Ion Channel Research Group
Studying the biophysics and regulation of membrane potassium channel proteins
Ion Channel Research Group
Our interest lies in studying the biophysics and regulation of membrane potassium channel proteins and their roles in the cellular physiology of human health and disease.
To date, our research has focused on the study of potassium channels from both mammals and human pathogenic fungi, defining protein structure-function relationships, interactions with accessory proteins, pharmacology and functional regulation by environmental cues (e.g. pH, pO2). Our studies employ an integrated electrophysiological, biochemical and molecular approach to answer key physiological questions.
What are ion channels?
Ion channels act as molecular portals for the passive diffusion of ions into and out of cells and their function is absolutely essential for controlling cellular electrical activity in almost all life forms.
Ion channels can be defined simply on the basis of their structure and ion discrimination. Potassium channels for example allow the selective movement of positively charged potassium ions (K+) across the cell membrane. The movement of K+ ions can lead to a change in the potential difference across the cell membrane which provides a driving force for signalling pathways needed for normal cellular function.
Structurally, potassium channels are formed through the assembly of multiple protein subunits encompassing a central water-filled pore or "conduction pathway" allowing for selective K+ diffusion across the lipid membrane. The opening and closing of the conduction pathway termed "gating" is tightly regulated by a variety of stimuli such as changes in membrane potential (voltage), second messenger stimulation, phosphorylation status and ligand binding. However, unregulated K+flux can be detrimental, ultimately leading to cell death. Thus understanding the molecular mechanisms underlying gating of potassium channels and the ability to activate or inhibit K+ flux is fundamental to manipulating normal cellular excitability and function and to revealing the pathological consequences of ion channel dyshomeostasis.
Our current laboratory research
Fungal potassium ion channels and their role in fungal physiology and virulence
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 the evolutionary conserved cell biology 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 search for novel anti-fungal targets is critical.
We have recently identified and cloned a number of potassium channels from several species of human pathogenic fungi which represent the primary sources of fatal infections in the immunosuppressed population (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans). These potassium channels, called TOK1 for Two-pore Outwardly rectifying K+ channel, are unique in that they have no known structural or functional homologues in either mammals or plants, and are the only potassium-selective ion channel expressed in yeast and fungi, making them ideal candidates for future antimicrobial compounds.
Each protein subunit comprises a core topology of eight transmembrane domains (TM) containing a pair of conserved semi-hydrophobic P regions (pore domains) between TM5-TM6 and TM7-TM8 respectively (see Figure 1), and functional channels are thought to form through subunit homodimerisation. Our strategy is to understand how these channels function and how they are regulated. This is achieved primarily using electrophysiological methods including two-electrode voltage clamp (TEVC), whole-cell or isolated patch clamp techniques (see Figure 2) to monitor changes in channel activity in real time and allows us to begin to probe some important and outstanding issues; How do they open and close? Why do they outwardly rectify? What molecular mechanisms underlie their unique functional behaviour? How do they impact fungal physiology?
Mammalian ion channels in health and disease
Our second research drive is directed towards understanding the role ion channels play in human health and disease. Our previous research has focused on structure-function relationships of potassium channels, particularly voltage-gated potassium channels (KV) and their accessory subunits specifically single transmembrane accessory proteins encoded by the KCNE gene family, mutations to which have been shown to underlie critical physiological disturbances including cardiac arrhythmias. More recently we have focussed on the intricacies of potassium channel “gating” and voltage-sensing employing a combined mutagenesis and electrophysiological approach to study structure-function behaviours in mammalian potassium channels.
We are currently collaborating with Professor Arthur Butt in a study characterising a novel potassium channel in neural cell development and disease and employs an ILSH-funded PhD student, Miss Maria Papanikolaou. Oligodendrocytes are the specialised glial cells in the CNS that make myelin, which insulates nerve fibres and is essential for rapid conduction of nerve impulses. Oligodendrocytes and myelin sheaths are primary targets of tissue destruction in numerous diseases, including multiple sclerosis (MS) and cerebral palsy. A full understanding of the mechanisms of myelination and demyelination is critical for the development of new therapeutic interventions in these diseases. Potassium channels are key proteins in both glia and neurons.
For example, potassium channels act as critical regulators of cell division and differentiation, and their loss causes membrane depolarization, increased seizure susceptibility, and the loss of myelin. We have identified a potassium channel that is expressed specifically during development of oligodendrocytes and neurons. The functions of this channel in neural cell development are completely unknown. Our project aims to investigate the functional expression of this potassium channel and how it regulates oligodendrocyte and neuronal development using a combined molecular, immunohistochemical and electrophysiological approach.
We are also collaborating with Professor Janis Shute in a study investigating the function of the CFTR chloride ion channel in human lung endothelial cells and its role in inflammation.