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Extracellular adenosine triphosphate (ATP) regulates a broad variety of physiological functions in a number of tissues partly via ionotropic P2X receptors. Therefore, P2X receptors are promising targets for the development of therapeutically active molecules. Bile acids are cholesterol-derived amphiphilic molecules; their primary function is the facilitation of efficient nutrient fat digestion. However, bile acids have also been shown to serve as signaling molecules and as modulators of different membrane proteins and receptors including ion channels. In addition, some P2X receptors are sensitive to structurally related steroid hormones. In this study, we systematically analyzed whether rat P2X receptors are affected by micromolar concentrations of different bile acids. The taurine-conjugated bile acids TLCA, THDCA, and TCDCA potently inhibited P2X2, whereas other P2X receptors were only mildly affected. Furthermore, stoichiometry and species origin of the P2X receptors affected the modulation by bile acids: in comparison to rat P2X2, the heteromeric P2X2/3 receptor was less potently modulated and the human P2X2 receptor was potentiated by TLCA. In summary, bile acids are a new class of P2X receptor modulators, which might be of physiological relevance.

Pflügers Archiv 10, (2021). doi:10.1007/s00424-021-02622-2, Published by Springer, Berlin ; Heidelberg

Background ADP-ribosylation is a ubiquitous post-translational modification that involves both mono- and poly-ADP-ribosylation. ARTD10, also known as PARP10, mediates mono-ADP-ribosylation (MARylation) of substrate proteins. A previous screen identified protein kinase C delta (PKCδ) as a potential ARTD10 substrate, among several other kinases. The voltage-gated K+ channel Kv1.1 constitutes one of the dominant Kv channels in neurons of the central nervous system and the inactivation properties of Kv1.1 are modulated by PKC. In this study, we addressed the role of ARTD10-PKCδ as a regulator of Kv1.1. Results We found that ARTD10 inhibited PKCδ, which increased Kv1.1 current amplitude and the proportion of the inactivating current component in HeLa cells, indicating that ARTD10 regulates Kv1.1 in living cells. An inhibitor of ARTD10, OUL35, significantly decreased peak amplitude together with the proportion of the inactivating current component of Kv1.1-containing channels in primary hippocampal neurons, demonstrating that the ARTD10-PKCδ signaling cascade regulates native Kv1.1. Moreover, we show that the pharmacological blockade of ARTD10 increases excitability of hippocampal neurons. Conclusions Our results, for the first time, suggest that MARylation by ARTD10 controls neuronal excitability.

Despite the identification of cholangiocytes in the liver and unipolar brush cells in the cerebellum as sites of expression, the physiological function of the bile acid-sensitive ion channel (BASIC) remains unknown. Rat BASIC (rBASIC) and mouse BASIC (mBASIC) share 97% of their amino acid sequence but show strikingly different biophysical properties. rBASIC is inactive at rest while mBASIC is constitutively active, when expressed in Xenopus oocytes. This conundrum rendered the identification of the physiological function even more difficult. In this study, we investigated the electrophysiological and pharmacological properties of BASIC from rat, mouse, and human in Hek293 cells using the patch clamp technique. Surprisingly, in Hek293 cells, rBASIC and mBASIC showed almost completely identical properties. Both are blocked by extracellular Ca2+ and thus are inactive at rest; both are selective for Na+, show similar affinities for extracellular Ca2+, were inhibited by diminazene, and activated by various bile acids. This is in contrast to previous results derived from Xenopus oocytes as expression system and suggests that the cell type is important for shaping the biophysical properties of BASIC. Furthermore, we compared hBASIC with rBASIC and mBASIC and observed similar properties between these channels with one exception: the bile acid sensitivity profile of hBASIC is different from rBASIC and mBASIC; hBASIC is more sensitive to bile acids which are abundant in human bile but not in rodent bile. Taken together, these results suggest similar physiological roles for BASIC in different species.

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na + channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and β -octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.

