Using Yeast to Study Potassium Channel Function and Interactions with Small Molecules

Abstract

Analysis of ion channel mutants is a widely used approach for dissecting ion channel function and for characterizing the mechanisms of action of channel-directed modulators. Expression of functional potassium channels in potassium-uptake-deficient yeast together with genetic selection approaches offers an unbiased, high-throughput, activity-based readout that can rapidly identify large numbers of active ion channel mutants. Because of the assumption-free nature of the method, detailed biophysical analysis of the functional mutants from such selections can provide new and unexpected insights into both ion channel gating and ion channel modulator mechanisms. Here, we present detailed protocols for generation and identification of functional mutations in potassium channels using yeast selections in the potassium-uptake-deficient strain SGY1528. This approach is applicable for the analysis of structure–function relationships of potassium channels from a wide range of sources including viruses, bacteria, plants, and mammals and can be used as a facile way to probe the interactions between ion channels and small-molecule modulators.

Tethered Protein Display Identifies a Novel Kir3.2 (GIRK2) Regulator from Protein Scaffold Libraries

“… 32 − 34 Previous studies demonstrated that different Kir3 family members were incapable of rescuing SGY1528 trk1 Δ trk2 Δ growth but that incorporation of Kir3 activating mutations could rescue the yeast under low potassium conditions. 19 , 20 Hence, we reasoned that it should be possible to devise a selection scheme to identify exogenous Kir3 activators by looking for a gain-of-function (GOF) effect under low potassium conditions similar to the strategy used to identify activating mutations of Kir3 19 , 20 and other potassium channels 14 , 16 …”

“… 6 A number of recent advances have aided in the prosecution of high-throughput small molecule screens against potassium channel targets, including development of a fluorescent dye that can detect thallium, an ion that permeates many potassium channels, 60 − 63 and the use of a yeast-based potassium transport strain. 16 , 22 , 64 , 65 Nevertheless, there remains a need to expand the range of systems that can be used to develop new ion channel modulators.…”

“…Positive colonies were grown in liquid -Ura-Met medium with 100 mM KCl; plasmids were isolated, retested, and sequenced. For detailed media compositions, see ref ( 16 ). The N5 clone was identified from the pZsurface-Kir3.2 library in which ∼48 668 independent transformants were screened.…”

“…Functional complementation of potassium transport deficient Saccharomyces cerevisiae strains lacking the Trk1p and Trk2p transporters, such as SGY1528, , has been a fruitful system for the study of a variety of eukaryotic − and bacterial potassium channels and the characterization of channel interactions with chemical modifiers. − Here, we use this system to develop a function-based selection that incorporates the basic principles of protein-based selections to search for a protein-based activator of the G-protein coupled inward rectifier, Kir3.2 (GIRK2) . Kir3 channels are a subfamily of inward rectifiers that are deeply involved in cardiovascular, sensory biology, and reward pathways .…”

Systematic analyses of structure/function variability of viral K+ channels for the development of synthetic channels

“…One aim of this study was to compare the results of yeast complementation assays with data from PLB measurements. Yeast complementation assays compare the growth of potassium uptake deficient (trk1/trk2) yeasts expressing heterologous ion channels versus growth of trk1/trk2yeasts without expression of heterologous ion channels on media with a low potassium concentration (Bagriantsev & Minor, 2013). Fig.…”