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Open Access

Peer-reviewed

Research Article

Identification and Analysis of Cation Channel Homologues in Human Pathogenic Fungi

Abstract

Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K+), calcium (Ca2+) and transient receptor potential (Trp) channels, but not sodium (Na+) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated Kv channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca2+ uniporter (MCU). In contrast to humans, which express many K+, Ca2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K+, Ca2+ and Trp channel homologues. Furthermore, the sequences of fungal K+, Ca2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

Citation: Prole DL, Taylor CW (2012) Identification and Analysis of Cation Channel Homologues in Human Pathogenic Fungi. PLoS ONE 7(8): e42404. https://doi.org/10.1371/journal.pone.0042404

Editor: Steven Harris, University of Nebraska, United States of America

Received: March 30, 2012; Accepted: July 5, 2012; Published: August 2, 2012

Copyright: © Prole, Taylor. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by a Meres senior research associateship from St. John’s College, Cambridge (to DLP), and by the Wellcome Trust (grant number 085295, to CWT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pathogenic fungi are widespread and cause a variety of diseases in humans, animals and plants, which are of huge medical and economic importance. In this study we focus on human fungal pathogens, which cause infections that are often difficult to treat and can be fatal [1], [2]. Fungal skin and nail infections such as tinea, which are caused most commonly by Trichophyton spp., affect more than twenty percent of the world’s population [3]. Various species of Candida are the most common cause of hospital-acquired fungal infections and cause opportunist infections in immunocompromised patients [1], [2], [4]. Airborne spores of Aspergillus spp. are widespread and these fungi cause disease via production of mycotoxins [5], induction of allergic reactions [6]–[8], or via localized and systemic infections [1], [2]. Systemic infections can also be caused by Blastomyces dermatitidis, Coccidioides spp. [9], [10] and Paracoccidioides brasiliensis; the latter affects more than 10 million people in South America [11]. Inhalation of airborne Histoplasma capsulatum is the most common cause of fungal respiratory infections [12], [13]. Cryptococcus neoformans and Cryptococcus gattii cause disease in around one million people each year, including immunocompetent individuals [14]–[16], and are estimated to cause more than 600,000 deaths [17]. The microsporidia Encephalitozoon spp. and Enterocytozoon bieneusi are an increasingly common cause of intestinal disease and diarrhoea in immunocompromised patients [18], [19]. Current therapies for many of the serious fungal diseases are inadequate or poorly tolerated, and resistance to therapeutic azole drugs is increasingly commonplace [20].

Ionic homeostasis within virtually all cells is maintained by an array of ion channels and transporters, which also allow rapid stimulus-evoked changes in cellular physiology. The diversity of cations (notably Na+, K+, H+ and Ca2+) with electrochemical gradients across biological membranes is much greater than for anions and there is a correspondingly diverse array of cation-selective channels [21]–[23]. Perturbing the activity of cation channels can profoundly affect cell function, and they are the targets of many clinically effective drugs [24]–[27]. This suggests that cation channels in fungal pathogens might play important roles in their physiology and may be targets for novel drugs.

Prominent cation channels include K+, Na+ and Ca2+ channels [22], the mitochondrial Ca2+ uniporter (MCU) [28], [29], many relatively non-selective channels such as Trp channels [22] and many ligand-gated channels [30]–[33]. The genome of the model fungal organism, Saccharomyces cerevisiae, encodes three homologues of mammalian cation channel subunits. These are the plasma membrane two-pore domain K+ (K2P) channel subunit TOK1 [34], [35]; the plasma membrane Ca2+ channel Cch1, which requires the additional Mid1 subunit for function [36], [37]; and the vacuolar membrane Trp channel subunit TrpY1 (also known as Yvc1) [38], [39]. The genome of S. cerevisiae does not encode homologues of MCU or Na+ channels, and also lacks genes encoding many other cation channel subunits (see Results). TOK1 homologues have been described in Candida albicans [40] and Neurospora crassa [41], [42], while genes encoding Ca2+ channels have recently been described in basal fungi [43], Aspergillus spp. [44] and C. neoformans [45]. In addition, purinergic P2X receptors, which are cation channels activated by adenosine triphosphate (ATP), have also been described in basal fungi [46]. However, there has been no systematic analysis of cation channels in many of the most important fungal pathogens.

Recent advances in genomics have resulted in whole-genome sequencing of many pathogenic fungi. In this study we examine these genomes comprehensively, using the sequences of diverse cation channel subunits from mammals, plants, fungi, bacteria and archaea, to search for genes that may encode cation channels. We identify genes likely to encode homologues of K+, Ca2+, Trp and MCU channels in many of the genomes examined. These genes are, however, less plentiful than in mammals and genes encoding homologues of many important mammalian cation channels, such as Na+ channels, are not present. Novel aspects of our findings include the identification of genes encoding previously undescribed homologues of K+, Ca2+ and Trp channel subunits in several pathogenic fungi; multiple homologues of Trp channel subunits in many fungi, including novel homologues more distantly related to TrpY1; novel homologues of voltage-gated K+ (Kv) channel subunits in Cryptococcus spp. and some other fungi; and homologues of MCU in Aspergillus spp. and Cryptococcus spp.

Results and Discussion

The genomes of most pathogenic fungi examined contain genes encoding homologues of K+, Ca2+ and Trp channel subunits, and some additionally have genes encoding homologues of MCU (Table 1). Many of these predicted proteins are not yet annotated as cation channels in fungal databases. In contrast, none of the fungal genomes examined contain genes encoding homologues of Na+ channels or the pore-forming subunits of many other cation channels, such as Orai1 (and its regulatory subunit, STIM1), purinergic receptors, cyclic nucleotide-gated (CNG) channels, hyperpolarization-activated cyclic nucleotide-sensitive non-selective (HCN) channels, N-methyl-D-aspartate (NMDA) receptors, nicotinic acetylcholine receptors, acid-sensing ion channels (ASICs), pannexins, two-pore Ca2+ (TPC) channels, mechanosensitive Piezo channels and voltage-gated Hv1 proton channels. It is also significant that genes encoding inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) subunits are absent from fungal genomes, despite the apparent importance of phospholipase C and IP3 in fungal physiology [47]–[49] and the ability of IP3 to elicit Ca2+ release from vacuolar vesicles of S. cerevisiae [50], N. crassa [51] and C. albicans [52]. The proteins responsible for the Ca2+-mobilizing effects of IP3 in fungi remain to be defined. No genes encoding homologues of any cation channel subunit were identified in the pathogenic microsporidia Encephalitozoon intestinalis, Encephalitozoon cuniculi and E. bieneusi, which have some of the smallest genomes known [53]. This is surprising given the importance of cation channels in most organisms. As Encephalitozoon spp. and E. bieneusi are obligate intracellular parasites, it may be that they do not require cation-selective channels to ensure ionic homeostasis, but rather rely on non-selective pathways that allow ionic continuity with the cytoplasm of the host cell. Other non-selective channels, ion transporters and exchangers are also likely to be present in fungi, which although beyond the scope of this study focussing on cation-selective channels, may also contribute substantially to cation fluxes and ion homeostasis.

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Table 1. Cation channel homologues in pathogenic fungi.

https://doi.org/10.1371/journal.pone.0042404.t001

K+ Channels

Genes encoding homologues of K+ channel subunits are found in the genomes of most pathogenic fungi examined, but are absent from Coccidioides spp. and H. capsulatum (Table 1). Most of these homologues are similar in predicted sequence and topological structure to the TOK1 channel subunit of S. cerevisiae (Table 1, Figure 1 and Figure 2). Surprisingly, in addition to genes encoding homologues of two-pore K+ channels, the genomes of C. neoformans and C. gattii also contain genes encoding homologues of voltage-gated Kv channel subunits, which form a separate fungal K+ channel family (Table 1 and Figure 1).

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Figure 1. Fungal homologues of K+ channel subunits.

Phylogram showing the relationship between the sequences of fungal and human K+ channel subunit sequences (see Methods: based on 44 high confidence positions from a multiple sequence alignment; gamma shape parameter 1.249; proportion of invariant sites zero). Branch length scale bar and branch support values >0.5 are shown. The predicted transmembrane topologies of the two distinct groups of putative K+ channel subunit (Kv and two-pore K2P channel subunits) homologues are also shown.

https://doi.org/10.1371/journal.pone.0042404.g001

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Figure 2. Fungal homologues of two-pore K+ (K2P) channel subunits.

(A) Predicted transmembrane topology of fungal K2P channel subunit homologues; (B) Multiple sequence alignment of the putative pore regions of fungal and human K2P channel homologues. The shaded bar indicates the highly conserved GXG motif within the selectivity filter.

https://doi.org/10.1371/journal.pone.0042404.g002

The putative two-pore K+ channel subunits contain a structure that is unique to fungal channels. Each subunit is predicted to contain eight transmembrane domains (TMDs), with two predicted selectivity filter regions, separated by two TMDs (Figure 2A) [34], [54], [55]. This predicted structure differs from the two-pore K+ channel subunits of other organisms, which have only four TMDs arranged like the last four TMDs of the larger fungal subunits [55]–[58]. Both types of two-pore channel are likely formed by dimerization of subunits [59], [60], which allows four pore-forming loops to create a central pore akin to that of mammalian K+ channels [56]. Multiple sequence alignments confirmed close sequence similarity of these proteins to the TOK1 two-pore K+ channel, and each contains the characteristic GXG selectivity filter motif of K+ channels within the putative pore regions (Figure 2B). Mutation of an aspartate residue immediately following the first GXG motif (D292N) dramatically alters the gating and K+ dependence of TOK1 [61]. Most TOK1 homologues have an aspartate residue at this locus, except for one homologue in Aspergillus flavus (EED45164) and another in Aspergillus fumigatus (XP_747058), which have an asparagine residue (Figure 2B). These homologues may therefore have substantially different gating properties to TOK1 and the other homologues. Another homologue which may have unique properties is that found in C. albicans (XP_712779), which has a VYG motif in place of a GXG motif in the second pore domain (Figure 2B). In contrast to the single gene encoding a two-pore K+ channel (TOK1) in S. cerevisiae, the genomes of Aspergillus spp. each contain two or three distinct genes (Table 1, Figure 1 and Figure 2B). This suggests that K+ channels with different properties may be formed by these subunits, and also that heteromerization of subunits may increase the diversity of K+ channels in Aspergillus spp.

The physiological roles of TOK1 homologues are largely unknown, but in S. cerevisiae TOK1 plays a role in setting the plasma membrane potential [62], [63]. TOK1 channels are blocked by extracellular divalent cations [34], and their activity is decreased at acidic cytosolic pH [64], [65], enhanced by cytosolic ATP [65] and altered by changes in temperature [66]. Physiological modulators of mammalian two-pore K+ channels include fatty acids, voltage, post-translational modification and membrane stretch [57]. Whether these stimuli similarly modulate fungal homologues of TOK1 is unknown.

The putative Kv channel subunits in Cryptococcus spp. are each predicted to have six TMDs, with TMD4 containing regularly spaced basic residues, similar to the voltage-sensing TMD4 domains of mammalian Kv channels [67], [68] (Figures 3A and 3B). They also have a single putative selectivity filter and pore-forming TMD6 region (Figures 3A and 3C). The fungal homologues have a conserved proline residue within TMD6, at a position equivalent to residue P405 of Kv1.2 (Figure 3C). In Kv channels, a proline residue at this position introduces a kink in the pore-lining TMD6 α-helix, which facilitates gating in response to movement of the TMD4 voltage sensor [69]–[71]. This characteristically kinked TMD6 of Kv channels differs from many other K+ channels such as KcsA, which lack the proline residue and have straighter pore helices [72] (Figure 3C). Using sequences of the Kv channel homologues of Cryptococcus spp. as bait in further BLAST searches revealed that the genomes of only a few other fungi encode similar homologues of Kv channel subunits (Table 1). To the authors’ knowledge this is the first description of homologues of Kv channels in fungi.

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Figure 3. Fungal homologues of voltage-gated K+ (Kv) channel subunits.

(A) Predicted topology of fungal Kv channel subunit homologues; (B) Multiple sequence alignment of the putative voltage sensor TMD4 regions of human Kv1.2 and fungal Kv channel homologues. Filled triangles above the alignment indicate the positions of conserved basic residues in Kv1.2; (C) Multiple sequence alignment of the putative pore regions of human Kv1.2 and fungal Kv channel homologues. Predicted pore-lining helices of each protein are underlined and the shaded bar indicates the highly conserved GXG motif within the selectivity filter.

https://doi.org/10.1371/journal.pone.0042404.g003

The identification of genes encoding novel homologues of Kv channels in Cryptococcus spp. and several other fungi is surprising. These genes appear to be confined to the genomes of fungi within the phyla Basidiomycota, Zygomycota and Chytridiomycota, and appear to be entirely absent in Ascomycota. The Kv channel homologues contain putative voltage-sensing TMD4 domains and hence may be regulated by transmembrane voltage. Most Kv channels are activated by membrane depolarization [73], while a few are activated by hyperpolarization [74], [75]. Both types share sequence similarity in their voltage sensor domains [76], which makes it difficult to determine the polarity of their voltage-dependence on the basis of sequence alone. Experimental studies will be necessary to define the voltage sensitivity of these homologues. The majority of Kv channels are present and functional in the plasma membrane, where the greatest changes in transmembrane potential usually occur. It therefore seems likely that the fungal Kv channel homologues reside in the plasma membrane, although this will also require experimental analysis. The existence of putative Kv channel homologues in fungi suggests that dynamic changes in membrane potential may occur in fungi. The plasma membrane potentials of some fungi have been estimated. For example, the plasma membrane potential of Pneumocystis jirovecii has been estimated as −78 mV [77], that of N. crassa as −200 mV [78], [79] and those of various yeast cells as −50 to −120 mV [80]. Membrane potentials of some fungi are dependent on extracellular K+ concentration [81] and dynamic changes in membrane potential occur in the hyphae of N. crassa [82]. However, whether the membrane potentials of fungi change in response to environmental stimuli, and whether the Kv channel homologues identified here respond to such changes is unknown.

In many organisms K+ channels are found predominantly in the plasma membrane, but they are also present in the membranes of intracellular organelles such as mitochondria [83], endoplasmic reticulum (ER) [84], secretory vesicles [85], nuclei [86]–[89], endosomes [90] and vacuoles [91]. Physiological functions of K+ flux are similarly varied and include regulating membrane potentials, facilitating osmolyte homeostasis, modulating enzyme activity, initiating mitogenesis or apoptosis, and aiding transmembrane transport [56], [92]–[96]. Experimental studies will be required to determine the expression, cellular location and function of fungal K+ channels.

Ca2+ Channels

The genomes of all fungi examined (except the microsporidia) contain a single gene encoding a homologue of the plasma membrane Ca2+ channel Cch1 found in S. cerevisiae (Table 1), which is similar in sequence and topological structure to human voltage-gated Cav channels [36], [37] (Figure 4A). The same fungal genomes also have a gene encoding a homologue of Mid1, a regulatory subunit similar to the α2δ-subunits of mammalian Cav channels [97], which is necessary for the function of Cch1 [36], [37] (Table 1). In Cav channels, a Ca2+-binding site that contributes to ionic selectivity is formed by four acidic residues (EEEE), one from the selectivity filter region of each of the four domains (Figures 4A and 4B) [98], [99]. Each of the fungal homologues of Cch1 has a similarly placed acidic motif but with three, rather than four, acidic residues (Figure 4B). Sequences of the surrounding pore domains in human Cav channels and fungal homologues of Cch1 also differ substantially (Figure 4B). The fungal homologues have several regularly spaced basic residues in the TMD4 region of each domain (Figure 5A and 5B). This suggests that these regions may act as voltage sensors similar to those of mammalian Cav channels [99], although the latter have more basic residues (more than 20) than their fungal counterparts (typically 13, except C. gattii and C. neoformans which have 16) (Figure 5B) [99]. This suggests that fungal homologues of Cch1 may have a less pronounced voltage dependence compared to mammalian Cav channels, although this will require experimental analysis.

