Cooling Down TRP Channel: Cold and Chemo-Sensitivity of the Human TRPA1

Abstract

Transient receptor potential (TRP) channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be activated and regulated by a variety of stimuli including temperature, mechano-stimulation and different kinds of molecules, which mediate the sensation of taste, as well as divalent cations and pH. Dysfunction of TRP channel can cause various pathological conditions, including an inherited pain syndrome, multiple kidney diseases and skeletal disorders. Hence, TRP channel become potential targets for the treatment of such disorders.Patch clamp electrophysiology remains the gold standard for studying ion channels. We have employed a planar patch clamp technology to study the purified human TRPA1 channel (hTRPA1). Solvent-free planar lipid bilayers can be formed in an automated fashion by positioning and subsequent bursting of giant unilamellar lipid vesicles containing ion channels, here hTRPA1, on the Port-a-Patch micron-sized apertures borosilicate glass substrate.In this study, the human TRPA1 was purified and reconstituted into lipid bilayers and single channel currents were recorded to understand the thermo- and chemosensory properties of the channel together with the role of the N-terminal ankyrin repeat domain (ARD). We report that hTRPA1with and without its N-terminal ARD is intrinsically cold-sensitive, and thus cold sensing properties of hTRPA1 reside outside the N-terminal ARD.Furthermore, hTRPA1 is activated by a range of environmental irritants, pungent compounds found in foods such as garlic, mustard and cinnamon, as well as metabolites produced during oxidative stress. Data will be presented showing activation and inhibition of the hTRPA1 channel reconstituted in bilayers as well as hTRPA1 expressed in HEK cells (Millipore), using the SyncroPatch 384PE planar patch clamp platform, by a variety of agonists and antagonists.