Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

Acid-sensing ion channels : modulation by serine-proteases and involvement in acid-induced action potential generation in hippocampal neurons

SUMMARY Acid-sensing ion channels (ASICs) are non-voltage gated sodium channels. They are activated by rapid extracellular acidification and generate an inactivating inward current. Four ASIC genes have been cloned: ASIC1, 2, 3 and 4, with variants a and b for ASIC1and AS1C2. ASICs are expressed in neurons of the central (CNS) and peripheral nervous system (PNS). In the CNS, ASICs have a role in learning, memory, as well as in neuronal death in ischemia. In the PNS, ASICs are involved in the perception of acid-induced pain, as well as in mechanoperception. In one part of my thesis project, we addressed the question of the mechanism of regulation of ASIC1 a by the serine protease trypsin at the molecular level. Trypsin modifies the function of ASIC1 a but not of ASIC1b. In order to identify the channel region responsible for this effect, we created chimeras between ASIC1 a and 1b. Subsequently, to identify the exact trypsin target(s), we mutated predicted trypsin sites in the region identified by the chimera. In the second part of a project, we investigated the role of ASICs at the cellular level, in neuronal signaling. Using the whole-cell patch clamp in hippocampal neuronal culture, we studied the potential involvement of ASICs in action potential (AP) generation. In the first part of the thesis work, we showed that trypsin modifies ASIC1a function: it shifts the pH activation and the steady-state inactivation curve towards more acidic values and accelerates the time course of the channel recovery from inactivation. We also showed that trypsin cleaves ASIC1a and that the functional effect and a channel cleavage correlate. In the inactivated state, channels cannot be modified by trypsin. Cleavage occurs in a channel region that is also important for inactivation of all ASICs; a part of this region is critical for the inhibition of ASIC1 a by the spider toxin Psalmotoxin1. In the second part of the thesis work, we showed that ASIC activity can modulate AP generation. ASIC activity by itself can induce trains of APs. In situations in which this activity by itself is not sufficient to induce APs, it can contribute to AP generation. During high neuronal activity, ASIC activity can block already existing trains of APs. In conclusion, depending on the activity of neuron in a particular moment, ASICs can differently modulate AP generation; they can induce, facilitate or inhibit APs. We also showed that trypsin changes the capability of ASICs to modulate AP generation by shifting the pH dependence to more acidic values, which adapts channel gating to pH conditions which may occur in pathological conditions such as ischemia. Our finding that trypsin modifies ASIC1 a function identifies a novel pharmacological tool, and proposes a mechanism of ASIC1a regulation that may have a physiological importance. The identification of the exact site of trypsin action gives insight to the molecular mechanisms of ASIC regulation. This work proposes a role in modulation of AP generation for ASICs in the CNS. RESUME Les canaux ASIC sont les canaux ioniques activés par l'acidification rapide extracellulaire. Activés, ils génèrent un courant entrant qui inactive en présence de stimulus acide. Quatre gènes ASIC ont été clonés, ASIC1, 2, 3 et 4, avec les variants a et b pour ASIC1 et 2. Les ASICs sont exprimés dans les neurones du système nerveux central (SNC) et périphérique (SNP). Dans le SNC, les ASIC ont un rôle dans le mémoire, apprentissage et la mort neuronale dans t'ischémie. Dans le SNP, ils ont un rôle dans la perception de la douleur et méchanosensation. Dans une partie de mon projet de thèse, nous avons étudié les mécanismes de la régulation d'ASIC1a par la sérine-protéase trypsine au niveau moléculaire. La trypsine modifie la fonction d'ASIC1a et pas ASIC1b. Nous avons créé les chimères entre ASIC1 a et 1 b, afin d'identifier la région du canal responsable pour l'effet. Pour identifier le(s) site(s) exactes de l'action de la trypsine, nous avons muté les sites potentiels de la trypsine dans la région identifiée par les chimères. Dans la deuxième partie du projet, nous avons étudié le rôle des ASICs au niveau cellulaire. En utilisant la technique du patch clamp dans les cultures des neurones de l'hippocampe, nous avons étudié l'implication des ASICs dans la génération des potentiels