A novel alternative splicing variant of mouse TRPA1 regulates its channel activity under inflammatory and neuropathic pain conditions 新規マウスTRPA1スプライシングバリアントは炎症性および神経障害性疼痛モデルにおいてTRPA1チャネル活性を制御する
A novel alternative splicing variant of mouse TRPA1 regulates its channel activity under inflammatory and neuropathic pain conditions
This Ph.D. thesis investigated the role of a novel mouse TRPA1 alternative splicing variant. The thesis is mainly divided into 9 chapters: abbreviations, introduction, materials and methods, results, discussion figure legends, references, tables and figures, and acknowledgements. Transient receptor potential (TRP) channels are non-selective cation channels that were originally identified in mutant Drosophila. The TRP channel superfamily is divided into seven subfamilies: TRPV (Vanilloid), TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipin), TRPN (NompC), TRPP (Polycystic) and TRPA (Ankyrin) with 27 TRP channels found in humans. The discovery and characterization of the TRP channel superfamily offers a superior tool for understanding the molecular mechanisms of sensations such as taste, pain, and temperature in the peripheral and central nervous systems. So far many studies have used different experimental methods and techniques to demonstrate that TRP proteins are indeed involved in many different forms of sensations. TRPA1 is the only member of the mammalian ankyrin subfamily of TRP channels. One intriguing property of the TRPA1 channel is the presence of up to 17 ankyrin repeat domains that could act as multiple binding sites for molecules that modulate channel functions. TRPA1 channel is expressed in many tissues and organs, including sensory neurons, astrocytes, the small intestine and colon. TRPA1 channel is predominately expressed in small-diameter neurons and functions as a nociceptive receptor in the peripheral nervous system in sensory neurons, such as those in the dorsal root ganglion (DRG) and trigeminal ganglion (TG). TRPA1 channel is believed to be involved in many forms of acute and chronic hyperalgesia, with some reports suggesting that nerve growth factor (NGF) regulates chronic inflammatory hyperalgesia by controlling TRPA1 gene expression in sensory neurons. Alternative splicing of pre-mRNA was discovered more than 30 years ago, when it was found that the same gene encoded two proteins, one was membrane-bound and another was secreted. In eukaryotes, alternative splicing is considered to be a complex cellular mechanism by which a small number of genes could encode much larger number of proteins. Indeed, more than 95% of human genes are estimated to undergo alternative splicing. The importance of alternative splicing as a regulatory process has been highlighted in many pre-mRNA splicing of regulatory and signaling proteins. Taking into account other post-translational modifications, the number of functional proteins encoded by the genome is surprisingly enormous. Thus, alternative splicing is one of the major sources to increase protein diversity. Recently, two individual studies identified novel Drosophila TRPA1 alternative splicing variants that were expressed in a tissue-specific manner and displayed different chemical and thermal sensitivities. To date there is no report of mammalian TRPA1 alternative splicing, even though there are many instances of alternative splicing variants of other mammalian TRP channels. For instance, an alternative splicing variant of TRPV1, a TRP channel expressed in sensory neurons and involved in nociception, is reported to be a dominant negative isoform and inhibits TRPV1 channel activity by forming a heteromer. However, whether alternative splicing variants of mammalian TRPA1 exist and their physiological and pathological significance in diseases remain to be elucidated. In this thesis, a novel mouse TRPA1 splicing variant has been found and cloned from mouse DRG neurons. This splicing variant, TRPA1b skips 30 amino acids compared with its full-length isoform, TRPA1a. Multiple experiment methods are applied to examine the physiological and pathological importance of TRPA1a and TRPA1b from mRNA level to protein level and finally to animal model level. Both isoforms, TRPA1a and TRPA1b are confirmed to be expressed in mouse dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons by RT-PCR analysis. When expressed in HEK293T cells, both isoforms can be translocate to the plasma membrane while co-immunoprecipitation showed that TRPA1a and TRPA1b physically interact with each other. Although TRPA1b do not respond to TRPA1 agonists, TRPA1a and TRPA1b co-expression significantly increases TRPA1a plasma membrane expression and current density in response to AITC, 2-APB, carvacrol and thymol without affecting its single-channel properties. Exogenous TRPA1b over-expression in WT DRG neurons also increases AITC responses, and expression of TRPA1a with TRPA1b produces larger ATIC responses compared with expression of TRPA1a alone in TRPA1KO DRG neurons. Moreover, expression levels of TRPA1a and TRPA1b mRNA changes dynamically in complete Freund’s adjuvant (CFA)-induced inflammatory and a partial sciatic nerve ligation (PSL)-induced neuropathic pain models. In contrast to TRPA1a, whose mRNA expression level changed in the early stages of CFA-induced inflammatory and PSL-induced neuropathic pain conditions, the TRPA1b expression level gradually increases throughout the two-week disease condition period. This up-regulated TRPA1b expression level could maintain the membrane expression of TRPA1a even with the rapidly changed TRPA1a mRNA expression. These results suggest that TRPA1 may be regulated through alternative splicing under inflammatory and neuropathic pain conditions. This thesis could explain how TRPA1 is involved in inflammatory and neuropathic pain and provide a possible mechanism to treat these diseases.