Thermo- and chemosensitive properties of Transient Receptor Potential Ankyrin 1 ion channels

Abstract: Popular Abstract in English All living things have the ability to detect and respond to changes in the surrounding environment and most of us take sensations caused by changes in light, sound, taste, temperature, and touch for granted. However, the process of detection and transmission of the signal generated to the brain is quite complex and some aspects not known. This thesis explores the sensors that detect the various changes and initiate this chain of events. In particular, one type of sensor named Transient Receptor Potential Ankyrin 1 (TRPA1) is studied. This sensor can detect a variety of unrelated dangerous conditions, such as toxic chemicals from nature or industry, chemicals produced in damaged tissue, harmful temperatures and mechanical stimuli, which lead to perception of pain and avoidance behaviour. Among the chemicals that activate TRPA1 are the plant derived spicy compounds in mustard, wasabi, cinnamon, garlic, mint, pepper, and oregano that many of us appreciate if the amount is right. Hence, TRPA1 is also called the wasabi receptor. This receptor is also found in chicken, lizards, snakes, insects, and worms but not in plants, and it helps the animals in finding appropriate temperatures. In insects and the vampire bat, the temperature sensitivity of the wasabi receptor helps in identification of a warm-blooded host or prey for feeding. However, the function as a temperature sensor in mammals is controversial ever since it was first proposed as a sensor of painful cold. In man, TRPA1 has been involved in several pathological conditions like pain, itch, and respiratory diseases and more, there by this receptor has been potential target for the novel drugs. Fortunately the principle of these sensors is simple, they work as a switch that when activated allows a current to pass the membrane of a nerve cell, generating an electrical signal which eventually transmits to the brain. In more detail, the switch is a protein based molecular machine that forms a gated channel in the membrane and only allow ions to pass when activated by a stimuli. The molecular mechanisms that allow temperature and all the diverse compounds to regulate the gate of the channel are not known. Only a small part of the wasabi receptor sits in the membrane, the bulk of the protein protrudes from the membrane into the cytosol. This protrusion is mainly folded by the first half of the protein that is named the N-terminal Ankyrin Repeat Domain (ARD). The function of the first half of the protein is not clear but changes here have suggested an involvement in regulation of the gating of the channel both by temperature and by binding of chemicals. The general aim of this thesis was to understand the functional role of TRPA1 as a sensor of temperature and chemicals. Genes from man and the malaria mosquito coding for TRPA1 and truncated versions lacking the first half, were used to transform a certain yeast, Pichia pastoris, into a protein production factory to achieve large amounts of these receptors. To examine the functionality of these extracted and purified proteins, a very sensitive technique was used measure currents passing through a single ion channel after activation by various means. In this purified and well-defined system, the human wasabi receptor responded to cold temperatures, even when lacking the first half of the protein. These results indicate that temperature sensation is intrinsic response of the channel and not dependent on intracellular molecules. Furthermore, the long N-terminal repeat structure is not needed for the activation by cold temperatures. Surprisingly, the human TRPA1 not only responded to cold but also to heat. We observed that the same isolated single ion channel can respond to both heat and cold and that these responses are modified by activators and the redox state of the channel. Similarly, we show that purified malaria mosquito TRPA1 is activated by heat and that this property is inherent to the channel and not dependent on the first half of the protein. Another technique, mass spectrometry was performed to identify binding sites of one key chemical activator and the results clearly show binding sites outside the N-terminal half of the human TRPA1. In conclusion for the first time we show that thermo- and chemosensitivity of the human TRPA1 is an inherent property of the receptor, irrespective of the N-terminal ARD. It is my hope that this thesis provides a better understanding of the function of TRPA1 that can be used for the development of novel treatments for human pain as well as control of insects transmitting diseases such as malaria.