Yr Athro Kenneth Wann

Ion channels and disease

Non-excitable cells, like excitable cells, possess plasma membrane ion channels. Evidence has slowly accumulated that these channels have surprisingly key roles to play in the life and death decisions a cell may make. Our work attempts to define the role of plasma membrane ion channels in cellular processes such as proliferation, apoptosis and differentiation in robust cell models. To this end we use a combination of single cell patch-clamp electrophysiology, reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR (Q-PCR) and functional assays of cell growth and viability. Proliferation assays (e.g. using commercially available MTS kits, and direct cell counting), apoptosis assays (e.g. using Annexin-V and propidium iodide labelling, and TUNEL labelling) and functional assays (e.g. mineralisation of osteoblast-like cells as assessed by Alizarin red staining) are used to delineate effects. Much of the work uses established cell lines, including osteosarcoma-derived cells such as osteoblast-like MG63 and SaOS2 cells, breast cancer (MCF-7), prostate cancer (LNCaP) cells, human brain astrocytoma (MOG-G-UVW), neuroblastoma (SH-SY5Y) and pluripotent embryonal carcinoma (NTERA-2).

In neuronal cell lines, we have focused on plasma membrane K⁺ channels and channels in subcellular organelles such as the endoplasmic reticulum (e.g. ryanodine receptor) and mitochondria. Understanding the links between modified channel function and activation of the cell death pathway remains the prime focus. Methods such as patch-clamp electrophysiology, planar bilayer work, RT-PCR and apoptotic assays are utilised. Cell lines (e.g. SH-SY5Y, MOG-G-UVW, NTERA-2) and primary cells are used.

The results of this work will hopefully both add to our understanding of the role of ion channels in fundamental cellular processes, and have relevance for diseases such as the degenerative illnesses (e.g. osteoporosis, neurodegeneration) and the cancers. The long term goal is to identify possible ion channel targets for therapeutic intervention in various diseases, including debilitating central nervous system disorders such as Alzheimer's disease.

Ionic signalling in Bacteria

Ca²⁺  is king of the messengers in eukaryotic cells and there is indirect evidence that Ca²⁺ plays a key role in shaping physiological processes in prokaryotes. These include tumbling and chemotaxis, the cell cycle, spore formation, virulence and pathogenesis, competence, and the regulation of protein synthesis. Clear definitive evidence has been lacking because of a lack of correlation of cytosolic free Ca²⁺ with bacterial growth and gene expression, and the lack of good candidates for a Ca²⁺ channel. The aim of this work is to show definitively that cytosolic free Ca²⁺ is a signal in bacteria controlling cell shape, growth and gene expression. One mechanism by which we are trying to do this is by bringing together electrophysiological and bioluminescent techniques in order to identify novel mechanisms for calcium influx/efflux. Current work involves the extensively studied bacterial mechanosensitive channels (MscS and MscL) and uses Ca²⁺ as signal for mechanosensitive channel opening to screen for lead compounds that could be employed as antimicrobials. 

Bacterial metabolic 'toxins' in diabetes

Bacteria in the gut, and in other hypoxic environments, produce hydrogen and methane, and small organic molecules when they metabolise carbohydrates such as lactose. The aim of this new work is to test the hypothesis that small organic toxins produced by anaerobic bacteria in the gut cause/contribute to diabetes and other major diseases. The strategy is to use a culture of pancreatic β -cells, already transformed with firefly luciferase and transfected with the photoprotein aequorin, enabling changes in cytosolic free Ca²⁺ and K⁺ channel conductance to be correlated with insulin secretion and cell growth.

Bioluminescent indicators

The measurement of the free Ca²⁺ inside cells has been the key to our understanding many aspects of intracellular signalling which underpin cellular processes as diverse as fertilization, secretion, contraction and movement, excitability and growth. Bioluminescence is one of the methods routinely used by cell biologists to measure free Ca²⁺ inside cells and in particular aequorin a photoprotein which emits light as a response to changes in physiological Ca²⁺. The goals of this project have been to devise a construct that would detect intracellular Ca²⁺ and also serve as a probe of ion channel function. Using genetic engineering techniques, we have  constructed a fusion protein between β2 (a modulatory subunit of the MaxiK channel) and aequorin. This construct does in principle monitor levels of [Ca²⁺]_i close to  ion channel microdomains and, provide information about the way the channel assembles and, if fully functional, will also indicate how the assembled channel responds to changes in local [Ca²⁺]_i. This recombinant protein will enable us to examine channel modulation and expression from a new perspective.

Origin of coelenterate bioluminescence

The aims of this research are first to establish which luminous marine species are present in Pembrokeshire. The goals are then to determine the biosynthetic pathway for coelenterazine in Obelia and to investigate how the kinetics of Obelia's flash are controlled by investigating calcium regulation within the photocyte. The long term objective is to develop Obelia as a model system to study new drugs which work on potassium and calcium ion channels.

Collaborators

Cardiff University

• Professor Tony Campbell (School of Pharmacy)
• Dr Bronwen Evans  (School of Medicine)
• Professor Tim Wess (School of Optometry)
• Dr Andrew Westwell (School of Pharmacy)
• Professor Gary Baxter (School of Pharmacy)

External

• Dr Michael Pasternack, Helsinki, Finland          
• Dr Katri Wegelius, Helsinki, Finland
• Dr Hugh Chapman Helsinki, Finland
• Dr Kid Tornquist Helsinki, Finland
• Professor Damian Bailey, Denver, USA
• Professor Peter Andrews, Sheffield, UK
• Professor Barry Holland, Orsay, France
• Dr Jen Hiller (Diamond Light Source, Oxfordshire, UK) www.diamond.ac.uk
• AssayMetrics (Cardiff, UK) www.assaymetrics.com