Kim Donghee, Cavanaugh Eric J
Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois 60064, USA.
J Neurosci. 2007 Jun 13;27(24):6500-9. doi: 10.1523/JNEUROSCI.0623-07.2007.
Pungent chemicals such as allyl isothiocyanate (AITC), cinnamaldehyde, and allicin, produce nociceptive sensation by directly activating transient receptor potential A1 (TRPA1) expressed in sensory afferent neurons. In this study, we found that pungent chemicals added to the pipette or bath solution easily activated TRPA1 in cell-attached patches but failed to do so in inside-out or outside-out patches. Thus, a soluble cytosolic factor was required to activate TRPA1. N-Ethylmaleimide, (2-aminoethyl)-methane thiosulfonate, 2-aminoethoxydiphneyl borate, and trinitrophenol, compounds that are known to activate TRPA1, also failed to activate it in inside-out patches. To identify a factor that supports activation of TRPA1 by pungent chemicals, we screened approximately 30 intracellular molecules known to modulate ion channels. Among them, pyrophosphate (PPi) and polytriphosphate (PPPi) were found to support activation of TRPA1 by pungent chemicals. Structure-function studies showed that inorganic polyphosphates (polyP(n), where n = number of phosphates) with at least four phosphate groups were highly effective (polyP4 approximately = polyP65 approximately = polyP45 approximately = polyP25 > PPPi > PPi), with K(1/2) values ranging from 0.2 to 2.8 mM. Inositol-trisphosphate and inositol-hexaphosphate also partially supported activation of TRPA1 by AITC. ATP, GTP, and phosphatidylinositol-4,5-bisphosphate that have three phosphate groups did not support TRPA1 activation. TRPA1 recorded from cell bodies of trigeminal ganglion neurons showed similar behavior with respect to sensitivity to pungent chemicals; no activation was observed in inside-out patches unless a polyphosphate was present. These results show that TRPA1 requires an intracellular factor to adopt a functional conformation that is sensitive to pungent chemicals and suggest that polyphosphates may partly act as such a factor.
辛辣化学物质，如异硫氰酸烯丙酯（AITC）、肉桂醛和大蒜素，通过直接激活感觉传入神经元中表达的瞬时受体电位A1（TRPA1）产生伤害性感觉。在本研究中，我们发现添加到移液管或浴液中的辛辣化学物质很容易激活细胞贴附式膜片中的TRPA1，但在内外翻式膜片中则无法激活。因此，需要一种可溶性胞质因子来激活TRPA1。N-乙基马来酰亚胺、（2-氨基乙基）甲硫代磺酸盐、2-氨基乙氧基二苯基硼酸盐和三硝基苯酚，这些已知可激活TRPA1的化合物，在内外翻式膜片中也无法激活它。为了鉴定一种支持辛辣化学物质激活TRPA1的因子，我们筛选了大约30种已知可调节离子通道的细胞内分子。其中，焦磷酸（PPi）和聚三磷酸（PPPi）被发现支持辛辣化学物质激活TRPA1。结构-功能研究表明，具有至少四个磷酸基团的无机多磷酸盐（polyP(n)，其中n = 磷酸基团的数量）非常有效（polyP4≈polyP65≈polyP45≈polyP25 > PPPi > PPi），K(1/2)值范围为0.2至2.8 mM。肌醇三磷酸和肌醇六磷酸也部分支持AITC激活TRPA1。具有三个磷酸基团的ATP、GTP和磷脂酰肌醇-4,5-二磷酸不支持TRPA1激活。从三叉神经节神经元细胞体记录的TRPA1在对辛辣化学物质的敏感性方面表现出类似的行为；除非存在多磷酸盐，否则在内外翻式膜片中未观察到激活。这些结果表明，TRPA1需要一种细胞内因子来采用对辛辣化学物质敏感的功能构象，并表明多磷酸盐可能部分充当这样一种因子。
