Glycyrrhiza glabra is one of 30 species of licorice which has been prescribed for centuries for a wide range of ailments and conditions. In particular, licorice root extract, as well as its main isoflavonoid constituent, glabridin, have been used to treat a variety of respiratory diseases, ranging from infection to asthma. As glabridin has been shown to modulate the activity of ion channels in other tissues, we determined the effect of glabridin on K+ and Cl⁻ secretion across primary human bronchial epithelial cells (HBEs), as these may represent therapeutic targets. Glabridin stimulated BKCa-dependent transepithelial potassium secretion across HBEs. In contrast, the glabridin derivative, vutiglabridin failed to stimulate K+ secretion and inhibited the glabridin-dependent K+ secretory current. Whole-cell patch-clamp studies on HEK cells expressing BKCa demonstrate that glabridin activates, whereas vutiglabridin inhibits, BKCa. We further demonstrate glabridin inhibits forskolin-mediated transepithelial Cl⁻ secretion across HBEs, while vutiglabridin has little effect. Using Fisher Rat Thyroid (FRT) cells stably expressing either wild type (FRT-WT) or F508del CFTR (FRT-F508del), we demonstrate glabridin neither potentiates WT CFTR nor corrects F508del CFTR. In contrast, whole-cell patch-clamp studies demonstrate glabridin inhibits KCa3.1 stably expressed in HEK cells. We previously demonstrated a role for KCa3.1 in forskolin-mediated Cl⁻ secretion across HBEs, likely explaining the inhibition of Cl⁻ secretion observed. In summary, we show that glabridin both stimulates BKCa-dependent K+ secretion and inhibits cAMP-mediated Cl⁻ secretion across HBEs. These findings support a role for BKCa in the therapeutic effects of glabridin in airway.

We previously demonstrated the CFTR correctors VX-445 (elexacaftor) and S-VX-121 (vanzacaftor) potentiate heterologously expressed BKCa channels, as well as in primary human bronchial epithelial cells (HBEs). This potentiation of BKCa resulted in altered vasoreactivity and neuronal excitability. We postulated novel compounds could be identified that would potentiate BKCa while not affecting CFTR. Herein, we demonstrate that the enantiomer of vanzacaftor, R-VX-121, possesses these attributes. Using Fisher rat thyroid (FRT) cells expressing F508del CFTR, we demonstrate S-VX-121 corrects F508del CFTR when incubated overnight, as assessed by an increase in transepithelial Cl- current (ICl) in response to forskolin, as well as the appearance of band C upon immunoblot (IB). In contrast, R-VX-121 failed to increase ICl and induce band C. Importantly, R-VX-121 competed with S-VX-121 to eliminate the correction of F508del CFTR observed during both ICl measurements and IB, indicating it associates with CFTR. Neither S- nor R-VX-121 potentiated CFTR, as assessed by changes in ICl. Distinct from our CFTR results, both S- and R-VX-121 potentiated BKCa in primary HBEs as well as during whole cell patch-clamp recording of heterologously expressed α-BKCa. Using wire myography, we demonstrate both S- and R-VX-12 vasodilate preconstricted mouse mesenteric arteries in a paxilline-dependent manner, confirming a role for BKCa. In contrast, the CFTR inhibitor, CFTRinh172 did not alter the effects of S- and R-VX-121 on vasoreactivity, confirming CFTR is not involved in this response. These data demonstrate R-VX-121 represents a novel BKCa potentiator that does not modulate CFTR function, suggesting R-VX-121 may be clinically useful as a BKCa agonist.NEW & NOTEWORTHY We previously demonstrated that the CFTR correctors, VX-445 and S-VX-121, are BKCa channel potentiators. These CFTR correctors altered vasoreactivity and action potential firing frequency effects, which may explain the adverse events (AEs) reported in cystic fibrosis (CF). We now demonstrate that the enantiomer of vanzacaftor, R-VX-121, potentiates BKCa, while not correcting or potentiating CFTR. Thus, we have identified a novel BKCa potentiator that may be useful in diseases where BKCa modulation is therapeutically proposed.

