In situ studies of localisation, structure and interactions of TRPC5 ion channels

Membrane proteins are crucial to cellular function, such as recognition of – and interaction with – the extracellular environment and neighbouring cells, and controlling membrane dynamics, cellular signalling and transport of molecules and ions across membranes. Membrane protein malfunction underlies many diseases and disorders (in humans, animals and plants), and ~60% of current drug targets are membrane proteins. Determination of membrane protein structures has provided detailed insights into their function and molecular interactions. However, membrane protein structures are typically determined after purification and stabilisation in non-native environments, hindering determination of physiologically relevant states necessary for understanding mechanism-of-action. The next revolution in membrane protein structural biology will be their structure determination in a native cellular environment by tomography, and many laboratories (including ours) are rapidly investing in tomography facilities for in-situ structural biology research. A major challenge in this field is how to detect and localise the membrane protein(s)-of-interest in a crowded membrane environment, and new technologies for this are urgently needed.

We propose to develop DogCatcher-quantum dot conjugates (DogDots), which will allow us to rapidly and specifically deliver quantum dots to membrane proteins that incorporate a short peptide (DogTag) within an extracellular loop. Because of the properties of the quantum dots (fluorescent, dense), DogDots will allow precise localisation of specific membrane proteins in cells and tissues through fluorescence and electron microscopy, thereby allowing in-situ studies of membrane proteins in their native environment. As a model system, we will use TRPC5 ion channels, which have important roles in health and disease, and for which we have all relevant tools, cell lines and expertise available.

This technology has the potential for translation to any membrane protein with an extracellular loop, and we will develop DogDots as a versatile technology for structural studies of membrane proteins, thereby transforming our understanding of these important biomolecules.