Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel

Abstract

Amiloride-sensitive Na+ channels are an important component of the Na+ reabsorption pathway in a number of epithelia. Here we report the cloning and characterization of cDNAs encoding two subunits of the human kidney epithelial Na+ channel (beta- and gamma-hENaC). Their predicted amino acid sequences were highly homologous (83-85% identical) to the corresponding subunits reported from rat colon (beta- and gamma-rENaC). Both beta- and gamma-hENaC mapped to human chromosome 16. Northern blot analysis showed high expression of beta- and gamma-hENaC in kidney and lung and differential expression of the three subunits in other tissues. Coexpression of beta- and gamma-hENaC with alpha-hENaC in Xenopus oocytes produced Na+ channels with high selectivity for Na+ and high sensitivity to amiloride. In addition, human subunits were able to substitute for the corresponding rat subunits in forming functional Na+ channels, suggesting conservation of function and suggesting that differences in sequence do not disrupt interactions between subunits. These results suggest that human alpha-, beta-, and gamma-ENaC together form Na+ channels with properties that are similar to those observed in epithelia, and will allow further investigation into the role that these channels may play in human disease.

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

“…To test this hypothesis, we used an airway cell line (JME cells) that had been extensively passaged and did not express ENaC, which we used to measure nonspecific binding after infection with a lentivirus containing an empty vector. To measure specific binding, we then infected these cells with a lentivirus containing yfp-tagged ␣ENaC, because the ␣ENaC subunit alone has previously been shown to form functional ENaCs, although with a significantly smaller conductance (25,26). Stable ␣ENaC expression was confirmed by Western blotting (Fig.…”

Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

“…DNA Constructs-Human ␣ENaC, ␤ENaC, and ␥ENaC were cloned in pMT3 as described previously (39,40). Mutations were generated by site-directed mutagenesis (QuikChange; Stratagene).…”

Identification of a single cytosine base insertion mutation at Arg-597 of the beta subunit of the human epithelial sodium channel in a family with Liddle's disease

“…The ENaC plays a major role in fluid absorption in the kidney, lung and colon and also constitutes the rate-limiting step for sodium absorption (8,(10)(11)(12); it is a multimer consisting of a, b and g subunits (2,(7)(8)(9). These three subunits have been cloned in rats and humans (6,10,13). Canessa et al (10) reported the structure and function of the b subunit of rat ENaC, and recognized them as important candidate genes for causing Liddle's syndrome.…”

“…Canessa et al (10) reported the structure and function of the b subunit of rat ENaC, and recognized them as important candidate genes for causing Liddle's syndrome. Subsequently, McDonald et al (13) cloned human b and g subunits, and demonstrated that b human ENaC (hENaC) DNA had an open reading frame of 1920 nucleotides encoding a protein of 640 amino acids with a predicted size of 73 kDa. Clinical investigators have recently demonstrated that some mutations of the cytoplasmic C-termimus of the b or g hENaC cause truncation of a part of the C-terminus, leading to increased activity of the ENaC (14)(15)(16)(17)(18).…”

“…Clinical investigators have recently demonstrated that some mutations of the cytoplasmic C-termimus of the b or g hENaC cause truncation of a part of the C-terminus, leading to increased activity of the ENaC (14)(15)(16)(17)(18). The activity of the a hENaC has been shown to be greatly augmented by the co-expression of abnormal b or g subunits (6,(13)(14)(15)(16)(17)(18). These results indicate that the cytoplasmic C-terminus of the b or g hENaC is involved in the normal negative regulation of channel activity and that these subunits may act independently (2)(3)(4)(5)15).…”