Bernard C. Rossier

Liddle's syndrome: Heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel

Liddles syndrome (pseudoaldosteronism) is an autosomal dominant form of human hypertension characterized by a constellation of findings suggesting constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel. We demonstrate complete linkage of the gene encoding the beta subunit of the epithelial sodium channel to Liddles syndrome in Liddles original kindred. Analysis of this gene reveals a premature stop codon that truncates the cytoplasmic carboxyl terminus of the encoded protein in affected subjects. Analysis of subjects with Liddles syndrome from four additional kindreds demonstrates either premature termination or frameshift mutations in this same carboxy-terminal domain in all four. These findings demonstrate that Liddles syndrome is caused by mutations in the beta subunit of the epithelial sodium channel and have implications for the regulation of this epithelial ion channel as well as blood pressure homeostasis.

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.

The heterotetrameric architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (α, β and γ) assemble to form a highly Na+‐selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC α, β and γ subunits shows that they assemble according to a fixed stoichiometry, with α ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each α, β and γ subunit are consistent with a four subunit stoichiometry composed of two α, one β and one γ. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two α subunits separated by β and γ subunits.

Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome

Liddle syndrome is an autosomal dominant form of hypertension, resulting from mutations in the cytoplasmic C‐terminus of either the beta or gamma subunits of the amiloride‐sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutations reported to date result in the elimination of 45–75 normal amino acids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced channel activity. To address this question, we have performed a systematic mutagenesis study of the C‐termini of the alpha, beta and gamma ENaC subunits of the rat channel and have analyzed their function by expression in Xenopus oocytes. The results demonstrate that a short proline‐rich segment present in the cytoplasmic C‐terminus of each subunit is required for the normal regulation of channel activity. Missense mutations altering a consensus PPPXY sequence of the alpha, beta or gamma subunits reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C‐termini are deleted. This proline‐rich sequence, referred to as the PY motif, is known to be a site of binding by proteins bearing a WW domain. These findings show that the three PY motifs in the C‐termini of ENaC are involved in the regulation of channel activity, probably via protein‐protein interactions. This new regulatory mechanism of channel function is critical for the maintenance of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.

Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5

Ca2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca2+ flux to the extracellular Ca2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca2+ channel in maintaining the Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca2+ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca2+ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca2+ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca2+ reabsorption and its essential role in the Ca2+ homeostasis.

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.

Genetic evidence supports a critical role for the epithelial sodium channel (ENaC) in both clearance of fetal lung liquid at birth and total body electrolyte homeostasis. Evidence from heterologous expression systems suggests that expression of the alphaENaC subunit is essential for channel function, whereas residual channel function can be measured in the absence of beta or gamma subunits. We generated mice without gammaENaC (gammaENaC -/-) to test the role of this subunit in neonatal lung liquid clearance and total body electrolyte balance. Relative to controls, gammaENaC (-/-) pups showed low urinary [K+] and high urinary [Na+] and died between 24 and 36 h, probably from hyperkalemia (gammaENaC -/- 18.3 mEq/l, control littermates 9.7 mEq/l). Newborn gammaENaC (-/-) mice cleared lung liquid more slowly than control littermates, but lung water at 12 h (wet/dry = 5.5) was nearly normal (wet/dry = 5.3). This study suggests that gammaENaC facilitates neonatal lung liquid clearance and is critical for renal Na+ and K+ transport, and that low level Na+ transport may be sufficient for perinatal lung liquid absorption but insufficient to maintain electrolyte balance by the distal nephron. The gammaENaC (-/-) newborn exhibits a phenotype that resembles the clinical manifestations of human neonatal PHA1.

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (INa) by a GPI-anchored serine protease (mouse channel–activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase INa 6–10-fold in the Xenopus oocyte expression system. INa and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I125-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases INa 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcPo). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances INa by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcPo. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in INa (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on INa was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCDcl4) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCDcl4 cell line either by Northern blot hybridization or reverse transcription–PCR. The hepatocyte nuclear transcription factor HNF-3-α (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel

Pseudohypoaldosteronism type 1 (PHA‐1) is an inherited disease characterized by severe neonatal salt‐wasting and caused by mutations in subunits of the amiloride‐sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC β subunit that causes loss of ENaC function and PHA‐1 replaces a glycine that is conserved in the N‐terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into α, β or γ subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild‐type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild‐type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (Po), suggesting that mutations of a conserved glycine in the N‐terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant α subunit (αG95S) directly demonstrate a striking reduction in Po. We propose that this mutation favors a gating mode characterized by short‐open and long‐closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Collecting duct–specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance

Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ASDN consists of the last portion of the distal convoluted tubule (late DCT), the connecting tubule (CNT), and the collecting duct (CD) (i.e., the cortical CD [CCD] and the medullary CD [MCD]). It has been proposed that the control of sodium transport in the CCD is essential for achieving sodium and potassium balance. We have tested this hypothesis by inactivating the alpha subunit of ENaC in the CD but leaving ENaC expression in the late DCT and CNT intact. Under salt restriction or under aldosterone infusion, whole-cell voltage clamp of principal cells of CCD showed no detectable ENaC activity, whereas large amiloride-sensitive currents were observed in control littermates. The animals survive well and are able to maintain sodium and potassium balance, even when challenged by salt restriction, water deprivation, or potassium loading. We conclude that the expression of ENaC in the CD is not a prerequisite for achieving sodium and potassium balance in mice. This stresses the importance of more proximal nephron segments (late DCT/CNT) to achieve sodium and potassium balance.