Cystic fibrosis (CF) is a genetic disorder resulting from mutations to the CF transmembrane regulator (CFTR) anion channel. CFTR correctors partially restore the folding and trafficking of mutant CFTR. We recently demonstrated that the correctors VX-445 and VX-121 directly potentiate large-conductance Ca2+-activated (BKCa) channels. We postulated that this could enhance the therapeutic potential of these drugs in the lung by increasing the driving force for transepithelial Cl- secretion. Herein, we evaluated the effect of acute addition of VX-445 on forskolin- and 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one-mediated Cl- secretion across primary human bronchial epithelial cells (HBEs) from wild type (WT) and F508del donors. Surprisingly, VX-445 (10 µM) induced a significant inhibition of forskolin-stimulated Cl- secretion in WT and F508del donor HBEs with corrected CFTR. We hypothesized that this was due to inhibition of the basolateral membrane Ca2+-activated K+ channel, KCa3.1 that maintains the driving force for Cl- secretion. Thus, we utilized patch-clamp techniques to evaluate VX-445 effects on isolated KCa3.1 currents. We demonstrate that VX-445 directly inhibits KCa3.1, as do similar molecules VX-659 and VX-121; however, only VX-659 inhibited KCa2.3 and KCa2.2 with a similar affinity to KCa3.1. To summarize, acute addition of CFTR correctors to HBEs reduces transepithelial Cl- secretion due to inhibition of KCa3.1.

Correct function of the Ca2+-activated K+ channel KCa2.3 (SK3) is essential for several physiological processes. Activity of KCa2.3 in neurons modulates the rate of action potential firing; in endothelial cells activation of KCa2.3 channels induces vasodilation, and accordingly, a decrease in blood pressure. KCa2.3 activity (i.e. currents) can be modulated by two main variables: the open probability ( Po) and the number of channels inserted into the plasma membrane ( N). Numerous pharmacological compounds have been identified and developed to alter KCa2.3 Po. In contrast, the mechanisms that control delivery of KCa2.3 to and from the plasma membrane are still poorly understood. After endocytosis, plasma membrane proteins are targeted to the endosomal network which is a key area for sorting membrane-bound proteins. Several endosomal-associated coat complexes have been identified that ‘rescue’ proteins from degradation and sequester proteins into recycling tubules for transport back to the plasma membrane. These complexes include Retromer, Retriever, and Commander. Associated with these complexes are members of the Sorting Nexin (SNX) family. SNX proteins bind to specific amino acid motifs to sort proteins for Retromer- or Retriever-mediated recycling. Interestingly, the Devor laboratory has demonstrated KCa2.3 can be rapidly recycled back into the plasma membrane after endocytosis. Based on those data, we hypothesised that the Retromer and Retriever complexes regulate KCa2.3 recycling. To investigate this, we are using biochemical [e.g. cell surface biotinylation, co-immunoprecipitation (co-IP)] and electrophysiological (e.g. patch-clamp) techniques. First, we determined the role of SNXs in KCa2.3 traffcking by using siRNA to knock down SNX protein expression in Fisher rat thyroid (FRT) cells expressing heterologous KCa2.3. Knockdown of Retromer-associated SNX3 or Retriever-associated SNX17 significantly decreased the cell surface population of KCa2.3 ( P<0.01, n=4, each). With protein-protein interaction experiments, KCa2.3 co-IPed with SNX17 and SNX3, but not Retromer-associated SNX27 ( n=3, each). Electrophysiology experiments are underway to determine if the knockdown of SNXs alters the functional expression of KCa2.3. and We are also investigating if KCa2.3 traffcking in primary endothelial cells requires SNX3 and SNX17. These results suggest that SNX3 and SNX17 are novel regulators of KCa2.3 traffcking. This work was supported by grants from NIH (HL083060, HL092157) and the Cystic Fibrosis Foundation (DEVOR20GO) to D.C.D.; Lottery Health New Zealand (R-LHR-2019-101706) to F.J.M.; and Aim Fund grant to K.L.H., and the Department of Physiology, University of Otago. M.J.E.L. was supported by a doctoral scholarship from the University of Otago. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.