Acid-sensing ion channels (ASICs) belong to the DEG/ENaC gene family. While ASIC1a, ASIC1b and ASIC3 are activated by extracellular protons, ASIC4 and the closely related bile acid-sensitive ion channel (BASIC or ASIC5) are orphan receptors. Neuropeptides are important modulators of ASICs. Moreover, related DEG/ENaCs are directly activated by neuropeptides, rendering neuropeptides interesting ligands of ASICs. Here, we performed an unbiased screen of 109 short neuropeptides (

Pentafluorosulfanyl-containing analogs of flufenamic acid have been synthesized in high yields. Computationally, pKa, LogP and LogD values have been determined. Initial bioactivity studies reveal effects as ion channel modulators and inhibitory activities on aldo-keto reductase 1C3 (AKR1C3) as well as COX-1 and COX-2.

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by extracellular acidification. Inhibiting ASICs is neuroprotective in mouse models of ischemic stroke. As inhalational anesthetics interact with many ion channels and as some of them have neuroprotective effects, we hypothesized that inhalational anesthetics modulate ASICs. We expressed different homo- and heteromeric ASICs heterologously in Xenopus oocytes. We co-applied with acidic pH the halogenated inhalational anesthetics sevoflurane, desflurane, and isoflurane and the noble gases xenon and argon at concentrations that are roughly equivalent to their minimal alveolar concentrations and analyzed their effect on current kinetics and amplitude. Sevoflurane, desflurane, and isoflurane as well as xenon and argon accelerated by a factor of ∼1.5 channel desensitization of the main ASICs of the central nervous system: homomeric ASIC1a and heteromeric ASIC1a/2a and ASIC1a/2b. Moreover, they decreased current amplitudes by ∼25%. For example, isoflurane accelerated desensitization of homomeric ASIC1a from 1.0 ± 0.4 s (mean ± SD) to 0.6 ± 0.2 s (n = 12; p = 0.0003) and decreased current amplitudes from 12.1 ± 7.5 μA to 9.3 ± 5.6 μA (n = 12; p = 0.0009). While inhalational anesthetics had similar effects on homomeric ASIC3, desensitization of ASIC1b was only accelerated by halogenated anesthetics but not noble gases; desensitization of homomeric ASIC2a was not modulated. In summary, we found a significant modulation of ASICs by different inhalational anesthetics. We conclude that ASICs should be considered as relevant targets of inhalation anesthetics.

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na(+) channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.

The human bile acid-sensitive ion channel (hBASIC) is a cation channel of the degenerin/epithelial Na(+) channel gene family that is expressed in the intestinal tract and can be activated by bile acids. Here, we show that in addition to its sensitivity for bile acids, hBASIC shares further key features with its rat ortholog: it is blocked by extracellular divalent cations, is inhibited by micromolar concentrations of the diarylamidine diminazene, and activated by millimolar concentrations of flufenamic acid. Furthermore, we demonstrate that two major bile acids present in human bile, chenodeoxycholic acid and deoxycholic acid, activate hBASIC in a synergistic manner. In addition, we determined the single-channel properties of hBASIC in outside-out patch clamp recordings, revealing a single-channel conductance of about 11 pS and a high Na(+) selectivity. Deoxycholic acid activates hBASIC in patch clamp recordings mainly by reducing the single-channel closed time. In summary, we provide a thorough functional characterization of hBASIC.

Cell death and differentiation 24(10), 1655-1671 (2017). doi:10.1038/cdd.2017.88, Published by Nature Publishing Group, Houndmills, Basingstoke

Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA.

TRPN1 is a candidate mechanotransduction channel in Drosophila and Caenorhabditis elegans, also present in hair cells of lower vertebrates. At its N-terminal cytoplasmic tail it contains 28 ankyrin repeats. We performed a yeast two-hybrid screen with the N-terminal ankyrin repeats of Xenopus TRPN1 as bait and identified the Penta-EF-hand protein peflin as a putative interaction partner. We confirmed this interaction by GST pulldown assays and by co-localization in an epithelial cell model. Collectively, our study identifies peflin as an interaction partner of TRPN1 in vitro.Structured summary of protein interactionsTRPN1 physically interacts with Peflin by two hybrid (View Interaction: 1, 2, 3)TRPN1 and Peflin colocalize by fluorescence microscopy (View Interaction: 1, 2)TRPN1 physically interacts with Peflin by pull down (View Interaction: 1, 2, 3)

Ion channels of the degenerin/epithelial Na(+) channel gene family are Na(+) channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na(+) channel (BLINaC) is an ion channel of the degenerin/epithelial Na(+) channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca(2+). Removal of Ca(2+) leads to robust currents and increases Na(+) selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca(2+) affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca(2+). In addition, mBLINaC is more selective for Na(+) and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na(+) channel.

