ROMK mutations from TOPMed and ClinVar show varying growth defects in yeast in low potassium medium.

(A) Schematic of a yeast-based assay to assess the activity of a human potassium channel. A yeast strain lacking endogenous potassium transporters, Trk1 and Trk2, is viable, yet unable to grow on medium containing low potassium, unless a human potassium channel (e.g., Kir2.1 or ROMK) is expressed. Because of impaired ROMK activity at low pH [84] and exaggerated steady-state residence in the ER [51], ROMK exhibits only a weak growth phenotype on low potassium in contrast to Kir2.1. The table shows the expected growth phenotype of yeast containing an empty vector, or expressing a related Kir channel, Kir2.1, or ROMK, and the predicted growth phenotype of yeast expressing a ROMK mutation from Fig 1. (B) Viability assays of yeast expressing 17 TOPMed/ClinVar mutations in medium containing low potassium (25 mM) grouped by growth defects. Yeast were transformed with an empty expression vector as a negative control, or with a plasmid expressing Kir2.1, ROMK, or the indicated ROMK mutation. An unstable Bartter mutant (Y314C) [15,54] was used as a negative control. In brief, yeast were grown overnight to saturation and diluted the next day to an OD₆₀₀ of 0.20 with medium supplemented with 25 mM KCl. OD₆₀₀ readings were recorded every 30 min for 48 hrs and normalized to wells containing medium. Graphs were made using GraphPad Prism (ver. 9.5.0), and data represent results from two replicates, ± S.E. (error bars). The growth defect categorization, e.g., “Severe defect”, was determined based on the normalized endpoint OD₆₀₀ values at t = 44 hrs and are also described in details in S3 Table. Briefly, we determined the break-points for each categorization as follows: No defect, OD ≥ 1 (or 100% WT); Slight defect, 0.9 ≤ OD < 1 (90–100% WT); Moderate defect: 0.8 ≤ OD < 0.9 (80–90% WT); Severe defect: OD < 0.8 (80% WT).