Biochemistry ARTICLE Figure 3. EM analysis of MG23 particles. (A) Raw EM images of recombinant MG23 particles. After adsorption to the glow-discharged carbon ﬁlm, negatively stained samples were imaged by EM. (B) Immunodecoration of MG23 particles. Recombinant MG23 bearing anti-His mAbs (top panels) and mAb-C (bottom panels) were imaged by EM. (C) Predicted membrane topology of the MG23 monomer from the hydropathicity proﬁle and limited proteolytic analysis (left) and the membrane position (blue line) deduced from the volume calculation of the reconstructed MG23 (right). Scale bars are 10 nm. images, we reconstructed the smaller-sized molecule assuming no intramolecular symmetry. The surface representation demon- strates that the smaller asymmetric structure has a roughly crescent-shaped appearance, 14 nm in height, 11 nm in side length, and 6 nm in narrow side length (Figure 5). The almost random orientation of the particles on the grid surface was also shown by a plot of the Euler angles of the 117 adopted class averages (Figure S3C of the Supporting Information). According to the FSC function, the resolution limit for the smaller-sized structure is 3.6 nm by the correlation coefficient of 0.5 criterion (Figure S3D of the Supporting Information). The final recon- struction included 1699 particle images (99% of all the selected images). Considering that the MG23 preparations were subjected to the EM analysis after aﬃnity puriﬁcation and size fractionation using sucrose gradient centrifugation, which were shown to be highly puriﬁed by SDS-PAGE, the concomitance of the large and small particles likely suggests that MG23 is easily disas- sembled and reassembled during puriﬁcation and storage. We considered that the smaller particles constitute a partial domain of the bowl-shaped assembly (see Discussion and Figure S4 of the Supporting Information). MG23 Conducts Cations. To investigate the possibility that MG23 plays a role in the transport of ions across the ER/SR, we reconstituted purified recombinant MG23 into planar lipid bilayers in symmetrical solutions of 260 mM KCl. Most incor- porations into the bilayer consisted of the apparent insertion of multiple channels; however, on rare occasions, we observed what appeared to be opening events to a single consistent single-channel level. A representative experiment is shown in Figure 6A. The current-voltage relationship of these channel events (Figure 6B) yielded a conductance of 85.1 ( 5.3 pS (n = 3; mean ( standard deviation). A similar conductance of 88.2 ( 2.9 pS (n = 5) was observed in symmetrical solutions of 260 mM K-PIPES (Figure 6C), indicating that MG23 preferentially conducts cations. MG23 is expressed in intracellular membranes, including the SR/ER Ca2þ-store membranes, and therefore, it is important to investigate if it is permeable to Ca2þ. To test this, we incorpo- rated recombinant MG23 into bilayers using solutions that are typically used to monitor Ca2þ currents through ryanodine receptor channels:27 250 mM Tris/HEPES and 15 μM Ca2þ on the cis side and 65 mM Ca2þ on the trans side. Under these conditions, we observed single-channel current ﬂuctuations indicating that Ca2þ is ﬂowing in the trans to cis direction through MG23 (Figure 6D). No channel activity was observed in solutions of symmetrical 250 mM Tris/HEPES, demonstrat- ing that the channel is not permeable to Tris or HEPES (Figure S5 of the Supporting Information). The current-voltage rela- tionship in these Ca2þ-containing solutions exhibits a unitary conductance of 62.5 ( 10.2 pS (n = 5). Our observations therefore demonstrate that MG23 can form an ion channel that is permeable to both Kþ and Ca2þ. After incorporation of MG23 into bilayers, current-voltage relationships were obtained under bi-ionic conditions (cis, 210 mM Kþ; trans, 65 mM Ca2þ). The reversal potential was calculated to be -6.9 ( -3.7 mV (n = 3) after correction for the junction potentials arising between the diﬀerent solutions (Figure S6 of the Supporting Information). Using the Fatt-Ginsborg equation, the PCa2þ/PKþ for MG23 was then calculated as 1.1 ( 0.2 (n = 3), which suggests that MG23 is approximately equally permeable to Ca2þ and Kþ. 2627 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632