[6] Studying ion channels using yeast genetics

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)

“…These plates were incubated for 4 days and imaged on days two, three, and four using a BioRad (Hercules, CA) Image Station. Medium to which no potassium was added ("0 mM") has been estimated to contain 7-10 mM potassium (42).…”

“…To this end, ROMK1S44D+K80M-expressing trk1Δtrk2Δ yeast were mated with the non-essential yeast deletion collection and arrays of triple mutant progeny (i.e., those that were deleted for TRK1, TRK2, and a nonessential gene; see Experimental Procedures) were generated ( Figure 4A). Potential negative effectors were identified by comparing growth on high (100 mM) versus low ("0" mM) potassium medium, in which the only available potassium derives from trace amounts in the media components (42). The initial screen revealed 40 very strong hits (z-score >3), 228 strong hits (z-score >2), and 519 weak hits (z-score >1) from the nearly 5,000 strains screened (Supplemental Table S1).…”

“…As deleting the genes encoding two plasma membrane potassium channels, TRK1 and TRK2, renders yeast inviable on low potassium media (42), an ectopically expressed potassium channel, such as ROMK, can rescue this phenotype, assuming that it is able to fold, assemble, and traffic to the plasma membrane ( Figure 1A). Previous studies by our group and other groups have exploited this technique for structure-function and genetic interaction studies of several human potassium channels (43)(44)(45)(46)(47)(48).…”

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

“…Kir1.1 was the first Kir channel expressed in this strain background and restored cell growth on low-potassium-containing media (123). This system was then optimized for genetic screens (87).…”

Identification of yeast proteins necessary for cell-surface function of a potassium channel

“…The mutation S177W (referred to as Kir*) renders Kir3.2 constitutively open in the absence of G protein signaling, permeable to Na ϩ and K ϩ , and does not disrupt functional expression of the channel at the cell surface of yeast or Xenopus oocytes (9,10). Expression of mutated K ϩ channels that are permeable to Na ϩ overwhelms the Na ϩ detoxification systems of yeast (11). Functional expression of Kir* can therefore be assayed on the basis of growth inhibition, reflected by small yeast-colony size, on media containing high Na ϩ concentrations.…”