Cell death & disease 5, e1290 (2014). doi:10.1038/cddis.2014.243, Published by Nature Publ. Group, London

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

The epithelial Na(+) channel (ENaC) is a key regulator of Na(+) absorption in various epithelia including the distal nephron and the distal colon. ENaC is a constitutively active channel, but its activity is modulated by a number of mechanisms. These include proteolytic activation, ubiquitination and cell surface expression, phosphorylation, intracellular Na(+) concentration, and shear stress. ENaC is related to the bile acid-sensitive ion channel (BASIC), a channel that is expressed in the epithelial cells of bile ducts. BASIC is activated by millimolar concentrations of extracellular bile acids. Bile acids are synthesized by the liver and secreted into the duodenum to aid lipolysis. A large fraction of the secreted bile acids is absorbed by the ileum and recirculated into the liver, but a small fraction passes the colon and is excreted. Bile acids can influence the ion transport processes in the intestinal tract including the colon. In this study, we show that various bile acids present in rat bile potently and reversibly increase the activity of rat ENaC expressed in Xenopus oocytes, suggesting that bile acids are natural modulators of ENaC activity.

Brain Liver Intestine Na+ Channel (BLINaC) is an ion channel of the DEG/ENaC gene family of unknown function. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. rBLINaC is potently inhibited by micromolar concentrations of extracellular Ca2+ and removal of Ca2+ leads to robust currents and increases Na+ selectivity. Recently we identified the fenamate flufenamic acid (FFA) and related compounds as agonists of rBLINaC. Application of millimolar concentrations of FFA to rBLINaC expressing oocytes induces a robust, Na+-selective current, which is partially blocked by amiloride. The identification of FFA as an artificial activator for BLINaC suggests the presence of an endogenous ligand.In rodents, the blinac mRNA is expressed mainly in brain, liver and intestine; in humans, it is mainly expressed in the small intestine. Immunhistochemical stainings of mouse liver revealed prominent expression of the BLINaC protein in the apical membrane of cholangiocytes lining the bile duct of mice, potentially implicating BLINaC in the generation of secondary bile. Guided by this localisation we tested whether the channel is affected by bile. The application of diluted bile to Xenopus oocytes expressing rBLINaC indeed induced a strong and reversible amiloride-sensitive unselective current. We identified bile salts as the BLINaC-activating molecules in bile and are currently trying to unravel the mechanistic basis of this activation. Bile salts are amphiphatic molecules that affect the structure, curvature and fluidity of membranes and might thus be activating BLINaC indirectly via a membrane dependent mechanism. Using a chimeric approach with ASIC1a, a related but bile salt-insensitive channel, we are currently defining the domains of BLINaC that are crucial for sensing membrane modulation induced by bile salts.

The brain liver intestine Na(+) channel (BLINaC) is a member of the degenerin/epithelial Na(+) channel gene family of unknown function. Elucidation of the physiological function of BLINaC would benefit greatly from pharmacological tools that specifically affect BLINaC activity. Guided by the close molecular relation of BLINaC to acid-sensing ion channels, we discovered in this study that rat BLINaC (rBLINaC) and mouse BLINaC are inhibited by micromolar concentrations of diarylamidines and nafamostat, similar to acid-sensing ion channels. Inhibition was voltage-dependent, suggesting pore block as the mechanism of inhibition. Furthermore, we identified the fenamate flufenamic acid and related compounds as agonists of rBLINaC. Application of millimolar concentrations of flufenamic acid to rBLINaC induced a robust, Na(+)-selective current, which was blocked partially by amiloride. The identification of an artificial agonist of rBLINaC supports the hypothesis that rBLINaC is opened by an unknown physiological ligand. Inhibition by diarylamidines and activation by fenamates define a unique pharmacological profile for BLINaC, which will be useful to unravel the physiological function of this ion channel.