Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Yuusuke Maruyama,Toshihiko Ogura,Kazuhiro Mio,Shigeki Kiyonaka,Kenta Kato,Yasuo Mori,Chikara Sato

doi:10.1074/jbc.m705694200

Abstract

Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose (ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.

Highlights

Transient receptor potential (TRP)2 channels, first described in the Drosophila phototransduction system [1], comprise a large family of cation channels [2,3,4,5]
Because site-directed mutagenesis demonstrates that gating persists in double mutation, which is critical for ADPR pyrophosphatase (ADPRase) activity of mitochondrial NUDT9, catalytic activity does not seem necessary for ADPRdependent gating [17]
The H2O2-induced intracellular Ca2ϩ rise was similar to that of the wild type (Fig. 1A), and the activation by adenosine diphosphate ribose (ADPR) was demonstrated to be similar by whole cell patch clamp experiment (Fig. 1, B–D): FLAG-tagged TRPM2 displays a linear voltage-current relationship that is characteristic of the TRPM2 channel (Fig. 1C)

Summary

Introduction

Transient receptor potential (TRP)2 channels, first described in the Drosophila phototransduction system [1], comprise a large family of cation channels [2,3,4,5]. We have reconstructed the three-dimensional structure of TRPM2 from negatively stained EM images. Formation of the Channel-Antibody Complex—Anti-FLAG antibodies were added to the purified TRPM2 protein in antibody binding buffer (Tris-buffered saline containing 1 mM DDM, 350 mM MgCl2, 15% glycerol, and 0.02% sodium azide), and incubated at 4 °C for 1 h.

Results

Conclusion