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Figure 4. The pore regions of human Cav channels, Cch1 and fungal homologues are similar.

(A) Predicted topology of Cav channels, with the pore loop regions of each domain highlighted in red; (B) Multiple sequence alignment of the putative pore loop regions from each domain of human Cav1.2 and fungal Cav channel homologues. The overall motif present at the putative Ca2+ binding site locus (indicated by asterisks above the alignments) is indicated.

https://doi.org/10.1371/journal.pone.0042404.g004

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Figure 5. The voltage sensor regions of human Cav channels, Cch1 and fungal homologues are similar.

(A) Predicted topology of Cav channels, with the voltage sensor TMD4 regions of each domain highlighted in red; (B) Multiple sequence alignment of the putative voltage sensor TMD4 regions from each domain of human Cav1.2 and fungal Cav channel homologues. The total number of basic arginine and lysine residues present in all four putative voltage sensor TMD4 regions is indicated.

https://doi.org/10.1371/journal.pone.0042404.g005

Plasma membrane Ca2+ channels are involved in many cellular processes. Ca2+ influx is a vital part of many physiological signalling pathways and it allows refilling of intracellular Ca2+ stores following release of intracellular Ca2+ [99]–[102]. The presence in all fungal genomes examined (except the microsporidia) of single genes encoding Cch1 and Mid1 homologues suggests a conserved function for Cch1/Mid1 Ca2+ channels, which are present in the plasma membrane in S. cerevisiae [36], [37]. Consistent with this, the fungal homologues of Cch1/Mid1 channels are involved in physiological processes such as mating [36], [37], [103], restoration of intracellular Ca2+ after release of Ca2+ from the ER [104], [105], growth, cell wall synthesis and virulence [106], [107], tolerance to cold stress and iron toxicity [108], high-affinity Ca2+ uptake during ionic stress [45], and hyphal growth [108]. Lack of Cch1 channels in S. cerevisiae impairs high-affinity Ca2+ uptake and leads to cell death in conditions of low Ca2+ concentration or when Ca2+ influx is required [36], [37]. The physiological regulators of Cch1/Mid1 channels are largely unknown, although charged TMD4 domains suggest possible regulation by voltage, and they are activated by mating pheromones [36], [37], [103] and by depletion of Ca2+ from the ER [104], [105].

Mitochondrial Ca2+ Uniporters

The genome of S. cerevisiae has been reported to lack genes encoding homologues of the recently described MCU, which provides a Ca2+ uptake pathway into mammalian mitochondria [28], [29]. This is consistent with a lack of effect of ruthenium red on mitochondrial Ca2+ uptake in S. cerevisiae [110]. In contrast, convincing homologues of MCU are encoded by the genomes of some pathogenic fungi (Table 1). As well as sequence similarity, the predicted topologies of fungal homologues are identical to MCU, with a single putative pore-loop region and the boundaries of the two predicted TMDs in identical positions (Figures 6A and 6B) [28], [29]. The sequences of MCU homologues in Aspergillus spp. and Cryptococcus spp. form a group that is phylogenetically distinct from plant and animal MCU homologues (Figure 6C). Like plant and human MCUs, most of the fungal homologues of MCU are predicted to contain cleavable N-terminal mitochondrial targeting sequences (MITOPROT; http://ihg.gsf.de/ihg/mitoprot.html) [111] (data not shown), suggesting that they may also be located in the inner mitochondrial membrane. Genes encoding homologues of MCU are present in pathogenic Ascomycetes (Aspergillus clavatus, A. flavus and A. fumigatus) and Basidiomycetes (C. gattii and C. neoformans) (Table 1). Genes encoding homologues of MCU are found in about 40% of all sequenced fungal genomes (data not shown). These include the genomes of various fungi in the Chytridiomycota, Basidiomycota and Ascomycota phyla (Table 1). Fungi that lack genes encoding homologues of MCU are also present in each phylum (Table 1). This absence of MCU homologues was in many cases confirmed in multiple, independently sequenced strains of fungi (see Methods), and by using the fungal homologues of MCU as bait in further BLAST searches. Those fungi that do have genes encoding homologues of MCU are closely related within their respective phyla (Table 1; [112], [113]. Together, these observations suggest that genes encoding homologues of MCU may have been lost on several independent occasions during the evolution of fungi.

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Figure 6. Homologues of MCU in pathogenic fungi.

(A) Predicted topology of MCU channels, with the putative pore loop indicated; (B) Multiple sequence alignment of the TMDs and putative pore loop regions of human MCU and fungal homologues, with the predicted TMDs of each protein underlined; (C) Phylogram showing the relationship between the sequences of fungal, animal and plant MCU homologues (see Methods: based on 89 high confidence positions from a multiple sequence alignment; gamma shape parameter 2.969; proportion of invariant sites 0.04). Branch length scale bar and branch support values >0.5 are shown.

https://doi.org/10.1371/journal.pone.0042404.g006

Sequence similarity between fungal and mammalian homologues of MCU may identify residues that are functionally important. The loop between the two TMDs of MCU has been proposed to form the selectivity filter [28], [29]. This region contains a 260WDXMEPVT267 motif in human MCU that is conserved in the fungal homologues (Figure 6B). Further alignment of MCU homologues from such diverse organisms as plants, Dictyostelium discoideum, trypanosomes, Monosiga brevicollis and other fungi (data not shown) [28], [29], [114] shows that a core 260WDXXEP265 motif is most highly conserved (numbered for human MCU). Conserved acidic residues within the selectivity filter of Cav channels coordinate Ca2+ ions [99]. This suggests a possible role for the acidic residues, D261 and E264, of human MCU, and their equivalents in the fungal homologues, in the binding of Ca2+. Mutation of D261 or E264 in MCU compromises function, while the S259A mutant is functional but resistant to the inhibitor, Ru360 [28]. Fungal homologues of MCU differ from human MCU at the position equivalent to residue 259 (they have leucine or alanine in place of serine), suggesting that they may have different pharmacological profiles.

We also searched the genomes of pathogenic fungi for genes encoding homologues of MICU1, a protein containing EF-hands that may form an auxiliary Ca2+-sensing subunit that modulates MCU activity [115]. Expression of MICU1 and MCU is highly correlated in many organisms and tissues [28], [29]. Indeed, this correlation was central to the comparative genomics approach that led to the molecular identification of MCU [28], [29]. We found that like genes encoding homologues of MCU, genes encoding homologues of MICU1 are present in Aspergillus spp. and Cryptococcus spp. but appear to be absent in Candida spp. and S. cerevisiae (Table 1). This further suggests that a MCU-MICU1 Ca2+ uptake pathway is present in some pathogenic fungi but not in others, and as reported previously [28], [29] it is absent in S. cerevisiae. It is intriguing that genes encoding homologues of MICU1, but not MCU, are present in some fungi (Table 1). It is unclear what role homologues of MICU1 might play in these fungi, which include T. rubrum, Coccidioides spp., P. brasiliensis, H. capsulatum and B. dermatitidis (Table 1). Mammalian MCU plays a role in processes such as metabolism, apoptosis and cell signalling [114]. The physiological implications of MCU channels and MICU1 in pathogenic fungi remain to be explored.

Trp Channels

Genes encoding homologues of Trp channel subunits are found in all fungal genomes examined, except those of the microsporidia (Table 1). Some species, including S. cerevisiae, Cryptococcus spp. and H. capsulatum, have a single gene (Table 1, Figure 7A), but others have two genes (T. rubrum, Aspergillus spp., Coccidioides spp., Paracoccidioides spp., Candida spp. and B. dermatitidis) (Table 1 and Figure 7A). Fungal homologues of Trp channel subunits form at least three distinct groups, here termed TrpY1-like (the largest group), Trp2 and Trp3 (Figure 7A). The fungal homologues have at least six predicted TMDs, suggesting that their topologies are similar to TrpY1 and human Trp channel subunits (Figure 7B) [22], [116].

