Genome mining yields putative disease-associated ROMK variants with distinct defects.

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal, and there is currently no cure. Bartter syndrome type II specifically arises from mutations in KCNJ1, which encodes the renal outer medullary potassium channel, ROMK. Over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified, yet their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined genomic data in both the NIH TOPMed and ClinVar databases with the aid of Rhapsody, a verified computational algorithm that predicts mutation pathogenicity and disease severity. Subsequent phenotypic studies using a yeast screen to assess ROMK function-and analyses of ROMK biogenesis in yeast and human cells-identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced cell surface expression. Another mutation (T300R) was ERAD-resistant, but defects in channel activity were apparent based on two-electrode voltage clamp measurements in X. laevis oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies to advance precision medicine

Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains*

Activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na+. We searched for determinants of Na+ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at γMet438 altered the Na+ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na+ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na+ self-inhibition. Mutations at the homologous site in the α subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na+ self-inhibition. Mutations at the homologous βAla422 resulted in minimal or no change in Na+ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing αG481Mβγ, αβγM438V, and αG481M/βγM438V, respectively. This pair of residues map to theα5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both αG481Cβγ and αβγM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that αGly481 and γMet438 are functional determinants of Na+ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery