Ahmed Chraibi

Phosphorylation of Nedd4‐2 by Sgk1 regulates epithelial Na+ channel cell surface expression

The epithelial Na+ channel (ENaC) plays an essential role in the regulation of whole body Na+ balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (α, β and γENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone‐induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4‐2 in a PY motif‐dependent manner and phosphorylates Nedd4‐2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4‐2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4‐2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone‐dependent regulation of ENaC.

An epithelial serine protease activates the amiloride-sensitive sodium channel

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to theprotease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodiumchannel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.

Na Self Inhibition of Human Epithelial Na Channel: Temperature Dependence and Effect of Extracellular Proteases

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na + channel lacking the pore-forming region of its α subunit

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

The Chemokine CCL2 Increases Nav1.8 Sodium Channel Activity in Primary Sensory Neurons through a Gβγ-Dependent Mechanism

Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Nav1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Nav1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Nav1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Nav1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gβγ inhibitor), indicating the involvement of Gβγ released from PTX-sensitive Gi/o-proteins in the cross talk between CCR2 and Nav1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Nav1.8 currents via a CCR2/Gβγ-dependent mechanism.

Epithelial Sodium Channel: A Ligand-Gated Channel?

The epithelial sodium channel (ENaC) is a key component of the transepithelial Na+ transport. In epithelia, it is responsible for the maintenance of Na+ balance (which in turn controls extracellular fluid volume and arterial blood pressure) and the regulation of airway surface fluid. While the regulation of channel synthesis and surface density have been well described, the control of channel opening is still poorly understood. The channel has a large extracellular domain of as yet unknown function; a number of extracellular factors have been shown to modulate ENaC activity, including extracellular Na+ itself (through a phenomenon called ‘self-inhibition’), several other organic or inorganic cations, which seem to interfere with self-inhibition, and serine proteases. Although a direct interaction with the extracellular domain of ENaC has not yet been demonstrated for each of these modulators, the available data strongly suggest that ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.

Regulatory Interdependence of Cloned Epithelial Na+ Channels and P2X Receptors

Epithelial Na+ channels (ENaC) coexist with a family of ATP-gated ion channels known as P2X receptors in the renal collecting duct. Although ENaC is itself insensitive to extracellular ATP, tubular perfusion of ATP can modify the activity of ENaC. To investigate a possible regulatory relationship between P2X receptors and ENaC, coexpression studies were performed in Xenopus oocytes. ENaC generated a persistent inward Na+ current that was sensitive to the channel blocker amiloride (I(am-s)). Exogenous ATP transiently activated all cloned isoforms of P2X receptors, which in some cases irreversibly inhibited I(am-s). The degree of inhibition depended on the P2X receptor subtype present. Activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptor subtypes inhibited I(am-s), whereas activation of P2X1, P2X3, and P2X5 receptors had no significant effect. The degree of inhibition of I(am-s) correlated positively with the amount of ionic charge conducted by P2X receptor subtypes. ENaC inhibition required Na+ influx through I(am-s)-inhibiting P2X ion channels but also Ca2+ influx through P2X4 and P2X(4/6) ion channels. P2X-mediated inhibition of I(am-s) was found to be due to retrieval of ENaC from the plasma membrane. Maximum amplitudes of ATP-evoked P2X-mediated currents (I(ATP)) were significantly increased for P2X2, P2X(2/6), and P2X5 receptor subtypes after coexpression of ENaC. The increase in I(ATP) was due to increased levels of plasma membrane-bound P2X receptor protein, suggesting that ENaC modulates protein trafficking. In summary, ENaC was downregulated by the activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptors. Conversely, ENaC increased the plasma membrane expression of P2X2, P2X(2/6), and P2X5 receptors.

Proven infection-related sepsis induces a differential stress response early after ICU admission

IntroductionNeuropeptides arginine-vasopressin (AVP), apelin (APL), and stromal-derived factor-1α (SDF-1α) are involved in the dysfunction of the corticotropic axis observed in septic ICU patients. Study aims were: (i) to portray a distinctive stress-related neuro-corticotropic systemic profile of early sepsis, (ii) to propose a combination data score, for aiding ICU physicians in diagnosing sepsis on admission.MethodsThis prospective one-center observational study was carried out in a medical intensive care unit (MICU), tertiary teaching hospital. Seventy-four out of 112 critically ill patients exhibiting systemic inflammatory response syndrome (SIRS) were divided into two groups: proven sepsis and non sepsis, based on post hoc analysis of microbiological criteria and final diagnosis, and compared to healthy volunteers (n = 14). A single blood sampling was performed on admission for measurements of AVP, copeptin, APL, SDF-1α, adrenocorticotropic hormone (ACTH), cortisol baseline and post-stimulation, and procalcitonin (PCT).ResultsBlood baseline ACTH/cortisol ratio was lower and copeptin higher in septic vs. nonseptic patients. SDF-1α was further increased in septic patients vs. normal patients. Cortisol baseline, ACTH, PCT, APACHE II and sepsis scores, and shock on admission, were independent predictors of sepsis diagnosis upon admission. Using the three first aforementioned categorical bio-parameters, a probability score for predicting sepsis yielded an area under the Receiver Operating Curve (ROC) curves better than sepsis score or PCT alone (0.903 vs 0.727 and 0.726: P = 0.005 and P < 0.04, respectively).ConclusionsThe stress response of early admitted ICU patients is different in septic vs. non-septic conditions. A proposed combination of variable score analyses will tentatively help in refining bedside diagnostic tools to efficiently diagnose sepsis after further validation.

A ubiquitous non-selective cation channel in the mouse renal tubule with variable sensitivity to calcium

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K+-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5±3.1 pS and 17.3±1.8 pS, respectively (n=4). In contrast, cell-attached patches were usually inactive when a Na+-rich solution filled the patch pipette. However, another type of channel with a conductance of 20–30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1±1.2 pS (n=17), excluded anions (PCl/PNa=0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa=1.5;PK/PNa=0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa=0.09;PBa/PNa≈0). The cation channel was moderately voltagedependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 μmol/l–0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition ofPo at 1.2 umol/l AMP. As in other cell models, 3′,5′-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.

Abstract. Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein.