR (band C) exits the trans-Golgi in exocytotic vesicles, whose insertion into the plasma membrane is facilitated by several Rab GTPase proteins that establish the interaction of the exosomes with myosin molecular motors and control the correct docking and fusion of vesicles with the correct target membranes. The association of CFTR with myosins appears to be cell type-specific. In the intestine, myosin 6 and 1a have been implicated in CFTR brush-border membrane expression in a segment-specific fashion. At different stages during its exocytosis/recycling/degradation pathway, CFTR also interacts with several proteins of the SNARE family, some of which even influence CFTR channel gating. In the intestine, syntaxin 3, together with SNAP23, seems particularly important in targeting CFTR to the apical membrane, and syntaxin 3 silencing reduced cAMP-induced cell surface CFTR expression by about 30%. Results from recent studies revealed that CFTR, mutant CFTR, and other membrane proteins can reach the plasma membrane via an unconventional alternative route that bypasses Golgi in specific cellular conditions. In this case, CFTR or CFTR mutants are only core-glycosylated, and this may explain why certain mouse strains express both band C and a little band B in even highly purified brush-border membrane preparations.
While biogenesis and maturation of CFTR are slow, its internalization from the plasma membrane is rapid. The recycling of internalized channels, in which Rab11 plays an essential role, is a key process in ensuring adequate CFTR availability. Shown in detail in intestinal cells for another anion transporter, but likely also crucial for CFTR, is the role of the PDZ domain ligand motif in the C terminus for determining whether the transporter is targeted to the recycling endosomes or the endosomal degradation pathway.
Only about half of the CFTR pool in the plasma membrane undergoes rapid recycling, the other half is stabilized in the membrane by tethering to filamentous actin by virtue of its PDZ interaction motif. This involves binding of CFTR to the PDZ domain adaptor Na + /H + exchanger regulatory factor-1 (NHERF1), which interacts via its ezrin/moiesin/radixin (ERM) domain with the actin-binding molecule ezrin. Indeed, we found that NHERF1-deficient mice showed a decrease in the small intestinal brush-border membrane abundance of CFTR and a marked reduction of cAMP-stimulated short circuit current response in isolated mucosa and in fluid secretion in vivo. Interestingly, overexpression of NHERF1 in airway cells was able to target even the trafficking mutant F508del to the plasma membrane and result in functional Cl − channel activity, a process that involved activation of RhoA, the Rho-kinase Rock, and phosphorylation of ezrin. In addition to interfering with the early degradation of misfolded CFTR mutants by interfering with the quality control system or by introducing artificial chaperones, the activation of endogenous small GTPase signaling and other modes of stabilizing mutant CFTR at the apical membrane are important strategies for targeted therapy in cystic fibrosis and have already achieved several breakthroughs. The use of rectal biopsies or intestinal organoids from CF patients allows a mutation-specific assessment of drug effectiveness in achieving these goals.
CFTR internalization is enhanced by the WNK (with no lysine kinase)/SPAK signaling pathway and counteracted by IRBIT/PP1. IRBIT has also been described to enhance CFTR gating, to orchestrate the interplay of apical and basolateral HCO 3 − transporters in the pancreatic duct cell, and to link the cAMP/Ca 2 + signaling to CFTR, thereby potentiating the individual second messenger signals. In the intestine, the importance of this signaling system has not yet been established.
