Biochemistry molecular size of 23 kDa.11 Although MG23 is abundantly expressed in the SR and nuclear membranes of striated muscle cells, its expression is also detected in a wide variety of cell types. The ubiquitous distribution suggests that MG23 may contribute to a common function in intracellular membrane systems. A recent study demonstrated that mutant thymocytes lacking MG23 became resistant to DNA damage-induced apoptosis, suggesting a role in the generation of ER-derived cell death signals.12 The physiological function of MG23, however, is still unknown. In this report, we provide biochemical and biophysical data suggesting that MG23 forms a massive homomultimeric complex, which can conduct cations, including Ca2þ, across the intracellular membrane systems. ’ MATERIALS AND METHODS Antibody and Topology Analysis. For producing mAbs, two synthetic peptides corresponding to the N-terminal and C-term- inal MG23 sequence were conjugated with a carrier protein and repeatedly injected into mice to generate hybridoma cells.11 Immunochemical experiments established two clones, mAb7 (mAb-N) and mAb251 (mAb-C), which specifically recognize the corresponding antigen epitopes. To examine the transmem- brane topology of MG23, we prepared SR vesicles from rabbit skeletal muscle13 and isolated ER vesicles from HEK293 cells transfected with MG23 expression plasmids.14 After the treat- ment of the vesicles with proteinase, the digestion profiles of native and recombinant MG23 were examined using mAbs as described previously.15 For further details, see the Supporting Information. Affinity Purification of Native and Recombinant MG23. For purification of native MG23, microsomal proteins were solubilized with NP-40 from rabbit muscle SR vesicles and reacted with affinity resin conjugated with mAb-N. After the resin had been extensively washed, MG23 was recovered with a buffer containing the epitope peptide. For production of recom- binant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence. The total microsome was prepared from yeast cells expressing His-tagged MG23, and the recombinant protein was solubilized with NP-40 or n-dodecyl β-D-maltoside (DDM) and purified using combined Ni and mAb-C affinity chromatog- raphy. For further details, see the Supporting Information. The purified MG23 preparations were used for chemical cross-link- ing, ultrastructural imaging, and channel recording. Particle Image Analysis. After affinity purification, MG23 was further purified by density gradient centrifugation. For immunodecoration of MG23, mAbs were reacted with the purified preparations. MG23 particles with or without the mAb decoration were applied to thin carbon films, negatively stained, and examined using electron microscopy (EM). For three- dimensional (3D) reconstruction of MG23 structures,7,16 parti- cle images for the large bowl-shaped and asymmetric small structures were automatically picked up from the electron micrographs.17,18 The particle images in each library were aligned rotationally and translationally19-21 and classified into clus- ters.22,23 The resulting class averages were used as new refer- ences, and the cycle from alignment to classification was repeated until convergence. On the basis of the symmetry of averaged images, 6-fold symmetry was imposed in the following computation of the large particles. No symmetry was assumed for the small ARTICLE particles. After convergence, a Euler angle was assigned to each average image,24 and a primary 3D model was reconstructed using simultaneous iterative reconstruction techniques.25 To optimize 3D reconstruction, reprojection images were created from the 3D model and used for realignment of the original particle images.26 The aligned particles were classified again, and a Euler angle of each average was assigned so as to be the best correlation to the 3D reprojection image. The computational steps were repeated until a stable 3D model was obtained. For further details, see the Supporting Information. Planar Phospholipid Bilayer Measurements. Native and recombinant MG23 proteins were solubilized and purified with either NP-40 or DDM. For detergent exchange in purified MG23 preparations, gel filtration chromatography was performed using a buffer solution containing 16 mM CHAPS and 16 mM 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Then, CHAPS was dialyzed using a dialysis membrane with a cutoff size of 12 kDa (Medicell International Ltd.) against a buffer containing 0.1 M NaCl, 0.1 mM EGTA, 25 mM PIPES (pH 7.4), 0.15 mM CaCl2, and 2.5 mM DTT (exchanged every 4 h for 16 h). Purified native and recombinant MG23 proteins were fused with planar phosphatidylethanolamine lipid bilayers as previously descri- bed.27 The trans chamber was held at ground and the cis chamber at potentials relative to ground. To examine if MG23 could behave as an ion channel permeable to monovalent cations or anions, we used symmetrical (in both cis and trans chambers) bathing solutions containing either 260 mM KCl, 20 mM HEPES, and 15 μM free Ca2þ (pH 7.2) or 260 mM K-PIPES and 15 μM free Ca2þ (pH 7.2). To investigate if MG23 is permeable to Ca2þ, we perfused the cis chamber with 250 mM HEPES, 80 mM Tris, and 15 μM free Ca2þ (pH 7.2) and filled the trans chamber with a solution containing 250 mM glutamic acid and 10 mM HEPES (pH 7.2) with Ca(OH)2 (free Ca2þ concentration of 65 mM). The free Ca2þ concentration and pH of solutions were measured at 22 C using a calcium electrode (Orion 93-20) and Ross-type pH electrode (Orion 81-55) as described previously.27 Single-channel events were recorded on digital audiotape. MG23 recordings were filtered at 600 kHz (-3 dB) and digitized at 20 kHz using Pulse (HEKA, Elektronik Lambrecht/Pfalz). The amplitude of channel openings was determined using WinEDR version 3.05 (J. Dempster, University of Strathclyde, Glasgow, U.K.) using manually controlled cur- sors. Noise analysis was performed with WinEDR version 3.05. The current fluctuations across the bilayer were subdivided into multiple segments in time with each segment containing N samples. The mean current for each segment was calculated with the formula Imean ¼ N ∑ IðiÞ i ¼ 1 N where I(i) is the amplitude of the ith current of the N samples in the segment. The mean current values were then plotted versus time. The relative Ca2þ/Kþ permeability ratio (PCa2þ/PKþ) of MG23 was calculated using the Fatt-Ginsborg equation:28 PCa2þ =PKþ ¼ ½Kþ =4½Ca2þ expðErevF=RTÞ½expðErev F=RTÞ þ 1 where R, T, and F have their usual meanings and Erev is the zero- current reversal potential. The value of RT/F used was 25.4 mV at 22 C. Erev was determined under bi-ionic conditions (cis, 210 mM Kþ; trans, 65 mM Ca2þ) and was taken as the voltage at 2624 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632