d'action (PA). Nous avons montré que la trypsine agit sur le canal ASIC1a ; elle décale l'activation et « steady-state » inactivation vers les valeurs plus acides, et elle raccourcit le temps du « recovery » du canal. La trypsine coupe ASIC1a sur le résidu K145 et l'effet fonctionnel et la coupure corrèlent. Nous avons identifié la région du canal responsable pour l'inactivation de tous les ASICs ; une partie de cette région est responsable pour ['inhibition d'ASIC1 a par la Psalmotoxinel . Nous avons montré que les ASICs peuvent moduler la génération des PAs. L'activité des ASICs peut induire les trains des PAs. Quand l'activité des ASICs n'est pas suffisante pour induire le PA, elle peut contribuer à sa génération. Pendant l'activité neuronale forte, l'activité des ASICs peut bloquer les trains des PAs qui existent déjà. En conclusion, dépendant de l'activité neuronale, les ASICs peuvent moduler la génération des PAs différemment ; ils peuvent induire, faciliter ou inhiber les PAs. La trypsine change la capacité des ASICs de moduler les PAs. Après l'action de la trypsine, les ASICs peuvent moduler la génération des PAs dans les conditions légèrement acides, suivies par les fluctuations du pH acide, qui peuvent exister dans l'ischémie. Le fait que la trypsine agit sur ASIC1a définit l'outil pharmacologique et propose le mécanisme de la régulation d'ASICI a qui pourrait avoir l'importance physiologique. L'identification du site de l'action de la trypsine éclaircit les mécanismes moléculaires de la régulation des ASICs. Cette étude propose un rôle des ASICs dans la modulation de la génération des PAs. Résumé pour le public large Les neurones sont les cellules de système nerveux dont la fonction est la signalisation. Comme toutes les autres cellules, les neurones ont une membrane qui sépare l'intérieur du milieu extérieur. Cette membrane est imperméable pour des particules chargées (ions). Dans cette membrane existent les protéines spécifiques, « canaux », qui permettent le transport des ions d'un côté de la membrane à l'autre, comme réponse aux stimuli différents. Ce transport des ions à travers la membrane génère un courant, qu'on peut mesurer. Ce courant est la base de la communication entre les neurones, ou, ce qu'on appelle la signalisation neuronale. Quand ce courant est suffisamment grand, il permet la génération du potentiel d'action, qui est le message principal de communication neuronale. Les canaux ASIC (acid-sensing ion channel), que nous étudions dans le laboratoire, sont activés par les acides. Les acides sont relâchés dans beaucoup de situations dans le système nerveux. Les ASIC ont été découverts récemment (en 1996), et nous ne connaissons pas encore très bien toutes les fonctions de ces canaux. Nous savons qu'ils ont un rôle dans le mémoire, apprentissage, la sensation de la douleur et l'infarctus cérébral. Dans la première partie de ce projet de thèse, nous avons voulu mieux comprendre comment fonctionnent ces canaux. Pour faire ça, nous avons étudié la régulation des ASICs par une protéine, trypsine, qui coupe le canal ASIC. Nous avons étudié ou exactement la trypsine coupe le canal et quels effets ça produit sur la fonction du canal. Dans la deuxième partie du projet de thèse, nous avons voulu mieux connaître comment le canal fonctionne au niveau de la cellule, comment il interagit avec les autres canaux et si il a un rôle dans la génération des potentiels d'action. Nous avons pu montrer que la trypsine change la fonction du canal, ce qui lui permet de fonctionner différemment. Nous avons aussi déterminé ou exactement ta trypsine coupe le canal. Au niveau de la cellule, nous avons montré que les ASIC peuvent moduler la génération des potentiels d'action, étant, dépendant de l'activité du neurone, soit activateurs, soit inhibiteurs. La trypsine est une molécule qui peut être libérée dans le système nerveux pendant certaines conditions, comme l'infarctus cérébral. A cause de ça, les connaissances que la trypsine agit sur le anal ASIC pourraient être important physiologiquement. La connaissance de l'endroit exacte ou la trypsine coupe le canal nous aide à mieux comprendre la relation structure-fonction du canal. La modulation de la génération des potentiels d'actions par les ASIC indique que ces canaux peuvent avoir un rôle important dans la signalisation neuronale.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A.

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Dysfunction of epithelial sodium transport: from human to mouse.