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Figure 7. Fungal homologues of Trp channel subunits.

(A) Phylogram showing the relationship between the sequences of fungal Trp channel subunit homologues (see Methods: based on 176 high confidence positions from a multiple sequence alignment; gamma shape parameter 1.209; proportion of invariant sites zero). The three distinct groups of Trp channel subunit homologue are shown. Branch length scale bar and branch support values >0.5 are shown; (B) Predicted topology of Trp channel subunits. The putative pore region responsible for mechanosensitivity, as well as the C-terminal acidic domain involved in Ca2+ sensitivity are indicated.

https://doi.org/10.1371/journal.pone.0042404.g007

In S. cerevisiae, Ca2+ release from vacuolar stores occurs via TrpY1 channels that are activated by membrane stretch [117], Ca2+ [117] and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) [118]. Activation by membrane stretch is likely mediated by the pore-forming domain [119] (Figure 7B), while activation by Ca2+ is dependent on a C-terminal region containing many acidic residues that may form a Ca2+-binding site [117] (Figure 7B). The sequences of the pore-forming regions divide the fungal homologues into their three major families (Figure 8). Sequence similarity between TrpY1 and the TrpY1-like homologues is pronounced in this region (Figure 8), suggesting that pore-mediated mechanosensitivity [119] may be a conserved feature of these channels. Most notable among the conserved residues are a glycine-phenylalanine motif in the middle of TMD5 (393GF394 in TrpY1), a phenylalanine in TMD6 (444F in TrpY1), and an acidic residue or motif following TMD6 (471DE472 in TrpY1) (Figure 8). These conserved residues in the pore domain of fungal Trp channels may play important roles in channel gating or conductance, although this will require experimental investigation. Many fungal homologues of Trp channel subunits contain highly acidic regions in their C-terminal domains (Figure 9), which are similar to the acidic region involved in activation of TrpY1 by Ca2+ [117]. The density of acidic residues is greatest for the TrpY1-like homologues (Figure 9) suggesting that they, like TrpY1, may be regulated by cytosolic Ca2+. There are fewer acidic residues in the Trp2 homologues and very few in the Trp3 homologues (Figure 9). Experimental studies will be required to assess the possibility that these regions confer differential Ca2+ regulation on fungal homologues of Trp channels. The regions of TrpY1 responsible for activation by PI(3,5)P2 have not been determined. Basic residues within the N-terminal region of mammalian TrpML channels are involved in activation by PI(3,5)P2 [118], but these residues are not conserved in either TrpY1 or the other fungal homologues of Trp channel subunits (data not shown). From these comparisons of sequences with known determinants of TrpY1 regulation, we suggest that the TrpY1-like group of homologues may form mechanosensitive and Ca2+-modulated channels. The physiological regulators of the Trp2 and Trp3 groups of homologues are more difficult to predict.

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Figure 8. Fungal homologues of Trp channel subunits show similarity to the pore region of TrpY1 involved in mechanosensitivity.

Multiple sequence alignment of the putative pore-domain TMDs and pore loop regions of fungal Trp channel subunit homologues, with the predicted TMDs of each protein underlined. The three distinct groups of Trp channel subunit homologue are indicated.

https://doi.org/10.1371/journal.pone.0042404.g008

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Figure 9. Fungal homologues of Trp channel subunits show similarity to the C-terminal acidic region of TrpY1 involved in Ca2+ sensitivity.

Multiple sequence alignment of the C-terminal acidic region of TrpY1 involved in Ca2+ sensitivity with fungal Trp channel homologues. A region of TrpY1 critical for Ca2+ sensitivity [117] is shown underlined. The total number of acidic residues present in this region for each homologue is indicated. Also indicated are the three distinct groups of Trp channel subunit homologues.

https://doi.org/10.1371/journal.pone.0042404.g009

Mammalian Trp channels play diverse roles both in release of Ca2+ and other ions from intracellular stores [23], [120]–[122], and in the influx of Ca2+ across the plasma membrane [116], [123], [124]. It is therefore interesting that genes encoding three distinct groups of Trp homologues are present in the genomes of several pathogenic fungi. One of these groups shares a high degree of sequence similarity with the vacuolar TrpY1 channel of S. cerevisiae, while the others are more distantly related. Further experimental work will be required to assess whether fungal Trp channel homologues form channels permeable to Ca2+ or other ions within the membranes of intracellular organelles such as vacuoles, ER or Golgi, or within the plasma membrane, and to define their physiological roles and regulation.

Relevance to Therapy

Currently used antifungal drugs include azoles, allylamines and the macrolides amphotericin and nystatin, all of which are thought to act mainly via effects on ergosterol [125], [126]. Other drugs include pyrimidine analogues which affect protein synthesis, and sulphonamides. These drugs often have limited efficacy together with substantial side-effects, and emergence of drug resistance is an increasing problem [20]. New drugs to treat fungal infections are therefore needed. In many organisms K+, Ca2+ and Trp channels are essential components of cellular signalling and homeostatic pathways, and they are drug targets in humans [24], [27]. While the human genome contains genes encoding at least 78 K+ channel subunits, 11 Cav channel α-subunits and more than 30 Trp channel subunits [22], the genomes of pathogenic fungi each contain only very small numbers of genes encoding homologues of cation channel subunits (Table 1). This striking lack of redundancy amongst cation channels in pathogenic fungi suggests that they might be effective therapeutic targets. Furthermore, some anti-fungal drugs affect K+, Ca2+ or Trp channel function. For example, azole drugs such as clotrimazole inhibit Trp channels [127], [128], K+ channels [129], [130] and Ca2+ channels [131].

Although they have sequence motifs similar to mammalian K+ channels, and two pore domains similar to human two-pore K+ (K2P) channel subunits, the fungal homologues of TOK1 have a topology and putative structure that is unique to fungi. They are also likely to have a unique gating mechanism [132]. These factors suggest that they may be attractive pharmacological targets. This suggestion gains some support from evidence that a viral toxin that activates TOK1 in S. cerevisiae causes cell death, due to excessive K+ flux [133], and a TOK1 homologue in C. albicans increases sensitivity to human salivary histatin-5 [40]. Activators or inhibitors of TOK1 homologues may therefore be novel anti-fungals.

A diverse range of agents affecting Ca2+ channels or Ca2+ signalling pathways are also toxic to fungi [134]–[138], and Ca2+ channels are involved in the survival of fungal cells after azole-induced stress [97], [138]. The differing pore sequences of human Cav channels and fungal homologues of Cch1 (Figure 4B) suggest that analogues of Cav channel modulators, which often bind within the pore region [139]–[143], may exhibit selectivity for fungal Cch1 homologues over Cav channels. Mitochondrial Ca2+ uptake may be involved in the anti-fungal effects of some peptides [144] and mitochondrial function is linked to drug sensitivity in several fungi [145]–[147], suggesting that fungal homologues of MCU may be attractive novel targets for anti-fungal drugs. Ru360 is a potent inhibitor of MCU [148], and analogues of this drug might possess selective anti-fungal properties against those fungi that contain genes encoding MCU homologues, such as Aspergillus spp. and Cryptococcus spp. Pharmacological modulators of Trp channel function, which are increasingly being developed as potential therapeutic drugs against human targets [27], may also show anti-fungal activity via effects on fungal homologues of Trp channels. Indole and other aromatic compounds such as quinoline and parabens activate TrpY1 [149] and may potentially have anti-fungal activity.

This study presents the opportunity for cloning and functional characterization of cation channels in pathogenic fungi, and suggests that rational design of drugs targeted against these channels may be an effective route to new therapies.

