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Here we provide the electrophysiology data for the manuscript "Two functional epithelial sodium channel isoforms are present in rodents despite pronounced evolutionary pseudogenization and exon fusion", published in Molecular Biology and Evolution (2021): msab271 (doi: 10.1093/molbev/msab271). Data are reported as current values in Excel format, sorted according to the appearance in Figures and supplemented by explanatory text on the procedures/data presentation.
Cysticfibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressiveand life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe theclinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individu-als harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made inprimary human bronchial epithelial cultures (HBEs) andXenopusoocytes. Molecular properties of R751L-CFTR were investigatedin the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotypeassociated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and asso-ciated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function inXenopusoocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall,R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR.This is thefirst report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological proper-ties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly,inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to al-ternative non-CFTR factors, which require further investigation.
The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, β, γ, δ), forming heterotrimeric αβγ- or δβγ-ENaCs. While the physiology of αβγ-ENaC is well understood, for decades the field has stalled with respect to δβγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analysed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma ago and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δβγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterisation at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αβγ- and δβγ-ENaC function as compared to human orthologues. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium around 10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). The most obvious explanation for this discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we use conventional and non-canonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that they contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work supports the notion that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. The results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.