Restored Physiology in Protein-Deficient Yeast by a Small Molecule Channel

Abstract

Deficiencies of protein ion channels underlie many currently incurable human diseases. Robust networks of pumps and channels are usually responsible for the directional movement of specific ions in organisms ranging from microbes to humans. We thus questioned whether minimally selective small molecule mimics of missing protein channels might be capable of collaborating with the corresponding protein ion pumps to restore physiology. Here we report vigorous and sustainable restoration of yeast cell growth by replacing missing protein ion transporters with imperfect small molecule mimics. We further provide evidence that this tolerance for imperfect mimicry is attributable to collaboration between the channel-forming small molecule and protein ion pumps. These results illuminate a mechanistic framework for pursuing small molecule replacements for deficient protein ion channels that underlie a range of challenging human diseases.

Figure 1

(a) The prospect of replacing missing protein channels with small molecule mimics. (b) Chemical structures of the archetypical ion channel-forming small molecule amphotericin B and its single atom-deficient and channel-inactivated derivative C35-deoxy amphotericin B (C35deOAmB).

Figure 2

(a) Restoration of cell growth with a small molecule mimic of a missing protein channel. (b) Disc diffusion with AmB on a plate of trk1Δtrk2Δ cells. (c) AmB restores cell growth at concentrations below its minimum inhibitory concentration, while C35deOAmB does not restore growth. (d) Tetraethylammonium diminishes AmB-mediated growth restoration in trk1Δtrk2Δ cells but has no effect on trk1Δtrk2Δ cells grown under permissive conditions (100 mM KCl). (e) AmB restores uptake of extracellular ⁸⁶Rb⁺, a surrogate for K⁺, in trk1Δtrk2Δ cells. (f) Vigorous restoration of cell growth is observed upon treating trk1Δtrk2Δ cells with AmB. (g) Similar to wild type cells, AmB-rescued trk1Δtrk2Δ cells show sustained growth over a period of >40 days. (h) A pre-formed AmB-ergosterol complex dramatically increases the range of concentrations over which physiology is restored. NS, not significant. *** P ≤ 0.0001. Graphs depict means ± SEM.

Figure 3

(a) A series of potassium-transporting polyene macrolide natural products restore vigorous cell growth in trk1Δtrk2Δ yeast, whereas no growth is observed upon treating with other small molecules that selectively transport other ions. (b–d) Nystatin, candicidin, and mepartricin were similarly able to restore physiology in a liquid broth dilution assay over a variety of concentrations. Graphs depict means ± SEM.

Figure 4

(a) AmB-treated wild type S. cerevisiae and trk1Δtrk2Δ cells are equally sensitive to the off-pathway microtubule inhibitor nocodazole. (b) AmB-rescued trk1Δtrk2Δ cells are substantially more sensitive to the V-ATPase inhibitor bafilomycin compared to wild type cells. (c) AmB-rescued trk1Δtrk2Δ cells are substantially more sensitive to the Pma1 inhibitor ebselen than wild type cells. (d–f) EC₅₀ values for various inhibitors of cell growth against wild type (black bars) and trk1Δtrk2Δ cells (white bars) treated with optimum rescue concentrations of potassium-transporting polyene macrolide natural products AmB, nystatin, candicidin, and mepartricin. NS, not significant. * P ≤ 0.05. ** P ≤ 0.001. *** P ≤ 0.0001. Graphs depict means ± SEM.