Materials and Methods

Genomes Analyzed

The genomes of the following pathogenic fungi were examined (NCBI and the Broad Institute of Harvard and MIT [150], February 2012): the Ascomycota Trichophyton rubrum CBS 118892, Aspergillus clavatus NRRL 1, Aspergillus flavus NRRL3357, Aspergillus fumigatus Af293 [151], Candida albicans SC5314 [152], Candida glabrata CBS 138, Candida tropicalis MYA-3404, Coccidioides immitis RS, Coccidioides posadasii C735 delta SOWgp, Paracoccidioides brasiliensis Pb01, Blastomyces dermatitidis ATCC 18188 and Histoplasma capsulatum H88; the Basidiomycota Cryptococcus gattii WM276 and Cryptococcus neoformans JEC21; and the Microsporidia Encephalitozoon intestinalis ATCC 50506, Encephalitozoon cuniculi GB-M1 and Enterocytozoon bineusi H348 [153]. The genome of S. cerevisiae S228c was also used. To corroborate the absence of genes encoding particular channel homologues, the genomes of additional strains were analyzed, including: S. cerevisiae CAT-1, A. fumigatus A1163, C. posadasii str. Silveira, P. brasiliensis Pb03, P. brasiliensis Pb18, C. albicans WO-1, H. capsulatum NAm1, B. dermatitidis ER-3, and C. neoformans var. neoformans B-3501A.

BLAST Searches, Alignments and Topology Analysis

Analysis of genomes, sequence alignments and topology analysis were conducted as reported previously [58], [154]. BLASTP and TBLASTN analyses to identify homologues of Ca2+, Na+ and non-selective cation channel subunits were carried out using the following human sequences (protein accession number in parentheses): full-length or pore sequences of IP3R1 (Q14643.2; pore region residues 2536–2608) or RyR1 (P21817.3; pore region residues 4877–4948), and sequences of human TrpA1 (NP_015628; N-truncated sequence residues 765-end), TrpV1 (NP_061197; N-truncated sequence residues 430-end), TrpC1 (P48995; N-truncated sequence residues 350-end), CNGA1 (EAW93049; transmembrane sequence residues 200–420), CNGB1 (NP_001288), HCN2 (NP_001185.3; full-length, and TMD residues 200–470), NMDA receptor NR1 (Q05586), NMDA receptor N2 (Q12879), AMPA receptor GRIA1 (P42261.2), kainate receptor GRIK1 (P39086), nAChR-alpha1 (ABR09427), purinergic receptor P2X4 (NP_002551.2), pannexin-1 (AAH16931), Orai1 (NP_116179.2), STIM1 (AAH21300), TPC1 (NP_001137291.1), TPC2 (NP_620714.2), TrpP1 (NP_001009944), TrpP2 (NP_000288), TrpM1 (NP_002411), TrpML1 (NP_065394), CatSper1 (Q8NEC5.3), acid-sensing ion channel-1 (ASIC1) (P78348.3), mitochondrial uniporter (NP_612366.1), Cav1.2 (NP_955630.2), Nav1.1 (NP_001189364), Piezo-1 (NP_001136336), Piezo-2 (NP_071351) and NALCN (AAH64343). Sequences of the S. cerevisiae Ca2+ channel Cch1 (CAA97244), Mid1 (NP_014108) and TrpY1 (NP_014730), as well as Arabidopsis thaliana TPC1 (AAK39554) were also used to search for fungal homologues. The sequence of the MCU auxiliary subunit MICU1 (NP_006068.2) was also used. Searches to identify K+ channel homologues were carried out using the following sequences of diverse human K+ channels (protein accession number in parentheses): Kv1.2 (NP_004965.1), Kv7.1 (NP_000209.2) and Kv11.1 (hERG1) (Q12809.1); Kir1.1 (ROMK1) (NP_000211.1), Kir2.1 (IRK1) (NP_000882.1), Kir3.1 (GIRK1) (NP_002230.1), Kir4.1 (P78508.1), Kir5.1 (Q9NPI9.1), Kir6.1 (KATP1) (Q15842.1), Kir6.2 (NP_000516.3) and Kir7.1 (CAA06878.1); K2P1.1 (TWIK1) (NP_002236.1), K2P2.1 (TREK1) (NP_001017425.2), K2P3.1 (TASK1) (NP_002237.1), K2P13.1 (THIK1) (NP_071337.2), K2P16.1 (TALK1) (NP_001128577.1) and K2P18.1 (TRESK2) (NP_862823.1); KCa1.1 (BK) (NP_001154824.1), KCa2.1 (SK1) (NP_002239.2), KCa2.2 (SK2) (NP_067627), KCa3.1 (IK/SK4) (NP_002241.1) and KCa4.1 (SLACK/KNa) (NP_065873.2). Other K+ channel sequences were also used to search for fungal homologues, including: bacterial KcsA (P0A334), bacterial cyclic nucleotide-gated MlotiK1 (Q98GN8.1), archaeal depolarization-activated KvAP (Q9YDF8.1), archaeal hyperpolarization-activated MVP (Q57603.1), archaeal Ca2+-activated MthK (O27564.1), and TOK1 from S. cerevisiae (CAA89386.1). Plant K+ channel sequences were also used, including: the vacuolar outwardly rectifying, Ca2+-regulated vacuolar two-pore TPK1 channel (NP_200374.1); vacuolar KCO3 (NP_001190480.1); the pollen plasma membrane TPK4 (NP_171752.1), the inward rectifier KAT1 (NP_199436.1), the outward rectifier SKOR (pore region of NP_186934.1, residues 271–340 to avoid ankyrin hits), and AKT1 (NP_180233.1). We also searched for homologues of Hv1 proton channel subunits (NP_115745.2). Default BLAST parameters for assessing statistical significance and for filtering were used (ie. an Expect threshold of 10, and SEG filtering).

Several procedures ensured that hits were likely homologues of cation channel subunits. Firstly, the presence of multiple transmembrane domains was confirmed using TOPCONS [155]. Secondly, reciprocal BLASTP searches (non-redundant protein database at NCBI) were made, using the identified fungal hits as bait, and only proteins that gave the original mammalian protein family as hits were analyzed further. Thirdly, the presence of conserved domains was confirmed using the Conserved Domains Database (NCBI). In addition, for homologues of K+ channel subunits, only hits with regions of sequence similarity that encompassed the selectivity filter sequence of the K+ channel subunit used as bait were acknowledged. Also, where possible, pore homology was confirmed by sequence alignment using ClustalW2.1 (European Bioinformatics Institute). Multiple sequence alignments were made using ClustalW2.1 and physiochemical residue colours are shown. Where shown, asterisks below the alignment indicate positions that have a single fully conserved residue, while colons indicate positions that have residues with highly similar properties (scoring >0.5 in the Gonnet PAM 250 matrix, ClustalW2). For phylogenetic analysis, multiple sequence alignments were made with MUSCLE v3.7 using default parameters. After using GBLOCKS at high stringency to remove regions of low confidence, and removing gaps, Maximum Likelihood analysis was carried out using PhyML v3.0 (WAG substitution model; 4 substitution rate categories; default estimated gamma distribution parameters; default estimated proportions of invariable sites; 100 bootstrapped data sets). Phylogenetic trees were depicted using TreeDyn (v198.3). MUSCLE, GBLOCKS, PhyML and TreeDyn were all functions of Phylogeny.fr (http://www.phylogeny.fr/) [156].

Author Contributions

Conceived and designed the experiments: DLP. Performed the experiments: DLP. Analyzed the data: DLP. Contributed reagents/materials/analysis tools: DLP. Wrote the paper: DLP CWT.