Macromolecular complex formation via PDZ-domain interactions : PDZ-domain interactions not only enhance the expression of both WT and mutated CFTR in the apical membrane but also modulate its functional activity and facilitate the formation of multiprotein complexes, so-called “signalosomes,” whereby the interaction partners are brought into close proximity so that they can interact with each other. Reviewed recently in detail, PDZ-domain protein-mediated interactions may bring receptor, signaling molecules, or other transporters into close proximity with the CFTR protein and thereby enable functional interaction. In the intestine of NHERF1-deficient mice, β 2 -receptor mediated CFTR-dependent luminal alkalinization is completely lost in vivo, while it is intact in NHERF2- and NHERF3-deficient animals. In contrast, the CFTR stimulation by the lysophosphatidic acid receptor subtype-2 was absent in NHERF2-deficient cells and in NHERF2-deficient mice. Shcheynikov and colleagues reported that the interaction of CFTR and anion transporters of the SLC26a family occurred only when their respective PDZ binding motifs were present. The resultant interaction of the STAS domain of the SLC26A members with the R domain of CFTR resulted in increased CFTR gating. Genetic deletion of NHERF3 (PDZK1) resulted in reduced agonist-stimulated jejunal HCO 3 − output (possibly mediated via Slc26-CFTR interaction (331) but no change in short circuit current response in isolated jejunum, suggesting that PDZK1, which is highly expressed in the small intestine, may be one of the PDZ-adaptors that confers such an interacting platform for the SLC26A members that are coexpressed with CFTR in the base of the villi. PDZK1 has also been reported to confer a molecular interaction between CFTR and the multidrug transport protein MRP3, which mediates cAMP efflux, and the absence of MRP3 was reported to result in increased susceptibility to CFTR-dependent diarrhea in mice. In our hands, PDZK1-deficient mice did not show this phenotype, possibly because the absence of PDZK1 interferes with CFTR dimerization or interaction with other transporters. In a CFTR mutant airway epithelial cell line, the absence of CFTR in the apical membrane disturbs the subapical cytoskeleton, results in a lack of the apical NHERF1-ezrin-actin complex with a failure to anchor PKA, and thus enables cAMP to diffuse away from the apical membrane and result in alternative, potentially harmful signaling (i.e., stimulating nuclear CREB, enhancing growth, etc.). CFTR corrector therapy rescues the apical cytoskeletal network and reestablishes compartmentalized cAMP signaling. A failure to form metabolically important AMP kinase and NDP kinase multiprotein complexes in the absence of CFTR has been related to detrimental consequences for cellular signaling and wasteful energy expenditure. It is unclear, however, if a disturbance of the apical cytoskeleton with dysfunctional compartmentalization of cAMP, etc. is also ongoing in intestinal cells, because the brush-border membrane contains many other ion transport proteins, even with much higher expression levels than CFTR, with PDZ domains that bind NHERF1 and assemble cytoskeletal-anchored multiprotein complexes. In addition to the NHERF proteins, other scaffolding proteins play a role in CFTR membrane expression and function, but their significance has not been not studied in the intestine yet.
NHERF PDZ-domain ligand interactions have been crystallized, and the design of small molecules that interfere with PDZ-domain interaction is considered an interesting strategy both for cystic fibrosis as well as for enterotoxin-mediated secretory diarrhea and modulation of drug transport.
CFTR Membrane Expression Within the Different Intestinal Segments and Along the Crypt to Villus Axis
Investigations into the molecular interactions between CFTR and other transporters, regulatory proteins, or receptors have mostly utilized heterologous expression systems for at least one of the interacting components. While this demonstrated that a molecular interaction is possible, a biological significance of these findings requires the demonstration of cellular or even subcellular colocalization of the interacting partners in the native tissue. In the intestine, the demonstration of the cellular expression pattern of CFTR and its potential interacting partners depends on the sensitivity and specificity of the antibodies, which can only be ascertained with certainty if null-deletion knockout tissue is available, and is incomplete at this time. Detailed segmental and crypt to villus axis expression studies for CFTR with demonstration of strong cryptal but not villous expression have been performed in human, and rodent intestine, and recently the crypt predominance of CFTR expression has also been described in pig intestine. While these studies demonstrated crypt-predominance of CFTR in all intestinal segments, substantial