Transgenic Chlamydomonas Expressing Human Transient Receptor Potential Ankyrin 1 (TRPA1) Channels to Assess the Effect of Agonists and Antagonists.

Kenjiro Yoshimura,Megumi Yoshida,Ryodai Yamamiya,Yuto Shimizu

doi:10.3389/fphar.2020.578955

Abstract

Transient receptor potential ankyrin 1 (TRPA1) channel is an ion channel whose gating is controlled by agonists, such as allyl isothiocyanate (AITC), and temperature. Since TRPA1 is associated with various disease symptoms and chemotherapeutic side effects, it is a frequent target of drug development. To facilitate the screening of TRPA1 agonists and antagonists, this study aimed to develop a simple bioassay for TRPA1 activity. To this end, transgenic Chlamydomonas reinhardtii expressing human TRPA1 was constructed. The transformants exhibited positive phototaxis at high temperatures (≥20°C) but negative phototaxis at low temperatures (≤15°C); wild-type cells showed positive phototaxis at all temperatures examined. In the transgenic cells, negative phototaxis was inhibited by TRPA1 antagonists, such as HC030031, A-967079, and AP18, at low temperatures. Negative phototaxis was induced by TRPA1 agonists, such as icilin and AITC, at high temperatures. The effects of these agonists were blocked by TRPA1 antagonists. In wild-type cells, none of these substances had any effects on phototaxis. These results indicate that the action of TRPA1 agonists and antagonists can be readily assessed using the behavior of C. reinhardtii expressing human TRPA1 as an assessment tool.

Highlights

Living organisms have developed sensors for detecting environmental conditions and the status of homeostasis
Antibodies against hTRPA1 detected a band at approximately 100 kDa in the detergent-soluble fraction from TA1 cells but not from wild-type cells (Figure 1C)
This study showed that C. reinhardtii cells expressing hTRPA1 exhibit negative phototaxis at temperatures of 15°C or lower (Figure 7)

Summary

Introduction

Living organisms have developed sensors for detecting environmental conditions and the status of homeostasis. Transient receptor potential (TRP) channels compose a large superfamily of ion channels that respond to chemical, thermal, and mechanical stimuli (Clapham, 2003). Mammalian TRP channels are classified into six families: TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV, which can be further classified into subfamilies, such as TRPV1 and TRPV2. TRPA1 responds to both temperature and various agonists, including allyl isothiocyanate (AITC, a component of wasabi) and bradykinin TRP channels help regulate the generation of action potentials in nerve cells and control various functions in nonexcitable cells. TRPP1 and TRPP2 (PKD1 and PKD2) are involved in detecting kidney urine flow (Nauli et al, 2003)

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