References

  1. 1. Gullo A (2009) Invasive fungal infections: the challenge continues. Drugs 69 Suppl 165–73.
  2. 2. Faguy DM (2011) Fungal pathogens: an overview. Radiol Technol 82: 321–340.
  3. 3. Havlickova B, Czaika VA, Friedrich M (2008) Epidemiological trends in skin mycoses worldwide. Mycoses 51 Suppl 42–15.
  4. 4. Miceli MH, Diaz JA, Lee SA (2011) Emerging opportunistic yeast infections. Lancet Infect Dis 11: 142–151.
  5. 5. Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P, Denning DW (2007) Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153: 1677–1692.
  6. 6. Marr KA, Patterson T, Denning D (2002) Aspergillosis. Pathogenesis, clinical manifestations, and therapy. Infect Dis Clin North Am 16: 875–894.
  7. 7. Agarwal R (2009) Allergic bronchopulmonary aspergillosis. Chest 135: 805–826.
  8. 8. Patterson K, Strek ME (2010) Allergic bronchopulmonary aspergillosis. Proc Am Thorac Soc 7: 237–244.
  9. 9. Deus Filho A (2009) Chapter 2: coccidioidomycosis. J Bras Pneumol 35: 920–930.
  10. 10. Borchers AT, Gershwin ME (2010) The immune response in Coccidioidomycosis. Autoimmun Rev 10: 94–102.
  11. 11. Brummer E, Castaneda E, Restrepo A (1993) Paracoccidioidomycosis: an update. Clin Microbiol Rev 6: 89–117.
  12. 12. Aide MA (2009) Chapter 4 - histoplasmosis. J Bras Pneumol 35: 1145–1151.
  13. 13. Nosanchuk JD, Zancope-Oliveira RM, Hamilton AJ, Guimaraes AJ (2012) Antibody therapy for histoplasmosis. Front Microbiol 3: 21.
  14. 14. Kronstad JW, Attarian R, Cadieux B, Choi J, D’Souza CA, et al. (2011) Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat Rev Microbiol 9: 193–203.
  15. 15. Kozubowski L, Heitman J (2012) Profiling a killer, the development of Cryptococcus neoformans. FEMS Microbiol Rev 36: 78–94.
  16. 16. Chaturvedi V, Chaturvedi S (2011) Cryptococcus gattii: a resurgent fungal pathogen. Trends Microbiol 19: 564–571.
  17. 17. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, et al. (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23: 525–530.
  18. 18. Didier ES, Weiss LM (2011) Microsporidiosis: not just in AIDS patients. Curr Opin Infect Dis 24: 490–495.
  19. 19. Anane S, Attouchi H (2010) Microsporidiosis: epidemiology, clinical data and therapy. Gastroenterol Clin Biol 34: 450–464.
  20. 20. Pfaller MA (2012) Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment. Am J Med 125: S3–13.
  21. 21. Hille B (2001) Ionic Channels of Excitable Membranes. Third Edition. Sunderland, Massachusetts: Sinauer Associates Inc. 814 p.
    • 22. Yu FH, Catterall WA (2004) The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. Sci STKE 2004: re15.
    • 23. Dong XP, Wang X, Xu H (2010) TRP channels of intracellular membranes. J Neurochem 113: 313–328.
    • 24. Kaczorowski GJ, McManus OB, Priest BT, Garcia ML (2008) Ion channels as drug targets: the next GPCRs. J Gen Physiol 131: 399–405.
    • 25. Wulff H, Castle NA, Pardo LA (2009) Voltage-gated potassium channels as therapeutic targets. Nat Rev Drug Discov 8: 982–1001.
    • 26. Alexander SP, Mathie A, Peters JA (2011) Guide to Receptors and Channels (GRAC), 5th edition. Br J Pharmacol 164 Suppl 1S1–324.
    • 27. Moran MM, McAlexander MA, Biro T, Szallasi A (2011) Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 10: 601–620.
    • 28. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, et al. (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476: 341–345.
    • 29. De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476: 336–340.
    • 30. Thompson AJ, Lummis SC (2007) The 5-HT3 receptor as a therapeutic target. Expert Opin Ther Targets 11: 527–540.
    • 31. Jarvis MF, Khakh BS (2009) ATP-gated P2X cation-channels. Neuropharmacology 56: 208–215.
    • 32. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, et al. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62: 405–496.
    • 33. Albuquerque EX, Pereira EF, Alkondon M, Rogers SW (2009) Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89: 73–120.
    • 34. Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK, Goldstein SA (1995) A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376: 690–695.
    • 35. Zhou XL, Vaillant B, Loukin SH, Kung C, Saimi Y (1995) YKC1 encodes the depolarization-activated K+ channel in the plasma membrane of yeast. FEBS Lett 373: 170–176.
    • 36. Paidhungat M, Garrett S (1997) A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect. Mol Cell Biol 17: 6339–6347.
    • 37. Fischer M, Schnell N, Chattaway J, Davies P, Dixon G, et al. (1997) The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating. FEBS Lett 419: 259–262.
    • 38. Palmer CP, Zhou XL, Lin J, Loukin SH, Kung C, et al. (2001) A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane. Proc Natl Acad Sci U S A 98: 7801–7805.
    • 39. Denis V, Cyert MS (2002) Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue. J Cell Biol 156: 29–34.
    • 40. Baev D, Rivetta A, Li XS, Vylkova S, Bashi E, et al. (2003) Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1. Infect Immun 71: 3251–3260.
    • 41. Roberts SK (2003) TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus. Eukaryot Cell 2: 181–190.
    • 42. Lew RR, Nasserifar S (2009) Transient responses during hyperosmotic shock in the filamentous fungus Neurospora crassa. Microbiol. 155: 903–11.
    • 43. Cai X, Clapham DE (2012) Ancestral Ca2+ signaling machinery in early animal and fungal evolution. Mol Biol Evol 29: 91–100.
    • 44. Bencina M, Bagar T, Lah L, Krasevec N (2009) A comparative genomic analysis of calcium and proton signaling/homeostasis in Aspergillus species. Fungal Genet Biol 46 Suppl 1S93–S104.
    • 45. Liu M, Du P, Heinrich G, Cox GM, Gelli A (2006) Cch1 mediates calcium entry in Cryptococcus neoformans and is essential in low-calcium environments. Eukaryot Cell 5: 1788–1796.
    • 46. Cai X (2012) P2X receptor homologs in basal fungi. Purinergic Signal 8: 11–13.
    • 47. Kozubowski L, Lee SC, Heitman J (2009) Signalling pathways in the pathogenesis of Cryptococcus. Cell Microbiol 11: 370–380.
    • 48. Cunningham KW (2011) Acidic calcium stores of Saccharomyces cerevisiae. Cell Calcium 50: 129–138.
    • 49. Silverman-Gavrila LB, Lew RR (2002) An IP3-activated Ca2+ channel regulates fungal tip growth. J. Cell Sci. 115: 5013–5025.
    • 50. Belde PJ, Vossen JH, Borst-Pauwels GW, Theuvenet AP (1993) Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. FEBS Lett 323: 113–118.
    • 51. Cornelius G, Gebauer G, Techel D (1989) Inositol trisphosphate induces calcium release from Neurospora crassa vacuoles. Biochem Biophys Res Commun 162: 852–856.
    • 52. Calvert CM, Sanders D (1995) Inositol trisphosphate-dependent and -independent Ca2+ mobilization pathways at the vacuolar membrane of Candida albicans. J Biol Chem 270: 7272–7280.
    • 53. Peyretaillade E, El Alaoui H, Diogon M, Polonais V, Parisot N, et al. (2011) Extreme reduction and compaction of microsporidian genomes. Res Microbiol 162: 598–606.
    • 54. Salkoff L, Jegla T (1995) Surfing the DNA databases for K+ channels nets yet more diversity.Neuron. 15: 489–492.
    • 55. Goldstein SA, Wang KW, Ilan N, Pausch MH (1998) Sequence and function of the two P domain potassium channels: implications of an emerging superfamily. J Mol Med (Berl) 76: 13–20.
    • 56. Miller C (2000) An overview of the potassium channel family. Genome Biol 1: REVIEWS0004.
    • 57. Enyedi P, Czirjak G (2010) Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev 90: 559–605.
    • 58. Prole DL, Marrion NV (2012) Identification of putative potassium channel homologues in pathogenic protozoa. PLoS One 7: e32264.
    • 59. Miller AN, Long SB (2012) Crystal structure of the human two-pore domain potassium channel K2P1. Science 335: 432–436.
