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

Sammanfattning: 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.