Chloride Channel Function in the Yeast TRK-Potassium Transporters

Abstract

The TRK proteins-Trk1p and Trk2p- are the main agents responsible for "active" accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration-qualitatively as expected for H(+) coupling to K(+) uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K(+) or H(+), but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K(+)-dependent and can be enhanced by K(+) starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K(0.5) at 46 micro M [K(+)](out) and 6.8 m M [K(+)](out), and saturating fluxes of approximately 5 m M/min and approximately 10 m M/min (referred to intracellular water). These numbers are compatible with the normal K(+)-transport properties of Trk1p and Trk2p, respectively.

The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5

“…All data were collected at 2 kHz and filtered at 250 Hz, and all current traces shown in Figs. 5 and 7 are original, whereas all plotted I-V relationships have been corrected for nonspecific leakage currents, as described in detail for S. cerevisiae (33). Preliminary data analysis was performed via the Heka Pulse software, but more detailed analysis was carried out with Microsoft Excel and/or Igor Pro (WaveMetrics, Inc.).…”

“…The upward currents (all six panels) represent K ϩ efflux through Tok1p, the K ϩ channel (34). Downward currents (top row) represent (Ͼ95%) chloride efflux through Trk1p, the K ϩ transporter (33,52). Vertical comparison (two left columns) demonstrates that reduction of intracellular chloride to submillimolar range nearly abolished the downward currents.…”

“…A Second Functional Assay: The Chloride Channel through Trk1p-A completely unexpected property of TRK proteins was recently identified by patch clamp experiments on whole protoplast membranes of S. cerevisiae; they conduct large inward currents when the cytoplasm is loaded with chloride ions (33,52). The molecular mechanism underlying such implied chloride effluxes is unknown, but it apparently serves as a voltagedriven escape for excess cytoplasmic chloride ions.…”

“…For each plot, maximal slope conductance was estimated from a straight line drawn through the Ϫ160-mV and Ϫ180-mV points (parallel dashed line, top left panel). I-V plots were corrected for small leakage currents, as already described (33). Inset, phase plot of Rb ϩ fluxes (from Fig.…”

“…This transporter, initially cloned in Saccharomyces by Gaber et al (29), is believed to operate as a proton-coupled system, having similar properties to the high affinity K ϩ uptake system in Neurospora (30,31), and has also been reckoned as a voltage regulator on the basis of dye distribution measurements (32). Finally, it has recently been shown to mediate chloride currents through Saccharomyces plasma membrane, which are made easily visible by raising intracellular Cl Ϫ above ϳ10 mM (33). The possible involvement of Tok1p in histatin toxicity was investigated by creating TOK1-disrupted mutants of C. albicans (34).…”