58.2.1.1

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.

58.2.1.2

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.

58.2.2.1

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.

Cellular model of active K ⁺ secretion in colon. Coordinated activation of apical large conductance K ⁺ (BK) channels, and basolateral Na ⁺ -K ⁺ -2Cl ⁻ cotransporter (NKCC) and Cl ⁻ channel-2 (CLC-2) channels, regulates the cAMP-induced K ⁺ secretion, which is inhibited by iberiotoxin (IbTX). ClC2 has been shown to be present in the basolateral membranes of mammalian colon. Direction of transcellular electrolyte movements ( ); cAMP-activation ( ); channel blocker ( ); electrolyte recycling ( ); driving force ( ).

(Reproduced from Sandle GI, Rajendran VM. Cyclic AMP-induced K ⁺ secretion occurs independently of Cl ⁻ secretion in rat distal colon. Am J Physiol Cell Physiol 2012; 303 :C328–33. PMC3423024.)

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.

58.2.2.2

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.

58.2.2.3

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.

Effect of sequential addition of mucosal and serosal DC-EBIO (an intermediate conductance K ⁺ (IK) channel opener) on active Cl ⁻ and K ⁺ secretion in normal rat distal colon. Active Cl ⁻ (measured as short circuit current ( Isc )) and ⁸⁶ Rb ⁺ (K ⁺ surrogate) secretion were measured in the presence of mucosal ortho-VO ₄ (H ⁺ ,K ⁺ -ATPase inhibitor) under voltage clamp condition. (A and B) Mucosal DE-EBIO stimulated both Cl ⁻ (A and B) and K ⁺ secretion. Serosal addition of DC-EBIO in the continued presence of mucosal DC-EBIO further stimulate the Cl ⁻ and K ⁺ secretion * P < .001 compared with basal; ** P < .001 compared with DC-EBIO (m); ^£ P < .05 compared with DC-EBIO (s).

(Reproduced from Nanda Kumar NS, Singh SK, Rajendran VM. Mucosal potassium efflux mediated via Kcnn4 channels provides the driving force for electrogenic anion secretion in colon. Am J Physiol Gastrointest Liver Physiol 2010; 299 :G707–14. PMC2950693.)

58.2.2.4

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.

58.2.5.1

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.

Effect of dextran sulfate sodium (DSS)-induced inflammation on Cl ⁻ and K ⁺ secretion in rat distal colon. (A) Increasing intracellular cAMP by forskolin (FSK, an adenylate cyclase activator) and Ca ^{2 +} by carbachol (CCH, adrenergic agonist) stimulate active Cl ⁻ secretion in normal colon. (B) DSS-induced inflammation diminished both cAMP- and Ca ^{2 +} -stimulated Cl ⁻ secretion. (C) In the presence of mucosal ortho-VO ₄ (H ⁺ , K ⁺ -ATPase inhibitor) neither K ⁺ absorption nor K ⁺ secretion is present, while cAMP induced the active K ⁺ secretion in normal colon. The cAMP-stimulated K ⁺ secretion is not further stimulated by CCH. (D) DSS-induced inflammation readily induced the active K ⁺ secretion in colon. The inflammation induced K ⁺ secretion is not altered by cAMP and CCH.

(Reproduced from Kanthesh BM, Sandle GI, Rajendran VM. Enhanced K ⁺ secretion in dextran sulfate-induced colitis reflects upregulation of large conductance apical K ⁺ channels (BK; Kcnma1). Am J Physiol Cell Physiol 2013; 305 :C972–80. PMC4042538.)

58.2.5.2

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.

58.4.1.1

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.

58.4.1.2

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.

58.4.1.3

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.

58.4.1.4

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.

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