Truncated beta epithelial sodium channel (ENaC) subunits responsible for multi-system pseudohypoaldosteronism support partial activity of ENaC

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Abstract

Aldosterone regulated epithelial sodium channels (ENaC) are constructed of three homologous subunits. Mutations in the α-, β- and γ-ENaC subunit genes (SCNN1A, SCNN1B and SCNN1G) are associated with multi-system pseudohypoaldosteronism (PHA), and mutations in the PY motif of carboxy-terminal region of β and γ subunits are associated with Liddle syndrome of hereditary hypertension. In this study we identified two frameshift mutations in the SCNN1B alleles of a female infant diagnosed with multi-system PHA inherited from her parents. This is the first case of PHA in an Ashkenazi family in Israel. The p.Glu217fs (c.648dupA in exon 4) and p.Tyr306fs (c.915delC in exon 6) mutations produce shortened β-ENaC subunits with 253 and 317 residues respectively instead of the 640 residues present in β-ENaC subunit. Expression of cRNAs carrying these mutations in Xenopus oocytes showed that the mutations drastically reduce but do not eliminate ENaC activity. The findings reveal that truncated β-ENaC subunits are capable of partially supporting intracellular transport of the other two subunits to the membrane and the final assembly of a weakly active channel together with normal α- and γ-ENaC subunits. Moreover, these results enhance our understanding of the long-term consequences of these types of mutations in PHA patients.

Introduction

Steroid hormone aldosterone that is synthesized and secreted from the zona glomerulosa of adrenal cortex [1] is one of the major regulators of electrolyte homeostasis and blood volume [2]. Secretion of aldosterone is regulated mainly by the renin-angiotensin system [3]. The major site of action of aldosterone in electrolyte regulation is epithelial cells in the distal tubule of kidney where aldosterone induces expression of epithelial sodium channel (ENaC) subunits increasing ENaC activity at the apical cell surface [4], [5]. Sodium ions that enter the epithelial cells via ENaC are pumped out into the interstitial space via Na/K ATPase located on the opposite basolateral membrane. Enhanced sodium transfer from the lumen of kidney tubule into epithelial cells increases interstitial fluid osmolarity. Water then freely flows in the same direction to maintain similar osmolarity of compartments. Consequently, aldosterone actions result in increases in blood volume and blood pressure [2], [6].

Epithelial sodium channels are constructed of three homologous subunits named as α-, β- and γ-ENaC that are embedded in the membrane with two trans-membrane segments [6], [7], [8]. The amino and carboxy-terminal domains of these subunits are located in the cytoplasm, while the bulk of their structure is exposed outside of the cell, forming part of the funnel that directs ions from the lumen into the pore of ENaC, and from there into the epithelial cell.

Mutations in the genes that encode for ENaC subunits are associated with two independent hereditary diseases: multi-system pseudohypoaldosteronism (PHA) [9], [10], [11], [12], [13], [14], [15], [16], [17], and Liddle syndrome of hereditary hypertension [18], [19]. Multi-system PHA is observed as an autosomal recessive disease, whereas Liddle syndrome shows autosomal dominant inheritance.

Multi-system PHA is manifested as a syndrome of aldosterone unresponsiveness that leads to severe salt-wasting in early infancy [20]. Most of the mutations responsible for PHA have been identified in the gene encoding the α subunit of ENaC [15]. There are only a few reports of PHA causing mutations in the genes encoding the β and γ subunits. Mutations responsible for the Liddle syndrome have been observed so far solely at the carboxy-terminal of the β and γ subunits. This region includes a PY (Pro-Pro-Pro-x-Tyr) motif that binds Nedd4-2, an ubiquitin ligase. Ubiquitylation of this motif eventually leads to removal of the protein from the membrane by endocytosis. Truncation of this motif by a nonsense mutation in the carboxy-terminal region, or a missense mutation in the PY motif, inhibits ubiquitylation of the subunit. As a consequence, channels accumulate at the membrane leading to enhanced activity of ENaC. Thus, while mutations associated with multi-system PHA reduce ENaC activity, mutations associated with the Liddle syndrome enhance ENaC activity [21].

In our examination of a multi-system PHA patient we identified two independent frame-shift mutations in the gene encoding the β-ENaC leading to drastic truncation of the carboxy-terminal region of β-ENaC. Previous studies indicated that the cell surface expression of functional ENaC is dependent on the presence of all three ENaC subunits. We undertook the present study to examine the question whether a severely truncated β-ENaC subunit missing more than 50% of its primary structure can partially support cell surface expression and activity of ENaC. For this purpose we generated the corresponding mutant forms of human β-ENaC by site-directed mutagenesis of normal human cDNAs and examined the activities of the mutant forms expressed in Xenopus oocytes that were microinjected with corresponding cRNAs.

Section snippets

Subjects

The female infant with multi-system PHA was born full term (gestational age: 40 weeks) weighing 3.385 kg as the second sibling of non-consanguineous Ashkenazi Jewish parents and presented with extreme lethargy at 8 days of age with a 1-day history of poor feeding. Laboratory evaluation on admission showed very high serum K+ levels and hyponatremia. Trans-tubular potassium gradient was 2.4, indicating very low renal potassium excretion. Sweat Cl concentration was also high, consistent with salt

PHA associated mutations in the β-ENaC gene

Patient's laboratory data on admission including hyperreninemia and hyperaldosteronism (Table 1) were highly suggestive of multi-system PHA. Subsequently she required large quantities of NaCl (60 mmol/kg/day), sodium bicarbonate and Kayexalate therapy to maintain the serum sodium and potassium within the normal range. Apart from electrolyte disturbances she also suffered from failure to thrive, chronic nasal discharge, seborrhea like skin eruptions, and recurrent respiratory infections. Thus,

Discussion

In this study we identified two heterozygous frame-shift mutations in the paternal and maternal alleles coding for β-ENaC subunit in a patient with multi-system PHA. This is the first case of PHA in an Ashkenazi family in Israel. Similar mutations were not detected in a sample of 50 alleles from Ashkenazi subjects. Thus the mutations, similar to other PHA causing mutations, do not appear to be frequent in this population. We know of only one previous report on mutations in the β-ENaC subunit of

Acknowledgments

This research was funded in part by the generous contribution of the Peter Simpson Family Fund and by a grant from the Chief Scientist of the Israel Ministry of Health. We are grateful to Prof. Elon Pras (Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel Hashomer, Israel) for providing the DNA samples for the control population. This work was carried out in partial fulfillment of the requirements for a PhD degree of OE at the Sackler Faculty of Medicine of Tel Aviv University.

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