The molecular basis of pH sensing by the human fungal pathogen Candida albicans TOK potassium channel

Two-pore domain, outwardly rectifying potassium (TOK) channels are exclusively expressed in fungi, with no homologs in humans, animals, plants, or insects. Recently, TOK channels have been cloned and characterized from several human fungal pathogens and have been posited as potential antifungal targets. TOK channel regulation by drugs and environmental factors is poorly understood. Here, we discovered that Candida albicans TOK (CaTOK) is regulated by extracellular pH, while TOK channels from other fungal species tested are relatively pH-insensitive. Low pH potentiated CaTOK channel outward currents (pKa = 6.0), hyperpolarized the voltage-dependence of activation by -31 mV, and increased pore selectivity for K+ over Na+, shifting the reversal potential (EREV) towards EK. Mutating H144 in the S1-S2 extracellular linker partially diminished pH sensitivity, suggesting H144 forms part of the CaTOK pH sensor. Functional analysis of chimeras with pH-insensitive Saccharomyces cerevisiae TOK and..., Channel subunit cRNA preparation and Xenopus laevis oocyte injection.
We generated cRNA transcripts encoding TOK channels from Candida albicans, Aspergillus fumigatus, Cryptococcus var. neoformans, and Saccharomyces cerevisiae, by in vitro transcription using the T7 mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA sub-cloned into plasmids (pMAX) incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability. Mutant TOK channel cDNAs were generated by Genscript (Piscataway, NJ, USA) or using QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), and cRNAs made as above. We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) with TOK cRNAs (1-5 ng). We incubated the oocytes at 16 oC in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 1 day prior to two-electrode voltage-clamp (TEVC) recording.
Two..., , # The molecular basis of pH sensing by the human fungal pathogen Candida albicans TOK potassium channel

The datasets included are the original Excel files used to generate each panel for figures 1-7 in this manuscript. The title of each Excel file is labeled to directly correspond to the figure in the manuscript:

Figure Number & panel > Channel Investigated > Condition > Parameter Measured

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The types of files included within this repository are as follows:

> Exemplar traces
> These data are the values used to generate exemplar traces of ion channel recordings. Data represents the current in microamps (uA) sampled every 100 microseconds (us) as a function of voltage (mV).

> IV
> These data were used to generate current-voltage relationships. Data in these files were measured from the peak of the prepulse current. All values are in microamps (uA).

> Gmax
> These data were used to generate conductance-voltage curves. Graphs were generated by taking ...