Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters

Abstract

In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca(2+) influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K(+) and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K(+) but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K(+) environments, suggesting they promote K(+) uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K(+) currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K(+) currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K(+) influx and that HACS may electrochemically respond to K(+) influx in much the same way as the homologous voltage-gated Ca(2+) channels in most animal cell types.

Fig 1

Kch1 and Kch2 regulate HACS activation in response to α-factor mating pheromone. ⁴⁵Ca²⁺ uptake into log-phase cultures of S. cerevisiae strains that include combinations of Kch1, Kch2, Cch1, and the secreted protease Bar1 (strains K432, NZY164, NZY138, NZY165, EMY113) was measured after 4 h of incubation in SC-100 medium in the presence or absence of 10 μM α-factor (A) plus 0.2 μg of FK506/ml (B), as indicated. Averages for three biological replicates (± the standard deviations [SD]) are shown. (C) The wild-type strain and kch1, kch2, and kch1 kch2 double mutants with or without the functional CCH1 gene (strains CS12, CS01, CS02, CS03, CS04, CS05, CS06, CS07) all expressing cytoplasmic aequorin from a plasmid (pEVP11/AEQ89) were monitored for luminescence in YPD medium before and after the sudden jump to pH 9.

Fig 2

Kch1 and Kch2 are essential for maintaining cell viability. (A) The strains listed in Fig. 1C were exposed to 25 μM α-factor in SC-100 media with or without additional 100 mM CaCl₂ and stained with propidium iodide. Live and dead cells were counted by flow cytometry. (B) Strains kch1 kch2 and cch1 kch1 kch2 (CS03 and CS07) were transformed with a control plasmid or plasmids that overexpress the KCH2-HA₃ or KCH1-HA₃ genes (pSM10, pCS01, and pCS02), exposed to 50 μM α-factor, and then analyzed for pheromone-induced cell death as in panel A. The averages of three biological replicates (± the SD) are shown.

Fig 3

Expression and localization of Kch1 and Kch2 in yeast cells responding to α-factor. (A) Western blots on whole-cell lysates of wild-type yeast strains (K601) expressing Kch2-MYC₁₃ and Kch1-MYC₁₃ from the chromosomal loci (CS34 and CS83) in the presence or absence of 1 μg of FK506/ml and 25 μM α-factor. Wild-type yeast cells (K601) expressing GFP-KCH1 (B) and GFP-KCH2 (C) from constitutive plasmids (pCS47, pCS48) were imaged after exposure to 10 μM α-factor or solvent. Pseudocolored images in panel B depict the scale of pixel intensity.

Fig 4

K⁺ is essential for the Ca²⁺ accumulation in response to α-factor mating pheromone. (A) Kch1/2-dependent ⁴⁵Ca²⁺ uptake was determined in SC-100 medium containing the indicated concentrations of KCl by subtracting the values obtained from kch1 kch2 double-mutant cultures (CS03) from those obtained from wild-type cultures (K601) with or without 4 h exposure to 25 μM α-factor plus 0.2 μg of FK506/ml. (B) β-Galactosidase activity of wild-type cells (K601) transformed with a FUS1-lacZ reporter gene (pJB207) in SC-100 medium in the presence or absence of 25 μM α-factor and 10 mM KCl. Bars indicate the averages (arithmetic means) of three biological replicates (± the SD).

Fig 5

Kch1 and Kch2 promote K⁺ uptake and K⁺-dependent activation of HACS. (A) A trk1 trk2 double mutant and a trk1 trk2 mid1 triple mutant (CS30 and CS163) bearing a control plasmid or plasmids overexpressing KCH1-HA₃ or KCH2-HA₃ genes (pCS42, pCS43, and pCS44) were serially diluted and then spotted onto SDAP pH 4.5 agar medium lacking methionine but containing 100 or 50 mM KCl as indicated. Colonies were photographed after 3 days incubation at 30°C. (B) ⁴⁵Ca²⁺ uptake into log-phase cultures of kch1 kch2 double mutants and cch1 kch1 kch2 triple mutants (CS03 and CS08) transformed with control plasmid or plasmids overexpressing KCH1-HA₃ and KCH2-HA₃ genes, in SC-100 medium supplemented with 1 μg of FK506/ml and incubated for 1.5 h. (C) ⁴⁵Ca²⁺ uptake into the kch1 kch2 double mutants expressing KCH1-HA₃ and KCH2-HA₃ genes was performed in SC-100 media that had been supplemented with 0.2 μg of FK506/ml plus the indicated concentrations of KCl, RbCl, or NaCl. The data points were normalized to corresponding plasmid controls to represent Kch1- and Kch2-dependent calcium uptake. Plots depict averages (± the SEM).

Fig 6

Kch1 and Kch2 mediate inward K⁺ currents. (A) Averaged patch-clamp traces from protoplasts of the wild-type strain (K432, n = 5, left panel) and the kch1 kch2 double mutant (NZY165, n = 6, right panel), recorded after 10 min incubation in Mg²⁺- and Ca²⁺-free recording buffer (see Materials and Methods). (B) Current-voltage plots for the steady-state currents from panel A, plus controls obtained from the same protoplasts, but with 10 mM extracellular Ca²⁺. Steady-state values calculated from the grand average data, over the interval 2.0 to 2.5 s, along each trace. (C) Plotted differences between corresponding traces of the two panels in panel A. These difference curves are well fitted by the sum of two simple exponentials plus an offset (see equation in Materials and Methods), with the same pair of rate constants for all six plots: α1 = 7.725 s, and α2 = 0.212 s, corresponding to half times of 90 ms and 3.27 s, respectively.

Fig 7

Heterologous expression of Kch1 in HEK293T cells induces inward K⁺ currents. (A) HEK293T cells transfected with the GFP-KCH1 gene (pCS50) were fixed, stained with DAPI, and imaged microscopically. (B) Representative traces of HEK293T cells transfected with plasmid or plasmid containing GFP-KCH1 gene (pEGFP-C3 and pCS50) subjected to whole-cell voltage-clamp measurements in buffers containing high- or low-divalent cations. (C and D) Current voltage traces summarizing raw data in panel B in high (C)- or low (D)-divalent cation buffers. The plots show averages (± the SEM) of 9 control cells (white symbols) and 19 GFP-Kch1 expressing cells (gray symbols) after normalization with the whole-cell capacitance.