Voltage-gated sodium (NaV) channels are responsible for the initiation and propagation of action potentials. In the heart, the predominant NaV1.5 α subunit is composed of four homologous repeats (I–IV) and forms a macromolecular complex with multiple accessory proteins, including intracellular fibroblast growth factors (iFGF). In spite of high homology, each of the iFGFs, iFGF11–iFGF14, as well as the individual iFGF splice variants, differentially regulates NaV channel gating, and the mechanisms underlying these differential effects remain elusive. Much of the work exploring iFGF regulation of NaV1.5 has been performed in mouse and rat ventricular myocytes in which iFGF13VY is the predominant iFGF expressed, whereas investigation into NaV1.5 regulation by the human heart-dominant iFGF12B is lacking. In this study, we used a mouse model with cardiac-specific Fgf13 deletion to study the consequences of iFGF13VY and iFGF12B expression. We observed distinct effects on the voltage-dependences of activation and inactivation of the sodium currents (INa), as well as on the kinetics of peak INa decay. Results in native myocytes were recapitulated with human NaV1.5 heterologously expressed in Xenopus oocytes, and additional experiments using voltage-clamp fluorometry (VCF) revealed iFGF-specific effects on the activation of the NaV1.5 voltage sensor domain in repeat IV (VSD-IV). iFGF chimeras further unveiled roles for all three iFGF domains (i.e., the N-terminus, core, and C-terminus) on the regulation of VSD-IV, and a slower time domain of inactivation. We present here a novel mechanism of iFGF regulation that is specific to individual iFGF isoforms and that leads to distinct functional effects on NaV channel/current kinetics.

TMEM16A is a Ca 2+ -activated Cl − channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca 2+ -activated Cl − currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders. NEW & NOTEWORTHY The Ca 2+ -activated Cl − channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Bile acids stimulate a bicarbonate-rich choleresis, in part, through effects on cholangiocytes. Because Cl- channels in the apical membrane of cholangiocytes provide the driving force for secretion and transmembrane member 16A (TMEM16A) has been identified as the Ca2+ -activated Cl- channel in the apical membrane of cholangiocytes, the aim of the present study was to determine whether TMEM16A is the target of bile-acid-stimulated Cl- secretion and to identify the regulatory pathway involved. In these studies of mouse, rat, and human biliary epithelium exposure to ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) rapidly increased the rate of exocytosis, ATP release, [Ca2+ ]i , membrane Cl- permeability, and transepithelial secretion. Bile-acid-stimulated Cl- currents demonstrated biophysical properties consistent with TMEM16A and were inhibited by pharmacological or molecular (small-interfering RNA; siRNA) inhibition of TMEM16A. Bile acid-stimulated Cl- currents were not observed in the presence of apyrase, suramin, or 2-aminoethoxydiphenyl borate (2-APB), demonstrating that current activation requires extracellular ATP, P2Y, and inositol 1,4,5-trisphosphate (IP3) receptors. TUDCA did not activate Cl- currents during pharmacologic inhibition of the apical Na+ -dependent bile acid transporter (ASBT), but direct intracellular delivery of TUDCA rapidly activated Cl- currents. CONCLUSION:Bile acids stimulate Cl- secretion in mouse and human biliary cells through activation of membrane TMEM16A channels in a process regulated by extracellular ATP and [Ca2+ ]i . These studies suggest that TMEM16A channels may be targets to increase bile flow during cholestasis. (Hepatology 2018;68:187-199).

TMEM16A is a newly identified Ca(2+)-activated Cl(-) channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing intracellular Ca(2+) concentration ([Ca(2+)]i), the regulatory pathways have not been elucidated. Protein kinase C (PKC) contributes to ATP-mediated secretion in BECs, although its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl(-) transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca(2+)-activated Cl(-) currents. Currents demonstrated outward rectification and reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl(-) currents with biophysical properties identical to TMEM16A in control cells but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca(2+)-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.

Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

TMEM16A is a Ca2+‐activated Cl− channel in the apical membrane of biliary epithelial cells (BECs), and contributes to biliary secretion and bile formation [Dutta et al. JBC, 2010]. Extracellular nucleotides activate TMEM16A via binding P2 receptors and increasing [Ca2+]i. While PKC contributes to ATP‐mediated secretion in BECs, the role in TMEM16A regulation is unknown.AimTo determine if PKC contributes to Cl− transport in BECs through regulation of TMEM16A.MethodsStudies were performed in human biliary Mz‐cha‐1 cells. Cl− currents were measured by whole‐cell patch clamp.ResultsTMEM16A Cl− currents were activated by exposure to extracellular ATP or increasing Ca2+ in the patch pipette. Currents had a reversal potential of 0 mV, time‐dependent activation and outward rectification. Currents were significantly inhibited by anti‐TMEM16A siRNA. In the presence of the PKC inhibitors Chelerithrine or Gö6976 Cl− currents were significantly inhibited. Conversely, activation by PMA resulted in large Cl− currents. Intracellular dialysis with recombinant PKCα activated Cl− currents with identical biophysical properties to TMEM16A.ConclusionPKCα couples ATP‐mediated signaling to TMEM16A activation in BECs. Modulation of kinase signaling pathways may represent a target for increasing biliary secretion and bile formation.[Studies are supported by AASLD/ALF Liver Scholar Award 2010, Children's Medical Center Foundation grant (Dutta), and NIH NIDDK DK078587]

In the liver, Cl− channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for biliary secretion. Extracellular nucleotides are important agonists for Ca2+‐activated Cl‐ channel activation. The molecular identity of the Ca2+‐activated Cl‐ channel is unknown.Aimto determine if TMEM16A represents the Ca2+‐activated Cl− channel in BECs.MethodsStudies were performed in human, mouse, and rat BECs. Membrane Cl− currents were measured by whole‐cell patch clamp and transepithelial secretion (Isc) by Ussing chamber.ResultsTMEM16A mRNA was detected by RT‐PCR in all BEC models. In polarized BEC monolayers, addition of ATP to the apical chamber increased Isc. Pre‐incubation with IL‐4 increased TMEM16A expression and was associated with an increase in Isc. The ATP‐stimulated Isc was completely inhibited by the Cl− channel blockers NPPB and niflumic acid, but insensitive to CFTR‐inh172. In single cells, exposure to ATP rapidly increased [Ca2+]i and activated whole‐cell Cl− currents with a reversal potential of 0 mV and outward rectification. Transfection with TMEM16A siRNA significantly decreased TMEM16A expression and inhibited the ATP‐stimulated whole‐cell currents.ConclusionThese results demonstrate that TMEM16A is functionally present in BECs and contributes to Ca2+‐activated Cl− secretion in response to extracellular nucleotides. These studies represent the first molecular identification of an alternate, non‐CFTR, Cl− channel in BECs.

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca(2+)-activated K(+) channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K(+) currents, increasing current density from 1.6 +/- 0.1 to 7.6 +/- 0.8 pA/pF. Currents were dependent on intracellular Ca(2+) and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K(+) currents had a unitary conductance of 46.2 +/- 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K(+) currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K(+) currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.

Bacterial toxin-mediated diarrheal disease is a major cause of morbidity and mortality worldwide. In this work we designed an on-bead library of protease-resistant, acid-stable peptoid molecules and screened for high affinity binding of cholera toxin. From 100000 compounds, we discovered a single sequence of residues that can bind and retain cholera toxin at high affinity when immobilized on a solid-phase particle. Furthermore, we demonstrate that these peptoid-displaying particles can sequester active cholera toxin from cell culture media sufficient to protect intestinal cells. We foresee this work as contributory to a potential adjunct therapeutic strategy against cholera infections and other toxin-mediated diseases.