ARTICLE pubs.acs.org/biochemistry Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel Elisa Venturi,† Kazuhiro Mio,‡ Miyuki Nishi,§ Toshihiko Ogura,‡ Toshio Moriya,‡,|| Samantha J. Pitt,† Kazutaka Okuda,§ Sho Kakizawa,§ Rebecca Sitsapesan,*,† Chikara Sato,*,‡ and Hiroshi Takeshima*,§ †School of Physiology and Pharmacology, Bristol Heart Institute and Centre for Nanoscience and Quantum Information, University of Bristol, Bristol, United Kingdom ‡Biomedical Research Institute and Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan §Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan G)raduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan bS Supporting Information ABSTRACT: Mitsugumin 23 (MG23) is a 23 kDa transmem- brane protein localized to the sarcoplasmic/endoplasmic reticu- lum and nuclear membranes in a wide variety of cells. Although the characteristics imply the participation in a fundamental function in intracellular membrane systems, the physiological role of MG23 is unknown. Here we report the biochemical and biophysical characterization of MG23. Hydropathicity proﬁle and limited proteolytic analysis proposed three transmembrane segments in the MG23 primary structure. Chemical cross-linking analysis suggested a homo-oligomeric assembly of MG23. Ultrastructural observations detected a large symmetrical particle as the predominant component and a small asymmetric assembly as the second major component in highly puriﬁed MG23 preparations. Single-particle three-dimensional reconstruction revealed that MG23 forms a large bowl-shaped complex equipped with a putative central pore, which is considered an assembly of the small asymmetric subunit. After reconstitution into planar phospholipid bilayers, puriﬁed MG23 behaved as a voltage-dependent, cation-conducting channel, permeable to both Kþ and Ca2þ. A feature of MG23 gating was that multiple channels always appeared to be gating together in the bilayer. Our observations suggest that the bowl-shaped MG23 can transiently assemble and disassemble. These building transitions may underlie the unusual channel gating behavior of MG23 and allow rapid cationic ﬂux across intracellular membrane systems. Tfunctional he endoplasmic/sarcoplasmic organelle responsible reticulum for important (ER/SR) cellular is a multi- pro- cesses, including protein maturation, lipid metabolism, Ca2þ signaling, and stress response. The ER/SR serves as an intracel- lular Ca2þ store, and activation of Ca2þ release channels, namely, inositol trisphosphate and ryanodine receptors, controls physio- logical functions such as muscle contraction, secretion, metabo- lism, and transcription.1,2 In addition, the ER is the site for synthesis and maturation of both membrane and secretory proteins, enforcing protein glycosylation, disulﬁde bridging, folding, and subunit assembly. When misfolded proteins accu- mulate within the lumen, the ER stress response is activated according to severity, leading to the recruitment of ER chaper- ones, inhibition of protein synthesis, and induction of apoptotic cell death.3,4 The activity of molecular chaperones, protein- processing enzymes, and metabolic enzymes of the ER largely depends upon the high luminal Ca2þ level. Uptake of Ca2þ into and release of Ca2þ from intracellular stores are electrogenic processes. Therefore, active Ca2þ ﬂuxes may be synchronized with the movements of other ionic species that compensate for charge imbalance across the ER/SR membrane.5,6 We have recently identiﬁed TRIC channel sub- types that function as monovalent cation channels and probably support release of Ca2þ from the ER/SR of various cell types.7-10 It is likely that the vital function of the ER/SR requires rapid and ﬂexible control of the ionic balance between the luminal and cytoplasmic sides. To understand the ionic home- ostasis across the ER/SR membrane, it is important to further characterize the functional properties of its constituent ion channels and transporters in the intracellular membrane system. Skeletal and cardiac muscle SR is specialized as the intracel- lular Ca2þ store for controlling contraction and abundantly contains Ca2þ-handling proteins such as Ca2þ-ATPase, calse- questrin, and ryanodine receptors.2 Muscle SR is, therefore, an ideal model system for studying Ca2þ store functions. To understand the molecular basis of Ca2þ stores, we have searched for novel SR proteins using monoclonal antibodies (mAbs) and previously identiﬁed mitsugumin 23 (MG23) with a mature Received: Revised: Published: December 7, 2010 February 15, 2011 March 07, 2011 r 2011 American Chemical Society 2623 dx.doi.org/10.1021/bi1019447 | Biochemistry 2011, 50, 2623–2632