The highly amiloride-sensitive epithelial sodium channel (ENaC) is an apical membrane constituent of cells of many salt-absorbing epithelia. In the kidney, the functional relevance of ENaC expression has been well established. ENaC mediates the aldosterone-dependent sodium reabsorption in the distal nephron and is involved in the regulation of blood pressure. Mutations in genes encoding ENaC subunits are causative for two human inherited diseases: Liddle's syndrome, a severe form of hypertension associated with ENaC hyperfunction, and pseudohypoaldosteronism (PHA-1), a salt-wasting syndrome caused by decreased ENaC function. Transgenic mouse technologies provide a useful tool to study the role of ENaC in vivo. Different mouse lines have been established in which each of the ENaC subunits was affected. The phenotypes observed in these mice demonstrated that each subunit is essential for survival and for regulation of sodium transport in kidney and colon. Moreover, the alpha subunit plays a specific role in the control of fluid absorption in the airways at birth. Such mice can now be used to study the role of ENaC in various organs and can serve as models to understand the pathophysiology of these human diseases.

Selective regulation of acid-sensing ion channel 1 by serine proteases.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line.

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

The cells and peripheral representation of sodium taste in mice.

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, whereas low concentrations of NaCl are typically attractive, particularly after sodium depletion. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, whereas the aversive one functions as a non-selective detector for a wide range of salts. Because amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt-taste-receptor system. Previously, we showed that four of the five basic taste qualities-sweet, sour, bitter and umami-are mediated by separate taste-receptor cells (TRCs) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses. Here we show that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCalpha in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.

An external site controls closing of the epithelial Na+ channel ENaC.

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid-sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the 'degenerin site' activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (betaS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a approximately 3-fold increase in the open probability. This effect is due to an 8-fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N-terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of betaS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state- and size-dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Licorice-induced hypertension and common variants of genes regulating renal sodium reabsorption.

AIM: To study if gene alterations affecting renal sodium reabsorption associate with susceptibility to licorice-induced hypertension.METHODS: Finnish subjects (n = 30) with a previously documented incident of licorice-induced hypertension were recruited for the study using a newspaper announcement. Their previous clinical and family histories as well as serum electrolyte levels were examined. DNA samples from all individuals were screened for variants of the genes encoding 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) and alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC).RESULTS: Upon licorice predisposition, the patients had a mean blood pressure of 201/118 mmHg. Circulating potassium, renin, and aldosterone levels were low. No significant DNA variations were identified in the 11betaHSD2 gene. Four subjects were heterozygous for beta- and gammaENaC variants previously shown to be associated with hypertension. Furthermore, a novel G insertion (2004-2005insG) in the SCNN1A gene encoding the alphaENaC was identified in two subjects. The frequency of these ENaC variants was significantly higher in subjects with licorice-induced hypertension (6/30 i.e. 20%) than in blood donors (11/301 i.e. 3.7%, P = 0.002).CONCLUSIONS: Defects of the 11betaHSD2 gene do not constitute a likely cause for licorice-induced hypertension. Variants of the ENaC subunits may render some individuals sensitive to licorice-induced metabolic alterations and hypertension.

Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate.

The epithelial Na+ channel ENaC mediates transepithelial Na+ transport in the distal kidney, the colon, and the lung and is a key element for the maintenance of Na+ balance and the regulation of blood pressure. Mutagenesis studies have identified residues alphaS583 and the homologous betaG525 and gammaG537 in the outer pore entrance that are critical for ENaC block by the K+-sparing diuretic amiloride. The aim of the present study was to determine first, whether these residues are part of the amiloride binding site, and second, whether they are general determinants of ENaC block by amiloride and its derivatives. Kinetic analysis of the association and dissociation rates of amiloride and benzamil to ENaC showed that mutation of residue alphaS583C and the homologous betaG525C increased the dissociation rate of the drugs from the binding site, with little changes in their association rate. Thus, these mutations destabilize the binding interaction between the blockers and the receptor on the channel, favoring the unbinding of the ligand. This strongly suggests that they are part of the binding site. Because mutations of alphaS583, betaG525, and gammaG537 have similar effects on amiloride, benzamil, and triamterene block, we conclude that these three ENaC blockers share a common receptor within the ion channel pore.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.