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

Abstract

The ability to sense and accommodate to an ever-changing environment is crucial for the survival of living organisms. Transient Receptor Potential (TRP) ion channels comprise a large superfamily of cation conducting membrane proteins that function as molecular sensors in diverse sensory processes including perception of light, taste, smell, sound, touch and temperature. The TRP Ankyrin 1 (TRPA1) ion channel is a unique member of the mammalian TRP superfamily, containing a large N-terminal ankyrin repeat domain (ARD) which constitutes half of the entire protein. TRPA1 responds to a variety of unrelated noxious stimuli such as chemicals, temperature and mechanical stress. It seems convincing that TRPA1 acts as a noxious chemical sensor and also plays a role in the detection of warm temperatures in non-mammalian species. The role of mammalian TRPA1 as a cold sensor is, however, controversial. The mammalian TRPA1 has been implicated in acute and inflammatory pain conditions and has been proposed as an important target for analgesics. If TRPA1 ion channels are intrinsically chemo-, thermo- and mechanosensitive proteins, regardless of species, remains to be shown.



The overall of aim of this thesis was to investigate possible inherent thermo- and chemosensitive properties of human TRPA1 (hTRPA1) and the malaria mosquito Anopheles gambiae TRPA1 (AgTRPA1), and the role of the N-terminal ARD, containing suggested key temperature elements as well as cysteines targeted by thiol reactive oxidants and electrophiles known to activate TRPA1. Difficulties to express and purify large amounts of proteins have hampered structural and functional studies of TRPs but were here overcome by heterologous expression in the yeast Pichia pastoris. Single-channel electrophysiological recordings of purified hTRPA1 reconstituted into artificial lipid bilayers indicated that cold- and chemosensitivity are inherent channel properties recognized by structures outside the N-terminal ARD. Surprisingly, hTRPA1 is also intrinsically sensitive to warm temperatures, and thus displays U-shaped thermosensitivity. Notably, redox state and ligands showed modulatory effects on the mammalian TRPA1 thermosensitivity. The purified AgTRPA1 was activated by heat and the electrophile allyl isothiocyanate (a major pungent ingredient in wasabi and mustard) independently of the N-terminal ARD. The TRPA1 proteins displayed fluorescence quenching upon exposure to temperature and ligands, suggesting that conformational changes occur in a membrane-independent manner and thus that TRPA1 is an intrinsic chemo- and thermosensitive protein. Mass spectrometry was used to map hTRPA1 binding sites of the frequently used electrophilic TRPA1 activator N-methyl maleimide. Our results indicate that thermal and chemical sensitivities of mammalian and non-mammalian TRPA1 are intrinsic channel properties and that the N-terminal ARD is not key but plays a modulatory role in TRPA1 chemo- and thermosensation. Our findings provide a better understanding of the function of hTRPA1, which can help to develop novel treatments for pain and illnesses/symptoms caused by sensory hypersensitivity.

Abstract (Swedish)

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.

• author: Moparthi, Lavanya
• supervisor: Urban Johanson; Peter Zygmunt
• opponent: Dr. Viana de la Iglesia, Félix, Universidad Miguel Hernández-CSIC, Instituto de Neurociencias, Alicante, Spain
• organization: Biochemistry and Structural Biology
• publishing date: 2016
• type: Thesis
• publication status: published
• subject: Biological Sciences
• keywords: TRP ion channels, TRPA1, ankyrin repeat domain, thermosensation, chemosensation, cold sensor, electrophiles, non-electrophiles, planar lipid bilayers, patch-clamp, fluorescence spectroscopy, mass spectrometry
• pages: 196 pages
• defense location: Lecture hall C at the Centre of Chemistry and Chemical Engineering, Naturvetarvägen 14, Lund
• defense date: 2016-04-08 09:30:00
• ISBN: 978-91-7422-432-0
• language: English
• LU publication?: yes
• id: f56938f7-f3e0-4302-b9ef-dbd68828a859 (old id 8837704)
• date added to LUP: 2016-04-04 10:07:35
• date last changed: 2020-04-22 15:14:42