Biophysical and physiological characterisation of potassium channels from pathogenic fungi

Opportunistic fungal pathogens are a major cause of life-threatening infections in individuals with a compromised immune system. An increase in the patient population at risk from the development of serious fungal infections, including HIV/AIDS patients, those undergoing blood and marrow transplant, major surgery or receiving chemotherapy has led to an associated rise in the frequency of invasive infections over the past two decades. Targeted anti-fungal therapies are often complicated by biological similarities between fungi and their mammalian hosts. Furthermore, fungal infections can be recalcitrant to therapy and resistance to traditional interventions such as fluconazole is a growing problem. Hence the need to identify novel anti-fungal targets is paramount.

The purpose of this research project is to characterise a fungal ion channel protein and its role in the fungal cell. Ion channels act as regulated "holes" in the cell membrane allowing ions such as sodium and potassium to pass in and out of the cell and their function is essential for maintaining the activity of cells in almost all forms of life. Ion channels can be defined simply on the basis of their structure and which ions they pass. Potassium ion channels for example allow only the movement of potassium into or out of the cell and are formed through the assembly of multiple proteins surrounding a "hole" or "ion pathway". The opening and closing of this pathway is tightly regulated by a variety of stimuli. Thus understanding the mechanisms of how these ion channel proteins open and close and how we could stimulate or inhibit potassium ion flux is fundamental to manipulating fungal cell function.

Using several species of human pathogenic fungi (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans) which represent the primary sources of fatal infections in the immunosuppressed population, I have identified a number of specific potassium channels (called TOK1). These TOK1 potassium channels potentially control the growth and function of fungal cells by managing the movement of potassium ions in and out of the cell. More importantly, if the ion channel pathway was held open and ion flow unregulated it could lead to excessive potassium loss and fungal cell death. TOK1 channels are found only in fungi and no similar protein exists in humans, animals or plants. As one of the limitations of existing therapies is that they do not distinguish between human and fungal cells, the unique fungal nature of these TOK1 channels makes them ideal targets for future anti-fungal therapies. The aim of this study is to understand the mechanism of how these channels open and close and begin to address the role these channels play in fungal cells. It is anticipated that data from this study will allow the future design of compounds specially targeted to induce unregulated potassium flow through TOK1 channels providing a unique strategy to combat and reduce the prevalence of dangerous fungal infections.

Fungal genomes express a gene encoding a potassium channel (TOK1) which has no known structural or functional homologues in either mammals or plants and is emerging as a potential therapeutic target for disrupting normal fungal cell physiology. TOK1 channel proteins are distinctive in topology with 2 pore-forming P domains and 8 transmembrane spans (2P/8TM) and unique in their strictly outwardly-rectifying function. I have recently cloned TOK1 homologues from multiple species of pathogenic fungi that represent the primary sources of fatal infections in immunosuppressed individuals (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans). Preliminary results establish that the TOK1 channel family can be functionally defined by their K+ selectivity, strict outward rectification above EK, voltage-dependence, time-dependent and -independent components to activation, non-inactivating current phenotype and pharmacology. Despite these advances, many key questions concerning the strict gating, physiological regulation and role of TOK1 channels in fungi remain to be addressed. The aim of the present study is to uncover the molecular mechanisms underlying the strict outward rectification of TOK1 channels using a combined molecular and electrophysiological strategy targeting specific residues lining the ion conduction pathway. I have also recently identified a pHO-sensing residue in the S1-S2 loop of the Candida albicans TOK1 homologue that modulates K+ selectivity. I will probe further the molecular and structural basis for this key finding and the role of CaTOK in morphological transitions of Candida albicans, a key virulence trait. These results, describing biophysical, regulatory, and physiological properties of TOK1 channels will provide a suitable platform for developing therapeutic strategies to reduce the prevalence of dangerous mycotic infections.

This project proposal represents a strategic investment in UK bioscience in terms of enhancing the knowledge economy and delivering and training highly skilled researchers. Data from this study will notably enhance our basic knowledge of ion channel proteins in terms of their protein structure-function relationships. Its long term potential impact is significant and broad ranging, impacting academia, the public sector, industry and the general public. Fungal TOK1 ion channels are unique to yeast and fungi and represent an exciting potential future target for antifungal therapies to treat the increasingly frequent incidence of fungal infections in humans, such as those caused by Candida, Aspergillus and Cryptococcus. Although not the focus of this proposal, cross-phyla conservation of TOK1 proteins will permit knowledge gained from this study to potentially impact strategies for managing agricultural fungal pathogens (i.e. Ustilago maydis, corn smut) which are responsible for significant agricultural and economic losses worldwide, endangering food and biofuel crops, and to animal fungal pathogens (i.e. Batrachochytrium dendrobatidis) which are decimating the planets frog and salamander population. Such broad ranging impact would be anticipated to attract global research and development investment leading to commercialisation and exploitation of scientific knowledge and investment in new technologies, therapeutic products and services with obvious direct societal and economic impact.

Ultimately it is anticipated that the general public will benefit in the long term from the proposed research, enhancing quality of life, health and well-being, a strategic objective of the BBSRC. Opportunistic fungal pathogens are a major cause of life-threatening infections in individuals with a compromised immune system, a susceptible population that is continuing to increase and is compounded by resistance to traditional antifungal therapies. This research will promote TOK1 proteins as potential future anti-fungal targets, increasing the profile of academic research into ion channel proteins and promoting collaboration between academia, the NHS, industry and other private sector businesses to investigate new avenues in drug discovery and their potential biological impact using animal models of fungal infection and patient samples. Thus the results of this project describing biophysical, regulatory and physiological properties of TOK1 channels will provide a suitable platform for developing potential therapeutic strategies to reduce the lethal consequences of pathogenic fungal infections across humans, animals and plants.