LETTERS TRIC channels are essential for Ca 21 handling in intracellular stores Masayuki Yazawa 1,2 , Christopher Ferrante 3 , Jue Feng 2 , Kazuhiro Mio 4 , Toshihiko Ogura 4,5 , Miao Zhang 1,2 , Pei-Hui Lin 3 , Zui Pan 3 , Shinji Komazaki 6 , Kazuhiro Kato 2 , Miyuki Nishi 1,2 , Xiaoli Zhao 3 , Noah Weisleder 3 , Chikara Sato 4 , Jianjie Ma 3 & Hiroshi Takeshima 1,2 Cell signalling requires efficient Ca 21 mobilization from intracel- lular stores through Ca 21 release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca 21 release 1–7 . Ca 21 release channels were cloned more than 15 years ago 8,9 , whereas the molecular identity of putat- ive counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are dif- ferentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysi- ological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca 21 handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K 1 permeability, as well as altered Ca 21 ‘spark’ signalling and voltage-induced Ca 21 release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchroniza- tion with Ca 21 release from intracellular stores. In the course of screening membrane proteins participating in cellular Ca 21 handling 10,11 , we identified a protein with a calculated molecular mass of 33,300 from rabbit skeletal muscle, and named it TRIC type A (TRIC-A; also known as mitsugumin 33). Homology searches in databases revealed an additional structural homologue named TRIC-B. TRIC subtypes show fragmentary sequence identit- ies, as determined by our complementary DNA and in silico cloning from various animal species (Supplementary Fig. 1). Northern blot- ting indicated that TRIC-A is preferentially expressed in excitable tissues, including striated muscle and brain, whereas TRIC-B is pre- sent in most mammalian tissues (Fig. 1a). Western blotting of frac- tionated muscle membranes suggested that TRIC-A is distributed throughout the sarcoplasmic reticulum (SR) but not in the cell- surface membranes (Fig. 1b). In further immunochemical studies, antibodies to TRIC-A decorated the SR and nuclear membranes in skeletal muscle, and TRIC-B behaved as an endoplasmic reticulum (ER)-resident protein in the brain tissues (Supplementary Fig. 1). Therefore, TRIC subtypes are localized to membrane systems assoc- iated with intracellular Ca 21 stores. TRIC subtypes show conserved hydropathicity profiles that sug- gest multiple transmembrane segments (Fig. 1c). In limited proteo- lysis analysis using membrane vesicles (Supplementary Fig. 2), we found that the amino terminus of TRIC-A is located in the SR/ER lumen, whereas the carboxy terminus of TRIC-A is exposed to the cytoplasm. Further analysis of epitope-tagged recombinant proteins predicted three transmembrane segments in TRIC-A, and also detected the hydrophobic loop as a candidate for an ion-conducting pore between the first and second transmembrane segments. The proposed topology of TRIC subtypes (Fig. 1d) bears an overall resemblance to that of glutamate receptor channels 12 . Affinity chromatography using monoclonal antibodies allowed us to obtain pure TRIC-A protein (.95% purity) from muscle micro- somal preparations solubilized with n-dodecyl b-D-maltoside (DDM) or digitonin (Supplementary Fig. 3). Treatment of TRIC-A with several chemical crosslinkers generated products with sizes corresponding to dimeric and trimeric assemblies (Fig. 1e and Supplementary Fig. 3). When purified TRIC-A was labelled with colloidal-gold-conjugated Fab fragments, electron microscopy fre- quently detected antigen–antibody complexes carrying three immu- nogold particles that protruded at near equal angular intervals (Fig. 1f). Combined computer algorithms, which collect, classify and average negatively stained electron-microscope images to recon- struct a three-dimensional volume 13–16 , and the tilt series of the particle images, demonstrated that TRIC-A forms an elongated tri- angular pyramidal structure (Fig. 1g and Supplementary Figs 4 and 5). Thus, TRIC subtypes form a homo-trimeric structure, as in the cases of the P2X (ref. 17) and bacterial porin channels 18 . Using lipid bilayer reconstitution studies, we observed a functional cation-selective channel with TRIC-A purified from skeletal muscle (Fig. 2a, b). In a recording solution composed of 200 mM KCl (cis)/50 mM KCl (trans) (see Methods), a slope conductance of 110 6 14 pS and a reversal potential of 220 6 1.7 mV were observed, indicating the cation-selective nature of the TRIC-A channel (Fig. 2f). Although the single channel conductance properties of TRIC-A are similar to those of the SR K 1 channel reported previously 5,19 , other characteristics suggest that the TRIC-A channel might represent a separate channel moiety. First, under bi-ionic conditions of 200 mM KCl (cis)/200 mM NaCl (trans), outward current was measured at a holding potential of 0 mV, which reverses at a potential of 210 mV (Fig. 2c). This negative reversal potential corresponds to moderate selectivity for K 1 over Na 1 (permeability ratio of P K /P Na 5 1.5), which contrasts to the high K 1 selectivity of the SR K 1 channel 5,19 . Second, the SR K 1 channel is highly sensitive to inhibition by dec- amethonium 5 , whereas the TRIC-A channel is insensitive to this drug at concentrations up to 100 mM (Fig. 2a). Furthermore, application of either CaCl 2 or MgCl 2 to the recording solutions did not signifi- cantly affect the reversal potential or open probability, indicating that the TRIC-A channel is impermeable and insensitive to divalent 1 Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan. 2 Department of Medical Chemistry, Graduate School of Medicine, Tohoku University, Miyagi 980-8575, Japan. 3 Department of Physiology and Biophysics, Robert Wood Johnson Medical School, New Jersey 08854, USA. 4 Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8568, Japan. 5 PRESTO, Japan Science and Technology Agency, Saitama 332- 0012, Japan. 6 Department of Anatomy, Saitama Medical University, Saitama 350-0495, Japan. Vol 448 | 5 July 2007 | doi:10.1038/nature05928 78 Nature ©2007 Publishing Group