Cloning and functional expression of a new epithelial sodium channel δ subunit isoform differentially expressed in neurons of the human and monkey telencephalon

Abstract

Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them.

Small Molecule Activator of the Human Epithelial Sodium Channel

“…Similar results were obtained with ␣2 hENaC, a splice variant of ␣ hENaC with 59 additional N-terminal amino acids (38); potencies of amiloride (IC 50 156 Ϯ 25 nM; n ϭ 3) and S3969 (EC 50 1.2 Ϯ 0.5 M; n ϭ 3) on ␣2␤␥ hENaC were not significantly different from ␣␤␥ hENaC. In addition, the potencies of amiloride (IC 50 2.1 Ϯ 0.2 M; n ϭ 3) and S3969 (EC 50 0.4 Ϯ 0.1 M; n ϭ 3) on ␦2␤␥ hENaC were comparable with ␦␤␥ hENaC; ␦2 hENaC is a splice variant of ␦ hENaC with a N-terminal extension because of an alternative transcriptional start site and inclusion of an alternative exon (39,40). Conversely, S3969 did not activate ␣␤␥ mENaC at concentrations yielding maximal activation of ␣␤␥ hENaC or ␦␤␥ hENaC (Fig.…”

Characterization of a novel splice variant of δ ENaC subunit in human lungs

“…In addition, ␦2 ENaC, a slicing variant of the first clone (defined as ␦1 ENaC here), was detected in the human lung (29) and central neuronal tissues (16,36). ␦2 ENaC encodes a protein of 704 amino acid residues, whereas a peptide of 638 proteinogenic amino acids is encoded by the ␦1 ENaC (16,44). ␦2 ENaC has a longer intracellular NH 2 terminus containing 66 more amino acid residues.…”

“…Biophysical and pharmacological studies such as these in Xenopus oocytes have shown that heteromultimeric ␦␤␥ ENaC channels exhibited high selectivity for Na ϩ over Li ϩ ions, less amiloride sensitivity than ␣␤␥ ENaC (25, 36, 44), and were regulated by changes in extracellular pH (24,36,53). In addition, ␦2 ENaC, a slicing variant of the first clone (defined as ␦1 ENaC here), was detected in the human lung (29) and central neuronal tissues (16,36). ␦2 ENaC encodes a protein of 704 amino acid residues, whereas a peptide of 638 proteinogenic amino acids is encoded by the ␦1 ENaC (16,44).…”

“…For example, a novel splice variant of the mouse ␣ ENaC subunit with deletion of the intracellular NH 2 -terminal domain, when coexpressed with the wild-type ␤ and ␥ ENaC subunits in Xenopus oocytes, showed lower single-channel activity (10). Similar differences in the functional domains of the NH 2 termini between ␦1 and ␦2 subunits may contribute differential regulation of the channel activity and/or trafficking by intracellular signals (16).…”

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro

“…We used and compared two primary anti--ENaC antibodies: a commercially available antibody raised against amino acids 81-310 near the N-terminus of human δ-ENaC (SC-21015, Santa Cruz Biotechnology, Heidelberg, Germany, termed anti-δ-N in this report) and an antibody raised against the C-terminus of the δ-ENaC protein (amino acids 563-638; this sequence is not conserved in other ENaC subunits, but common to both described δ-ENaC isoforms [20], termed anti-δ-C in this report). For immunofluorescence microscopy, matching Alexa Fluor-488-labelled F(ab') 2 fragments were supplied by Invitrogen (Biosciences, Dun Laoghaire, Ireland).…”