DOI: 10.5061/dryad.t76hdr893
title: The molecular basis of pH sensing by the human fungal pathogen Candida albicans TOK potassium channel

description: 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 point mutants revealed that V462 and S466
 in the final transmembrane segment complete the CaTOK pH-responsive
 elements. A tripartite network of residues thus endows CaTOK with the
 ability to functionally respond to pH.
description: <strong><em>Channel subunit cRNA preparation and
 Xenopus laevis oocyte injection.</em></strong>
 We generated cRNA transcripts encoding TOK channels
 from <em>Candida albicans</em>, <em>Aspergillus
 fumigatus</em>, <em>Cryptococcus var. neoformans</em>,
 and <em>Saccharomyces cerevisiae</em>, by <em>in
 vitro</em> transcription using the T7 mMessage mMachine kit (Thermo
 Fisher Scientific), after vector linearization, from cDNA sub-cloned into
 plasmids (pMAX) incorporating <em>Xenopus laevis</em> 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 <em>Xenopus laevis</em> oocytes
 (Xenoocyte, Dexter, MI, US) with TOK cRNAs (1-5 ng). We incubated the
 oocytes at 16 <sup>o</sup>C in ND96 oocyte storage solution
 containing penicillin and streptomycin, with daily washing, for 1 day
 prior to two-electrode voltage-clamp (TEVC) recording.
 <strong><em>Two-electrode voltage clamp (TEVC)
 </em></strong> We performed TEVC at room
 temperature using an OC-725C amplifier (Warner Instruments, Hamden, CT)
 and pClamp10 software (Molecular Devices, Sunnyvale, CA) 1 day after cRNA
 injection. Oocytes were placed in a small-volume oocyte bath (Warner) and
 viewed with a dissection microscope.  Oocytes expressing TOK channels were
 recorded in 4 mM extracellular KCl bath solution containing (in mM): 96
 NaCl, 4 KCl, 1 MgCl<sub>2</sub>, 1 CaCl<sub>2
 </sub>unless otherwise indicated. Extracellular 4 mM KCl solutions
 were buffered (in mM) with: 10 MES (pH 4.0 to 5.5), 10 HEPES (pH 6.0 to
 7.5), 5 CHES (pH 8.0 to 9.5). Pipettes were of 1-2 MΩ resistance when
 filled with 3 M KCl.  We recorded currents in response to voltage pulses
 between -120/-80 mV and +40 mV at 10 mV intervals from a holding potential
 of -80 mV, to yield current-voltage relationships and examine activation
 kinetics. We plotted raw or normalized tail currents for CaTOK versus
 prepulse voltage and fitted with a single Boltzmann function. We analyzed
 data using Clampfit (Molecular Devices) and Graphpad Prism software
 (GraphPad, San Diego, CA, USA), stating values as mean ± SEM.
 <strong><em>Relative permeability
 calculations</em></strong> Values were
 calculated using the Goldman-Hodgkin-Katz (GHK) equation where
 <em>V<sub>m</sub></em> is the absolute reversal
 potential and <em>P</em> is the permeability. By knowing the
 intracellular and extracellular concentrations of each ion, one can modify
 this equation to calculate the relative permeability of each ion. Here, we
 modified the equation to determine the relative permeability of two ions
 in a system in which only the extracellular ion concentration was known.
 By plotting the I/V relationships for CaTOK in the presence of 100 mM
 Rb<sup>+</sup>, Na<sup>+</sup>, and
 Cs<sup>+</sup> we can compare their relative permeability to
 100 mM K<sup>+</sup>. Permeability ratios for each ion
 (<em>x</em>) compared to K<sup>+</sup> were
 calculated.              <strong><em>CaTOK
 structural analysis </em></strong> We used
 AlphaFold to generate a predicted structure for CaTOK. We visualized the
 CaTOK channel structure in UCSF Chimera
 (https://www.rbvi.ucsf.edu/chimera), which we also used
 to generate figures. <strong><em>Statistics
 and Reproducibility</em></strong> All
 values are expressed as mean ± SEM.  Comparison of two groups was
 conducted using a t-test; all <em>p</em> values were
 two-sided. One-way ANOVA was applied to all tests comparing more than two
 groups; all <em>p</em> values were two-sided.
description: # 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 &gt; Channel
 Investigated &gt; Condition &gt; Parameter Measured --- The types of files
 included within this repository are as follows: &gt; Exemplar traces &gt;
 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). &gt; IV &gt; 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). &gt; Gmax &gt; These data were used to generate
 conductance-voltage curves. Graphs were generated by taking measurements
 from the tail current (-30 mV) immediately following the prepulse current.
 &gt; Resting membrane potential &gt; These data points represent the
 resting membrane potential (EM) of unclamped Xenopus laevis oocytes
 expressing the channel of interest at pH 7.5 subsequent pH ranges. Resting
 membrane potential values are in millivolts (mV). &gt; Dose responses
 (current density) &gt; These data were taken from values measured at 0 mV
 from the prepulse for pH 7.5 and each subsequent pH. Raw data values are
 in microamps (uA). &gt; Dose responses (Resting membrane potential) &gt;
 These data were taken from unclamped oocytes for pH 7.5 and each
 subsequent pH. Raw data values are expressed as millivolts (mV). &gt; Dose
 responses (voltage dependent activation) &gt; These data were taken from
 values measured in the Gmax graphs for pH 7.5 and each subsequent pH. Raw
 data values are expressed as millivolts (mV). &gt; Relative current
 comparisons &gt; These data were taken at 0 mV from IV curves generated in
 response to switching to pH 7.5 to subsequent pH ranges. &gt; Wash-in &gt;
 These data were taken from a continuous recording protocol where oocytes
 expressing CaTOK were held at 0 mV and their currents measured in response
 to switching from pH 7.5 to pH 5.0. Raw data values expressed as microamps
 (uA). &gt; Statistics &gt; All statistical analysis were conducted as
 either paired t-test or one-way ANOVA with Dunnett's correction for
 multiple comparison. &gt; Additional Information &gt; Excel files where
 cells contain 'n.a' means not applicable, i.e. No data was
 obtained for this measurement.