The intermediate-conductance Ca 2+ -activated K + channel KCa3.1 ( KCNN4) regulates many physiological processes, including, water and electrolyte transport in polarised epithelia and vascular tone. Crucial for KCa3.1 function is the regulation of the number of channels at the plasma membrane. Even though, the mechanism of trafficking of KCa3.1 to and from the basolateral membrane (BLM) of epithelial cells has been advanced recently, there is opposing evidence suggesting that KCa3.1 is recycled back to the leading edge of migrating cells; while others have reported that KCa3.1 does not enter recycling endosomes, but is targeted for degradation. The highly conserved multi-protein complex Retromer has been demonstrated to regulate the membrane retrieval and recycling of many membrane-bound proteins, including ion channels. Using mass-spectrometry, it was reported that when VPS35, a core protein of the Retromer complex, was knocked-down, the membrane expression of KCa3.1 was reduced 1.4 fold in HeLa cells; suggesting a link between Retromer and trafficking of KCa3.1. In contrast, our data indicate KCa3.1 was not affected by knockdown of VPS35 in our FRT (Fischer rat thyroid)-KCa3.1 stable cell line. Here, we have examined other key components (sorting nexins SNX1, SNX4, SNX27; cargo binding proteins) of the Retromer complex to determine if those proteins aid in trafficking of KCa3.1 directly to the BLM. We conducted immunoblot and Ussing chamber experiments to examine the effects of knockdown of SNX 1, SNX2 or SNX27 on the BLM population of KCa3.1 and functional expression of the channel (K + current), respectively. Using our FRT-KCa3.1 cell line, we demonstrate knockdown of SNX1, SNX4 or SNX27 decreases KCa3.1 current. Further, we demonstrate knockdown of SNX4 or SNX27 decreases BLM expression of KCa3.1, consistent with the decrease in current observed.These data, along with our previous data, strongly indicate that the role of Retromer in the trafficking of KCa3.1 to the BLM of epithelial cells is quite dynamic. Reducing SNX4 or SNX27 have notable effects on trafficking of KCa3.1. While knockdown of SNX1 might affect the trafficking of KCa3.1 by altering other proteins of the Retromer complex. This work was supported by the National Institutes of Health grants HL083060 and HL092157 to DCD, Lottery Health New Zealand (R-LHR-2019-101706) to FJM, Aim Fund grant to KLH, and the Department of Physiology, University of Otago. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Clsecretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl- equivalent current (ICl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (Ra) and basolateral membrane (BLM; Rb) resistances. In WT CFTR HBEs, DCEBIO stimulated ICl, which was increased by forskolin. Similarly, forskolin stimulated ICl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited ICl. DCEBIO decreased Rb, whereas TRAM-34 increased Rb, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate ICl, although the subsequent addition of forskolin + VX-770 increased ICl. Importantly, following stimulation of ICl with forskolin + VX-770, DCEBIO induced a further significant increase in ICl. As above, DCEBIO reduced Rb, whereas TRAM-34 increased Rb, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on ICl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate ICl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled ICl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl- secretion across CF airway.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl − secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl − secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl − equivalent current (I Cl ) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (R a ) and basolateral membrane (BLM; R b ) resistances. In WT CFTR HBEs, DCEBIO stimulated I Cl , which was increased by forskolin. Similarly, forskolin stimulated I Cl , and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited I Cl . DCEBIO decreased R b , whereas TRAM-34 increased R b , consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate I Cl , although the subsequent addition of forskolin + VX-770 increased I Cl . Importantly, following stimulation of I Cl with forskolin + VX-770, DCEBIO induced a further significant increase in I Cl . As above, DCEBIO reduced R b , whereas TRAM-34 increased R b , consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on I Cl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate I Cl . However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled I Cl . Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl − secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl − secretion across CF airway.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

The small‐ and intermediate‐conductance Ca2+‐activated K+ channels KCa2.3 (KCNN3) and KCa3.1 (KCNN4) regulate a plethora of physiological processes: including, water and electrolyte transport in polarised epithelia, neuronal firing, and vascular tone. Critical for KCa2.3 and KCa3.1 function is the regulation of the number of channels at the plasma membrane; however, the mechanisms that regulate the trafficking of KCa2.3 and KCa3.1 to and from the plasma membrane are still poorly understood. There are several marked differences in the trafficking pathways of these two channels, e.g. KCa2.3 has previously been established to recycle back to the plasma membrane after endocytosis; however, there are differences in opinion if KCa3.1 recycles in the literature. The highly conserved multi‐protein complex retromer has been demonstrated to regulate the retrieval and recycling of many membrane‐bound proteins, including ion channels. We hypothesised that retromer was required for the recycling of both KCa2.3 and KCa3.1. To test this hypothesis, we utilised a combination of biochemical and electrophysiological methods. Stabilisation of retromer with the pharmacological chaperone R55 increased the KCa2.3 population at the cell surface. Additionally, siRNA‐induced knockdown of the retromer subunit VPS35 decreased KCa2.3 levels at the cell surface. Surprisingly, even though KCa2.3 and KCa3.1 are in the same gene family, similar biochemical and Ussing chamber electrophysiological experiments demonstrated that R55 did not have an effect on KCa3.1 cell surface levels or current; suggesting, that retromer does not regulate the trafficking or recycling of KCa3.1. Cumulatively, these data suggest, for the first time, that retromer is involved in the recycling of KCa2.3, but not KCa3.1, a member of the same gene family.

Epithelial tissues play many roles in maintaining homeostasis of the human body. These tissues separate the body from the external environment (e.g., skin which protects the body), and of course, epithelial tissues separate body compartments, line the surfaces of organs, and line the inner surfaces of many hollow organs. Epithelial cells are polarized as there are specific transport proteins (ion channels and ion transporters) residing in the apical and basolateral membranes of the epithelial cells. Different epithelial cells perform specific functions in the regulation of absorption and secretion of ions, solutes, nutrients, and water. Understanding how these tissues (cells) function has been challenging and a number of techniques have been developed and/or adapted to study the functions of epithelial tissues and cells. Our ability to understand the physiology and the disease pathophysiology of epithelial tissues and cells is really reduced down to determining the fundamental characteristics and basic biology/physiology of the specific ion channels and ion transporters participating in overall epithelial transport physiology. This chapter provides a historical overview of various experimental techniques which have been instrumental and are still employed to discover intriguing aspects of epithelial ion transport physiology.

