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Functional- and structural characterization of Calmodulin-dependent TRPV channel inactivation

Principal Supervisor: Prof. Geerten W. Vuister, Department of Molecular and Cell Biology

Co-supervisor: Dr. John Mitcheson

PhD project title: Functional- and structural characterization of Calmodulin-dependent TRPV channel inactivation

University of Registration: University of Leicester

Project outline:

The Vanilloid subfamily of Transient Receptor Potential (TRPV) ion channels have diverse physiological roles including nociception, synaptic plasticity and calcium reabsorbtion. Mutations of TRPV genes are associated with neurodegenerative disorders, bone growth abnormalities, chronic pain and cancer. The ubiquitous calcium sensor protein calmodulin (CaM) is a proven crucial regulator of TRPV1,-4,-5,-6 and inactivates the channels through interactions with their intracellular C-terminal tails (C-tails). This CaM inactivation process is a vital Ca2+-dependent feedback mechanism required for tight regulation of Ca2+ entry in the cell. Prof. Vuister’s group has made substantial new progress and gained structural insights on the CaM-mediated mechanism of inactivation of TRPV6 that suggests a novel ‘two-tail’ mechanism that involves a crucial role of the CaM N-lobe differentially interacting with two individual chains of the C-terminus of the channel.

The project aim: to establish the validity of the two-tail model of CaM dependent inactivation of TRPV6 and to determine if the same mechanism extends across other members of the TRPV family. The project will use a combination of functional studies using electrophysiology, calcium imaging and mutagenesis techniques that are guided by insights from structure based studies.

Approach: NMR studies have already established the boundaries of the relevant regions of the C-termini in TRPV4, -5 and -6 channels. The PhD project will use biophysical techniques to identify crucial residues driving the formation of TRPV4/5/6:CaM complexes. The functional role of these residues in regulating inactivation will then be tested in the fully intact channels, under physiologically relevant conditions, using whole-cell electrophysiology approaches. In addition, mechanistic insights of how CaM regulates the inactivation process by applying CaM mutants directly to the intracellular surface of TRPV channels and using the inside-out conformation of the patch-clamp technique to measure their effect on channel gating.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Expression, modification and purification of protein constructs
  • Biophysical characterisation of protein stability and interactions
  • NMR sample preparation and hetero-nuclear multi-dimensional NMR data collection and data analysis
  • Electrophysiology sample preparation and data collection
  • Bioinformatics approaches
  • Protein modelling and structure calculations

Contact: Professor Geerten Vuister, University of Leicester