Welsh School of Pharmacy

Ion channels and disease

Non-excitable cells, like excitable cells, possess plasma membrane ion channels. Over the last decade evidence has accumulated that these channels have perhaps 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) and functional assays of cell growth and viability. Proliferation assays (e.g. using commercially available MTT 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. Our principal focus is on the characterisation and pharmacology of K⁺ and TRP channels. Much of the work uses established cell lines, including osteosarcoma-derived cells such as osteoblast-like MG63 and SaOS cells, breast cancer (MCF-7) and prostate cancer (LNCaP) cells. Clinical samples are available locally to enable similar work to be undertaken on primary cultures.

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. There is the added prospect of novel targets for drug therapy.

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.

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 are constructing a fusion protein between β2 (a modulatory subunit of the MaxiK channel) and aequorin. This construct will 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.

Bacterial 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 diabetes and other major diseases. The strategy is to use a culture of pancreatic b 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.

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 Medicine)

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