The advent of intestinal organoid culture in 2009 was a fortuitous development in the search for a valid marker of intestinal stem cells, and provided proof of murine intestinal stem cell regenerative potential. Intestinal organoid culture was preceded by key discoveries of the Wnt/β-catenin signaling pathway and the development of 3D culture matrices. The latter, involving a laminin-rich gel to provide an artificial basement membrane, was instrumental to primary intestinal epithelial culture by preventing anoikis, an immediate apoptotic event when intestinal epithelial cells detach from the basement membrane. One of the first physiological studies using 3D murine “mini-gut” structures showed cystic fibrosis transmembrane conductance regulator (CFTR) expression and anion channel activity in the crypt-like structures projecting from the epithelial-lined central cavity. Detailed investigations of ion transport physiology using human intestinal organoids, both primary and iPSC-derived, found close similarities to existing knowledge of ion transport physiology and included the development of the forskolin-induced swelling assay (FIS). The FIS assay using organoids cultured from rectal biopsies of cystic fibrosis patients provided an avenue for personalized medicine to test small-molecule modulators on different CFTR mutations. More recent research has led to the development of 2D primary intestinal epithelial monolayers, which provide easy access to the apical, lumen-facing membrane and the opportunity for traditional ion transport studies with Ussing chambers. Human 2D primary intestinal monolayers also demonstrate the dominance of CFTR in anion secretion and provide a quantitative H. R. de Jonge · M. J. C. Bijvelds Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands A. M. Strubberg · L. L. Clarke (*) Dalton Cardiovascular Research Center, Columbia, MO, USA Department of Biomedical Sciences, University of Missouri, Columbia, MO, USA e-mail: ClarkeL@missouri.edu J. Liu Dalton Cardiovascular Research Center, Columbia, MO, USA © The American Physiological Society 2020 K. L. Hamilton, D. C. Devor (eds.), Ion Transport Across Epithelial Tissues and Disease, Physiology in Health and Disease, https://doi.org/10.1007/978-3-030-55310-4_1 1 evaluation of its chloride and bicarbonate secretory conductances. These aspects of ion transport physiology using 2D and 3D intestinal cultures are discussed along with the relative advantages and disadvantages of each culture method with respect to technical aspects and recapitulation of native intestinal epithelium.

While epithelial solute transport predates recorded history, our understanding of epithelial function has risen from the most basic level only recently. This chapter provides an historical perspective of epithelial electrophysiology and an initial foundation for much of the information contained in this three volume 2nd edition. Epithelial cell models are presented in their contemporary contexts to demonstrate the philosophical breakthroughs that they heralded along with the novel techniques that made them possible. The text touches on the roles that unique physiological systems such as eel gill, frog skin, rabbit intestine, and cultured cell lines have contributed to our understanding. Two examples of diseases associated with abnormal Cl− transport, cholera diarrhea and cystic fibrosis, are discussed and underlying mechanisms that contribute to the pathology are identified. A hypothetical cell model with the minimal complement of transport proteins that are required for Cl− secretion (Na+/K+-ATPase, Na+/K+/2Cl− cotransporter, K+ channel, and Cl− channel) along with their required localization to the mucosal (apical) or serosal (basolateral) membrane is presented. Selected examples of these transport mechanisms are presented and discussed in the light of their discoveries, biophysical characteristics, pharmacology, genetic identities, and their molecular partners. A recently published comprehensive cell model is presented as the climax of the chapter that sets the stage for distinct components that are presented in greater detail in various chapters of this volume and the other volumes of this 2nd edition.

We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

This book sheds new light on the physiology, molecular biology and pathophysiology of epithelial ion channels and transporters. It combines the basic cellular models and functions by means of a compel

Renal failure is a medical condition in which the kidneys are not working properly. There are two types of kidney failure: 1) acute kidney failure, which is sudden and often reversible with adequate treatment; and 2) chronic renal failure, which develops slowly and often is not reversible. The last stage of chronic renal failure is fatal without dialysis or kidney transplant. The treatment for chronic renal failure is focusing on slowing the progression of kidney damage. Several reports have described a promising approach to slow the loss of renal function through inhibition of the basolateral membrane, Ca 2+ -activated K + (KCa3.1) channel with a selective and nontoxic blocker TRAM-34. This review summarizes pathophysiological studies that describe the role of KCa3.1 in kidney diseases.