    • 60. Brohawn SG, del Marmol J, MacKinnon R (2012) Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. Science 335: 436–441.
    • 61. Roller A, Natura G, Bihler H, Slayman CL, Eing C, et al. (2005) In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance. Pflugers Arch 451: 362–370.
    • 62. Bertl A, Ramos J, Ludwig J, Lichtenberg-Frate H, Reid J, et al. (2003) Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations. Mol Microbiol 47: 767–780.
    • 63. Maresova L, Urbankova E, Gaskova D, Sychrova H (2006) Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance. FEMS Yeast Res 6: 1039–1046.
    • 64. Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, et al. (1996) A pH-sensitive yeast outward rectifier K+ channel with two pore domains and novel gating properties. J Biol Chem 271: 4183–4187.
    • 65. Bertl A, Bihler H, Reid JD, Kettner C, Slayman CL (1998) Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ. J Membr Biol 162: 67–80.
    • 66. Loukin SH, Saimi Y (1999) K+-dependent composite gating of the yeast K+ channel, Tok1. Biophys J 77: 3060–3070.
    • 67. Papazian DM, Timpe LC, Jan YN, Jan LY (1991) Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature 349: 305–310.
    • 68. Liman ER, Hess P, Weaver F, Koren G (1991) Voltage-sensing residues in the S4 region of a mammalian K+ channel. Nature 353: 752–756.
    • 69. del Camino D, Holmgren M, Liu Y, Yellen G (2000) Blocker protection in the pore of a voltage-gated K+ channel and its structural implications. Nature 403: 321–325.
    • 70. Webster SM, Del Camino D, Dekker JP, Yellen G (2004) Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges. Nature 428: 864–868.
    • 71. Long SB, Campbell EB, Mackinnon R (2005) Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309: 903–908.
    • 72. Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, et al. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280: 69–77.
    • 73. Yellen G (2002) The voltage-gated potassium channels and their relatives. 419: 35–42.
    • 74. Schachtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gaber RF (1992) Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. 258: 1654–8.
    • 75. Sesti F, Rajan S, Gonzalez-Colaso R, Nikolaeva N, Goldstein SA (2003) Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel. Nat Neurosci 6: 353–61.
    • 76. Mannikko R, Elinder F, Larsson HP (2002) Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. Nature 419: 837–41.
    • 77. VanderHeyden N, McLaughlin GL, Docampo R (2000) Regulation of the plasma membrane potential in Pneumocystis carinii. FEMS Microbiol Lett 183: 327–330.
    • 78. Slayman CL (1965) Electrical properties of Neurospora crassa. Effects of external cations on the intracellular potential. J Gen Physiol 49: 69–92.
    • 79. Ermolayeva E, Sanders D (1995) Mechanism of pyrithione-induced membrane depolarization in Neurospora crassa. Appl Environ Microbiol 61: 3385–3390.
    • 80. Vacata V, Kotyk A, Sigler K (1981) Membrane potentials in yeast cells measured by direct and indirect methods. Biochim Biophys Acta 643: 265–268.
    • 81. Slayman CL, Slayman CW (1962) Measurement of membrane potentials in Neurospora. Science 136: 876–877.
    • 82. Slayman CL, Long WS, Gradmann D (1976) “Action potentials” in Neurospora crassa, a mycelial fungus. Biochim Biophys Acta 426: 732–744.
    • 83. Szewczyk A, Jarmuszkiewicz W, Kunz WS (2009) Mitochondrial potassium channels. IUBMB Life 61: 134–143.
    • 84. Ng KE, Schwarzer S, Duchen MR, Tinker A (2010) The intracellular localization and function of the ATP-sensitive K+ channel subunit Kir6.1. J Membr Biol 234: 137–147.
    • 85. Geng X, Li L, Watkins S, Robbins PD, Drain P (2003) The insulin secretory granule is the major site of KATP channels of the endocrine pancreas. Diabetes 52: 767–776.
    • 86. Mazzanti M, DeFelice LJ, Cohn J, Malter H (1990) Ion channels in the nuclear envelope. Nature 343: 764–767.
    • 87. Quesada I, Rovira JM, Martin F, Roche E, Nadal A, et al. (2002) Nuclear KATP channels trigger nuclear Ca2+ transients that modulate nuclear function. Proc Natl Acad Sci U S A 99: 9544–9549.
    • 88. Yamashita M, Sugioka M, Ogawa Y (2006) Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release. FEBS J 273: 3585–3597.
    • 89. Chen Y, Sanchez A, Rubio ME, Kohl T, Pardo LA, et al. (2011) Functional Kv10.1 channels localize to the inner nuclear membrane. PLoS One 6: e19257.
    • 90. Bao L, Hadjiolova K, Coetzee WA, Rindler MJ (2011) Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits. Am J Physiol Heart Circ Physiol 300: H262–270.
    • 91. Gobert A, Isayenkov S, Voelker C, Czempinski K, Maathuis FJ (2007) The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis. Proc Natl Acad Sci U S A 104: 10726–10731.
    • 92. Kuo MM, Haynes WJ, Loukin SH, Kung C, Saimi Y (2005) Prokaryotic K+ channels: from crystal structures to diversity. FEMS Microbiol Rev 29: 961–85.
    • 93. Loukin SH, Kuo MM, Zhou XL, Haynes WJ, Kung C, et al. (2005) Microbial K+ channels. J Gen Physiol 125: 521–7.
    • 94. Ward JM, Maser P, Schroeder JI (2009) Plant ion channels: gene families, physiology, and functional genomics analyses. Annu Rev Physiol 71: 59–82.
    • 95. Gajdanowicz P, Michard E, Sandmann M, Rocha M, Correa LG, et al. (2011) Potassium K+ gradients serve as a mobile energy source in plant vascular tissues. Proc Natl Acad Sci U S A 108: 864–9.
    • 96. Remillard CV, Yuan JX (2004) Activation of K+ channels: an essential pathway in programmed cell death. Am J Physiol Lung Cell Mol Physiol 286: L49–67.
    • 97. Martin DC, Kim H, Mackin NA, Maldonado-Baez L, Evangelista CC Jr, et al. (2011) New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast. J Biol Chem 286: 10744–10754.
    • 98. Cibulsky SM, Sather WA (2000) The EEEE locus is the sole high-affinity Ca2+ binding structure in the pore of a voltage-gated Ca2+ channel: block by Ca2+ entering from the intracellular pore entrance. J Gen Physiol 116: 349–362.
    • 99. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16: 521–555.
    • 100. Verret F, Wheeler G, Taylor AR, Farnham G, Brownlee C (2010) Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium-based signalling. New Phytol 187: 23–43.
    • 101. Wheeler GL, Brownlee C (2008) Ca2+ signalling in plants and green algae - changing channels. Trends Plant Sci 13: 506–14.
    • 102. Parekh AB, Putney JW Jr (2005) Store-operated calcium channels. Physiol Rev 85: 757–810.
    • 103. Iida H, Nakamura H, Ono T, Okumura MS, Anraku Y (1994) MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Mol Cell Biol 14: 8259–8271.
    • 104. Locke EG, Bonilla M, Liang L, Takita Y, Cunningham KW (2000) A homolog of voltage-gated Ca2+ channels stimulated by depletion of secretory Ca2+ in yeast. Mol Cell Biol 20: 6686–6694.
    • 105. Hong MP, Vu K, Bautos J, Gelli A (2010) Cch1 restores intracellular Ca2+ in fungal cells during endoplasmic reticulum stress. J Biol Chem 285: 10951–10958.
    • 106. Bormann J, Tudzynski P (2009) Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence. Microbiology 155: 3922–3933.
    • 107. Cavinder B, Hamam A, Lew RR, Trail F (2011) Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascopore discharge in the filamentous fungus Gibberella zeae. Eukaryot. Cell 10: 832–41.
    • 108. Peiter E, Fischer M, Sidaway K, Roberts SK, Sanders D (2005) The Saccharomyces cerevisiae Ca2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity. FEBS Lett 579: 5697–5703.
    • 109. Brand A, Lee K, Veses V, Gow NA (2009) Calcium homeostasis is required for contact-dependent helical and sinusoidal tip growth in Candida albicans hyphae. Mol Microbiol 71: 1155–1164.
    • 110. Uribe S, Rangel P, Pardo JP (1992) Interactions of calcium with yeast mitochondria. Cell Calcium 13: 211–217.
