The role of CaBP4 in human epilepsy

Epilepsy is a relatively common (~1 in 100) neurological disorder affecting all ages. It is characterised by sudden unusual bursts of excessive or synchronous neuronal activity that can lead to characteristic seizures. A significant percentage of epilepsy sufferers have a refractory form of the disease and do not respond to medication. Unfortunately, most of these individuals are also unsuitable for surgery and therefore their symptoms can be extremely difficult to treat. There is a pressing need to identify new molecular pathways involved in epilepsy which could reveal new therapeutic targets for the disease.
Calcium controls or influences almost all aspects of mammalian cell behaviour and is essential for normal neurotransmission. Calcium signals in the mammalian central nervous system (CNS) are detected by families of specific calcium sensing proteins [1]. These proteins then interact with downstream effectors to drive processes as diverse as neurotransmitter release, alterations in neuronal gene expression, changes in neuronal excitability and regulation of neuronal architecture and connectivity. As such, they are involved in high-level functions including learning, problem solving and memory acquisition. Dysregulated CNS calcium signalling has been linked to epilepsy in humans and rodents [2]. A recent genotyping study of a family having a rare inherited form of the disease identified a single point mutation in the calcium sensor, Calcium Binding Protein 4 (CaBP4G155D) as a possible underlying cause [3].
Although CaBP4 has important functions in the regulation of voltage gated calcium channels essential for neurotransmission in the mammalian auditory system, no CNS localised functions for CaBP4 have been reported. The G155D mutation suggests that CaBP4 may have a yet undiscovered CNS specific function that is linked to epilepsy.
This project is designed to investigate this possibility by examining the structure and function of the protein to explain the epilepsy phenotype. The student will employ a diverse range of experimental techniques, routine in our laboratory [4], that will provide excellent training in modern protein biochemistry and cell biological approaches. They will determine the structure of CaBP4 wild-type (CaBP4WT) and CaBP4G155D through protein crystallisation (collaboration with Dr Andrew Lovering, University of Birmingham, UK). The effect of the mutation on the chemical stability, calcium and target peptide binding properties of CaBP4 will be examined using a combination of biochemical and biophysical approaches including: Protease sensitivity, intrinsic fluorescence, circular dichroism spectroscopy and isothermal titration calorimetry. The project will then address the functional differences imparted by the G155D mutation. HEK cells expressing CaBP4WT or CaBP4G155D and transfected with known CaBP4 regulated voltage gated calcium channels will be loaded with calcium dye and depolarisation induced calcium fluxes monitored by confocal microscopy. These combined approaches will allow us to link structure to function and will provide insights into the molecular basis of a unique form of human epilepsy. This in turn could reveal novel potential therapeutic targets for future investigation.
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