CFTR expression was also seen in murine and rat villus cells, particularly in the duodenum. In the large intestine, a strong crypt-predominance of CFTR, known from functional studies on cAMP-activated anion efflux from colonocytes isolated along the crypt-surface axis, was ascertained. In rat and human jejunum, “CFTR high expresser cells” are interspersed as single “secretory” enterocytes with a very high CFTR staining in the brush-border membrane among the absorptive enterocytes along the lateral villus surface. These cells highly express an apical V-ATPase and basolateral NKCC1, but no NBCe1, Slc26a members or NHE3, suggesting that their functional role is exclusively one of fluid secretion, with no absorptive function. They resemble the cells of the Brunner’s glands in the transporter expression profile, but are completely different from the pancreatic ducts cells, suggesting that they secrete mostly Cl − and fluid, and less HCO 3 − than the pancreatic duct cells (which is consistent with what we measure from the crypt openings). This suggests that the transport principles of epithelial HCO 3 − secretion are profoundly different in the small intestine and the pancreas, at least in rodents. Throughout the intestine, NBCe1 and NBCn1 (if expressed in the respective segment) are predominantly expressed in the villi, suggesting that both transporters counteract enterocyte acid loads and import HCO 3 − to be exchanged for luminal Cl − rather than for HCO 3 − secretion via CFTR. Consistent with the presence of the “CFTR high expresser cells” in the villi, functional investigations also suggest that human duodenal villi can secrete Cl − . In the colon, CFTR mRNA and protein expression are crypt-predominant and rather uniform along its entire length (own unpublished observations), whereas Slc26a3 (DRA) and NHE3 are surface cell predominant, and their expression is strongly segmental. This is in accordance with the observation that during the digestive process, absorptive and secretory functions of the intestinal epithelium need to occur in parallel, rather than in an alternative fashion such as in the pancreatic or salivary ducts.
CFTR-Dependent and -Independent Intestinal Electrogenic Anion Secretion
Traditionally, cAMP-stimulated Isc responses have been attributed to CFTR activation, while Isc responses activated by Ca 2 + -dependent agonist have been attributed to activation of a putative calcium-dependent chloride channel (CACC). In isolated CFTR null murine intestine (i.e., from cftr tm1Cam , cftr m1UNC mice), which expresses no functional CFTR protein, the apical or basolateral addition of Ca 2 + -mobilizing agonists fails to elicit an Isc response that has the appearance of an anion channel-mediated response, except in the distal colon, where we occasionally found a small carbachol-induced Isc response, depending on the mouse background strain and the age of the mouse. This suggests that Ca 2 + -activated Cl − channels are either not present in the brush border membrane of murine intestine, or cannot be activated, possibly due to a high apical membrane potential. Wilschanski et al. found a rectal PD response to UTP in Cftr m1HSC/m1HSC “long term survivor” mice on a mixed CD1/129SvJ background, which was not observed in the CFTR m1UNC/m1UNC mice that die early. Gyömörey et al. studied the small intestine of these mice (because they die of intestinal obstructions in the small intestine), and found a higher basal PD in the long-term survivors than in a CFTR-deficient strain with early lethality. Because basal PD is a sum signal of many electrogenic processes in the apical and basolateral membranes and in the tight junctions, this finding does not necessarily indicate the presence of a non-CFTR Cl − conductance. We performed bilateral 36 Cl − flux studies in CFTR-deficient murine jejunum of the Cftr tm1Cam/tm1CAM mice on the NMRI background, on which they survive well, and found a slightly reduced mucosal to serosal (m-s) Cl-flux and no increase in serosal to mucosal (s-m) Cl − flux upon stimulation in the CFTR −/− jejunum ( Fig. 58.10 ). Interestingly, the heterozygote jejunal mucosa, which did not display a significant reduction in Isc response to stimulation, nevertheless had a lower s-m Cl − flux, suggesting that under maximal stimulation, the Cl − flux may show a better correlation with synthetized CFTR protein than the peak Isc response. Indeed, Wilke et al. had demonstrated that after low temperature rescue of F508del in isolated mouse jejunum, a 20% increase of CFTR mutant protein in the plasma membrane resulted in a full Isc response to agonists. An in vivo jejunal perfusion study in cystic fibrosis pediatric patients also did not show an increase in the transepithelial PD upon theophylline application, although in CF patients, who express mutant but not necessarily completely dysfunctional CFTR, variable PD responses have been observed in various epithelia.