    • 111. Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241: 779–786.
    • 112. Wang H, Xu Z, Gao L, Hao B (2009) A fungal phylogeny based on 82 complete genomes using the composition vector method. BMC Evol Biol 9: 195.
    • 113. Fungal Genome Initiative (2012) Broad Institute of Harvard and MIT (2012) http://www.broadinstitute.org/scientific-community/science/projects/fungal-genome-initiative/fungal-genome-initiative.
      • 114. Docampo R, Lukes J (2012) Trypanosomes and the solution to a 50-year mitochondrial calcium mystery. Trends Parasitol 28: 31–37.
      • 115. Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, et al. (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature 467: 291–296.
      • 116. Montell C (2005) The TRP superfamily of cation channels. Sci STKE 2005: re3.
      • 117. Su Z, Zhou X, Loukin SH, Saimi Y, Kung C (2009) Mechanical force and cytoplasmic Ca2+ activate yeast TRPY1 in parallel. J Membr Biol 227: 141–150.
      • 118. Dong XP, Shen D, Wang X, Dawson T, Li X, et al. (2010) PI(3,5)P2 Controls Membrane Traffic by Direct Activation of Mucolipin Ca2+ Release Channels in the Endolysosome. Nat Commun 1: 38.
      • 119. Su Z, Anishkin A, Kung C, Saimi Y (2011) The core domain as the force sensor of the yeast mechanosensitive TRP channel. J Gen Physiol 138: 627–640.
      • 120. LaPlante JM, Falardeau J, Sun M, Kanazirska M, Brown EM, et al. (2002) Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway. FEBS Lett 532: 183–187.
      • 121. Dong XP, Cheng X, Mills E, Delling M, Wang F, et al. (2008) The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 455: 992–996.
      • 122. Kiselyov K, Colletti GA, Terwilliger A, Ketchum K, Lyons CW, et al. (2011) TRPML: transporters of metals in lysosomes essential for cell survival? Cell Calcium 50: 288–294.
      • 123. Taylor CW, Prole DL, Rahman T (2009) Ca2+ channels on the move. Biochemistry 48: 12062–12080.
      • 124. Gees M, Colsoul B, Nilius B (2010) The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2: a003962.
      • 125. Zhang YQ, Rao R (2010) Beyond ergosterol: linking pH to antifungal mechanisms. Virulence 1: 551–554.
      • 126. Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, et al. (2012) Amphotericin primarily kills yeast by simply binding ergosterol. Proc Natl Acad Sci U S A 109: 2234–2239.
      • 127. Hill K, McNulty S, Randall AD (2004) Inhibition of TRPM2 channels by the antifungal agents clotrimazole and econazole. Naunyn Schmiedebergs Arch Pharmacol 370: 227–237.
      • 128. Meseguer V, Karashima Y, Talavera K, D’Hoedt D, Donovan-Rodriguez T, et al. (2008) Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole. J Neurosci 28: 576–586.
      • 129. Alvarez J, Montero M, Garcia-Sancho J (1992) High affinity inhibition of Ca2+-dependent K+ channels by cytochrome P-450 inhibitors. J Biol Chem 267: 11789–11793.
      • 130. Tian M, Dong MQ, Chiu SW, Lau CP, Li GR (2006) Effects of the antifungal antibiotic clotrimazole on human cardiac repolarization potassium currents. Br J Pharmacol 147: 289–297.
      • 131. Thomas GP, Karmazyn M, Zygmunt AC, Antzelevitch C, Narayanan N (1999) The antifungal antibiotic clotrimazole potently inhibits L-type calcium current in guinea-pig ventricular myocytes. Br J Pharmacol 126: 1531–1533.
      • 132. Loukin SH, Lin J, Athar U, Palmer C, Saimi Y (2002) The carboxyl tail forms a discrete functional domain that blocks closure of the yeast K+ channel. Proc Natl Acad Sci U S A 99: 1926–1930.
      • 133. Ahmed A, Sesti F, Ilan N, Shih TM, Sturley SL, et al. (1999) A molecular target for viral killer toxin: TOK1 potassium channels. Cell 99: 283–291.
      • 134. Xu T, Feng Q, Jacob MR, Avula B, Mask MM, et al. (2011) The marine sponge-derived polyketide endoperoxide plakortide F acid mediates its antifungal activity by interfering with calcium homeostasis. Antimicrob Agents Chemother 55: 1611–1621.
      • 135. Binder U, Chu M, Read ND, Marx F (2010) The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa. Eukaryot Cell 9: 1374–1382.
      • 136. Rodrigues AA, Pina-Vaz C, Mardh PA, Martinez-de-Oliveira J, Freitas-da-Fonseca A (2000) Inhibition of germ tube formation by Candida albicans by local anesthetics: an effect related to ionic channel blockade. Curr Microbiol 40: 145–148.
      • 137. Inoue I, Seishima M, Osada K, Kitajima Y (1996) Different effects of azole-antifungal agents on the regulation of intracellular calcium concentration of Trichophyton rubrum. J Dermatol Sci 12: 156–162.
      • 138. Kaur R, Castano I, Cormack BP (2004) Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob Agents Chemother 48: 1600–1613.
      • 139. Peterson BZ, Tanada TN, Catterall WA (1996) Molecular determinants of high affinity dihydropyridine binding in L-type calcium channels. J Biol Chem 271: 5293–5296.
      • 140. Kraus R, Reichl B, Kimball SD, Grabner M, Murphy BJ, et al. (1996) Identification of benz(othi)azepine-binding regions within L-type calcium channel α1 subunits. J Biol Chem 271: 20113–20118.
      • 141. Striessnig J, Glossmann H, Catterall WA (1990) Identification of a phenylalkylamine binding region within the α1 subunit of skeletal muscle Ca2+ channels. Proc Natl Acad Sci U S A 87: 9108–9112.
      • 142. McDonough SI, Boland LM, Mintz IM, Bean BP (2002) Interactions among toxins that inhibit N-type and P-type calcium channels. J Gen Physiol 119: 313–328.
      • 143. Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J (2005) International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev 57: 411–425.
      • 144. Lupetti A, Brouwer CP, Dogterom-Ballering HE, Senesi S, Campa M, et al. (2004) Release of calcium from intracellular stores and subsequent uptake by mitochondria are essential for the candidacidal activity of an N-terminal peptide of human lactoferrin. J Antimicrob Chemother 54: 603–608.
      • 145. Sanglard D, Ischer F, Bille J (2001) Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob Agents Chemother 45: 1174–1183.
      • 146. Shingu-Vazquez M, Traven A (2011) Mitochondria and fungal pathogenesis: drug tolerance, virulence, and potential for antifungal therapy. Eukaryot Cell 10: 1376–1383.
      • 147. Hallstrom TC, Moye-Rowley WS (2000) Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae. J Biol Chem 275: 37347–37356.
      • 148. Matlib MA, Zhou Z, Knight S, Ahmed S, Choi KM, et al. (1998) Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes. J Biol Chem 273: 10223–10231.
      • 149. John Haynes W, Zhou XL, Su ZW, Loukin SH, Saimi Y, et al. (2008) Indole and other aromatic compounds activate the yeast TRPY1 channel. FEBS Lett 582: 1514–1518.
      • 150. Origins of Multicellularity Sequencing Project and Fungal Genome Initiative, Broad Institute of Harvard and MIT (2012) http://www.broadinstitute.org.
        • 151. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, et al. (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438: 1151–1156.
        • 152. Jones T, Federspiel NA, Chibana H, Dungan J, Kalman S, et al. (2004) The diploid genome sequence of Candida albicans. Proc Natl Acad Sci U S A 101: 7329–7334.
        • 153. Akiyoshi DE, Morrison HG, Lei S, Feng X, Zhang Q, et al. (2009) Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi. PLoS Pathog 5: e1000261.
        • 154. Prole DL, Taylor CW (2011) Identification of intracellular and plasma membrane calcium channel homologues in pathogenic parasites. PLoS One 6: e26218.
        • 155. Bernsel A, Viklund H, Hennerdal A, Elofsson A (2009) TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 37: W465–468.
        • 156. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, et al. (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36: W465–469.