October 27
@
4:00 PM
–
5:00 PM
Susan Daniel
Distinguished Professor
Department of Chemical and Biomolecular Engineering
Cornell University
Toward objective pain monitoring and treatment: Cell-free biosensors based on nociceptive ion channels
Effectively monitoring and managing pain remain major challenges in the clinic and constitute active areas of research today. Given the subjective nature of the pain response and the ongoing opioid crisis, there is a critical need to both identify new non-opioid drug targets for chronic pain management and provide more objective ways to measure pain. Using pain receptors themselves to monitor biomarkers of pain and screen potential channel blockers remains challenging, with current drug screening protocols lacking quantitative and direct measures of receptor function and dysfunction. To improve the treatment and measurement of pain, we describe here the development of prototypical pain biosensors. We utilize in vitro transcription and translation cell-free methods to rapidly express and integrate two distinct and pharmacologically relevant types of pain receptors into membrane-based sensing platforms: the Transient Receptor Potential Vanilloid 1 (TRPV1) channel and pro-algesic P2X purinergic receptors (P2X2), avoiding all the downsides of cell-based expression, isolation, and reconstitution into membranes and sensor platforms. Because these receptors are ligand-gated ion channels activated by different stimuli, we then assess both channels’ responsiveness using electrical or optical methods to monitor changes in ionic flux across the membranes.
In our first system, we show that devices containing P2X2 receptors show an increase in ionic flux following ATP administration, indicating ligand-gated channel opening. This ion flux is abolished in the presence of a known P2X2 inhibitor, but not the P2X7 inhibitor, demonstrating the screening potential of this platform to identify new subtype specific therapeutics for pain management. Using the same in vitro expression system, we then cell-free synthesized and integrated the human nociceptor TRPV1 channels into membrane vesicles containing a fluorescence-based ion flux reporter. In these vesicles TRPV1 channel opening could be stimulated by two distinct pain-causing stimuli – heat and capsaicin, showing that in our minimal system, this receptor subtype is responsive to distinct channel activators. The modular nature of these two platforms enables isolation and assessment of individual pain receptors in a way that is not possible using native cells in a convenient, tailorable device compatible with rapid, high throughput screening. Both electrical and optical platforms, which contain cell free synthesized pain receptors, provide new ways to study receptor function and advance our detection and treatment of pain.