The molecular basis of pH sensing by the human fungal pathogen Candida albicans TOK potassium channel
Data files
Sep 24, 2024 version files 254.52 MB
Abstract
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.
README: 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
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 measurements from the tail current (-30 mV) immediately following the prepulse current.Resting membrane potential
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).Dose responses (current density)
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).Dose responses (Resting membrane potential)
These data were taken from unclamped oocytes for pH 7.5 and each subsequent pH. Raw data values are expressed as millivolts (mV).Dose responses (voltage dependent activation)
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).Relative current comparisons
These data were taken at 0 mV from IV curves generated in response to switching to pH 7.5 to subsequent pH ranges.Wash-in
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).Statistics
All statistical analysis were conducted as either paired t-test or one-way ANOVA with Dunnett's correction for multiple comparison.Additional Information
Excel files where cells contain 'n.a' means not applicable, i.e. No data was obtained for this measurement.
Methods
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-electrode voltage clamp (TEVC)
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 MgCl2, 1 CaCl2 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.
Relative permeability calculations
Values were calculated using the Goldman-Hodgkin-Katz (GHK) equation where Vm is the absolute reversal potential and P 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+, Na+, and Cs+ we can compare their relative permeability to 100 mM K+. Permeability ratios for each ion (x) compared to K+ were calculated.
CaTOK structural analysis
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.
Statistics and Reproducibility
All values are expressed as mean ± SEM. Comparison of two groups was conducted using a t-test; all p values were two-sided. One-way ANOVA was applied to all tests comparing more than two groups; all p values were two-sided.