The ability to detect and respond to harmful conditions in the environment is a skill essential for survival. As a result, many organisms have specialized systems for accurately detecting variables such as temperature or the presence of noxious chemicals. TRPA1, an ion channel in pain-sensing neurons, is involved in those processes. TRPA1 is activated by a wide range of electrophiles as well as by temperature. TRPA1 is a particularly interesting target for study due to its associations with pain and inflammation. The importance of the N-terminal cytoplasmic region of TRPA1 for temperature and chemical sensing is well established, but relatively little is known about the molecular mechanisms. This project focuses on the temperature-sensing mechanism of insect TRPA1. In insects, two TRPA1 isoforms differ only at a small region at the extreme N-termini, but the channels have very different temperature sensitivities12-13. The overall goal for this project is t understand how the N-termini of those insect TRPA1 isoforms interact with the ankyrin repeat domain to modulate temperature sensitivity. By combining biochemical, biophysical, and structural analyses of TRPA1 N-terminal fragments, I aim to elucidate the molecular mechanism of temperature sensing.
The first aim of the project is investigate potential interactions between the isoform specific-regions of insect TRPA1 and the ankyrin repeat domain. This requires pure, soluble fragments of the N-terminal region of TRPA1, so l will put forth a large effort to produce the fragments necessary to search for interactions. Isothermal calorimetry and mutagenesis will be used to measure binding properties and identify regions important for interaction. In addition, pairs of corresponding fragments from the different insect TRPA1 isoforms will be produced for comparison in aim 2.
The second aim i s to identify biophysical and biochemical differences of the fragments that are responsible for the different temperature sensitivities of insect TRPA1 isoforms. Circular dichroism and limited proteolysis will be used to compare the secondary structure composition and thermal stability of construct pairs. The third and final aim is to solve the structures of TRPA1 N-terminal fragments through X-ray crystallography. By combining the information gained from aims 2 and 3, we aim to determine the role of the N-termini in the molecular mechanism of temperature sensing. Structures of the N-terminal domains of TRPA1 will provide a useful framework for further investigation of TRPA1 temperature sensing through biochemistry and electrophysiology experiments. Structural information will also be useful for rational drug design. TRPA1 antagonists may have analgesic and anti-inflammatory effects. These types of compounds may therefore be useful for treating neuropathic pain or chronic inflammation disorders.
Detecting and responding to conditions in the environment is essential for survival. We have specialized systems to accurately detect environmental conditions. One important protein is Transient Receptor Potential Ankyrin 1 (TRPA1), found in pain-sensing neurons that detect both temperature and harmful chemicals. The goal of this research is to determine the three-dimensional structure of the temperature sensor of TRPA1, to understand how TRPA1 is able to respond to temperature. The information could be used in the design of drugs to suppress abnormal TRPA1 function for pain-relief or anti-inflammatory effects.
|Brewster, Monique S J; Gaudet, Rachelle (2015) How the TRPA1 receptor transmits painful stimuli: Inner workings revealed by electron cryomicroscopy. Bioessays 37:1184-92|