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A yeast-based assay to assess the activity of a heterologously expressed potassium channel.

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posted on 2020-04-06, 17:36 authored by Luca Ponzoni, Nga H. Nguyen, Ivet Bahar, Jeffrey L. Brodsky

(A) Schematic of the yeast-based assay. A yeast strain lacking its endogenous potassium transporters, Trk1 and Trk2, is unable to grow on medium containing low potassium but can be rescued by the expression of a human potassium channel. (B) Controls for yeast viability assays on solid (top panel) and in liquid (bottom panel) medium. Yeast cultures were transformed with an empty expression vector as a negative control, or with a plasmid expressing Kir2.1, WT ROMK1, or ROMK1 with the V140M or K80M mutation. Kir2.1, ROMK1 V140M and K80M were used as controls, and V140M and K80M exhibit increased growth compared to cells expressing the WT channel. For the viability assay on solid medium (top panel), saturated overnight cultures of yeast were serially diluted and spotted on SC-Leu medium supplemented with dextrose and containing the indicated concentration of potassium. Plates shown were imaged after a two-day incubation at 30°C and are representative of three independent experiments. For the viability assay in liquid medium (bottom panel), yeast grown overnight to saturation were diluted to an OD600 of 0.20 with medium supplemented with 25mM KCl. OD600 readings were recorded, normalized to wells containing only medium, and these values were subtracted from the reading at t = 0 (see Materials and Methods). Graphs were made using GraphPad Prism (v8. 1. 2), and data represent results from four independent experiments (n = 2–3 each), ± the range of the data. The growth of K80M on 100mM KCl is reduced because K80M enhances potassium uptake and intracellular potassium concentration, which is toxic.

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