Fischer Michael J M, Balasuriya Dilshan, Jeggle Pia, Goetze Tom A, McNaughton Peter A, Reeh Peter W, Edwardson J Michael
Institute of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Universitätsstrasse 17, 91052, Erlangen, Germany,
Pflugers Arch. 2014 Dec;466(12):2229-41. doi: 10.1007/s00424-014-1497-z. Epub 2014 Mar 19.
Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.
瞬时受体电位阳离子通道亚家族V成员1（TRPV1）在感知环境危害以及炎症后疼痛感觉增强方面发挥关键作用。相当一部分表达TRPV1的细胞也表达瞬时受体电位阳离子通道亚家族A成员1（TRPA1）。有证据表明TRPV1与TRPA1之间存在相互作用，这种相互作用主要依赖钙，并且有人提出这两种蛋白可能形成异源通道。在此，我们构建了亚基串联体以寻找这种相互作用的直接证据。我们发现TRPV1::TRPV1串联体和TRPV1形成了具有相似特性的通道。TRPV1::TRPA1串联体对TRPV1激动剂辣椒素、酸性pH和乙醇有反应，但对TRPA1激动剂无反应。通过原子力显微镜（AFM）成像的分离的TRPV1和TRPV1::TRPA1分子体积均与四聚体通道的形成一致。抗体以四重对称方式修饰TRPV1上的表位标签，这是同四聚体所预期的。相比之下，成对抗体主要在180°修饰TRPV1::TRPA1上的标签，表明形成了由两个面对面排列的TRPV1::TRPA1串联体组成（的二聚体）通道。TRPV1::TRPA1被蛋白激酶C（PKC）激活致敏，并且可被TRPV1拮抗剂抑制。TRPV1::TRPA1被热激活，并且显示出与TRPV1相似的阈值和温度系数。然而，由TRPV1::TRPA1形成的通道仅具有两个辣椒素结合位点，并且与TRPV1::TRPV1或TRPV1相比，显示出的总电流更小，辣椒素诱导的电压依赖性门控变化也更小。我们得出结论，TRPA1的存在对TRPV1发挥功能抑制作用。
