Biochemistry Figure 4. Reconstruction of the larger-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations of the 3D reconstruction; fourth row, projections of the 3D reconstruction. Consistency among data sets is very high in size, shape, and inner structure, indicating successful 3D reconstruction from the original images of recombinant MG23 particles. (B) Surface representation of two MG23 structures from top (left) and oblique (right) views. Dimensions of the larger-sized bowl-shaped structure are 12 nm (height), 16 nm (wide side length), and 17 nm (diagonally at the widest transmembrane region). The scale bars are 10 nm. Distinctive Characteristics and Voltage Dependence of MG23 Channel Gating. In the majority of experiments with MG23 preparations, multiple active channels were observed in the bilayer. This occurred even when we saw what appeared to be single fusion events of the liposomes into the bilayer. The presence of multiple channels and the frequent brief open events usually produced “messy-looking” or “flickery” single-channel recordings. Another distinctive characteristic of MG23 gating was the fact that multiple channels appeared to gate in unison so that many channels opened and closed together in a coupled or coordinated manner. Both the flickery gating and the coordinated channel openings can be seen in the two types of experiments: one in which Kþ is the permeant ion (Figure 7A) and another in which Ca2þ is the permeant ion (Figure 7B). In both channel measure- ments, we observed single opening events (asterisks), coordinated gating of multiple channels (arrowheads), and long sojourns of coordinated gating (bar). We have not identified any tendency for a preferred number of MG23 channels to gate together in synchrony; this seems to be a random process. For example, two channels may open and close together (as in Figure 7B), or greater numbers of channels may gate in a synchronous fashion. ARTICLE Because coordinated gating of multiple channels occurred randomly and gating was usually very rapid giving rise to ﬂickering events, it was very diﬃcult to measure the single- channel open probability (Po) of MG23 channels. To examine the voltage dependence of MG23 channel gating, we therefore used noise analysis (see Figure 7C for a typical example). At 10 mV (channel openings are downward deﬂections), Po was fairly low, although there were several coordinated channel opening events. At -10 mV (channel openings are upward deﬂections), there were more channel openings, and at -20 mV, the channels became very active and it was obvious that there were many chan- nels present in the bilayer. The strong voltage dependence of MG23 is an important distinguishing feature of this channel and was observed in every experiment. Typically, channel activity was low around 0 mV but rapidly increased as the holding potential was made more negative. Holding potentials above -30 mV tended to break the bilayer because of the large currents. ’ DISCUSSION We successfully puriﬁed MG23 proteins from both rabbit muscle SR and recombinant yeast using aﬃnity chromatography. The original EM images of the MG23 preparations themselves clearly suggested that the predominant large particles are mor- phologically identical in native and recombinant MG23 prep- arations. However, even after the size fractionation of highly puriﬁed MG23 by sucrose gradient centrifugation, MG23 parti- cles were not uniform in size. In the MG23 preparations, the predominant component was the bowl-shaped particle with 6-fold symmetry (Figure 4), but we also detected the smaller crescent-shaped particles as the second most common compo- nent (Figure 5). The application of chemical cross-linkers to MG23 preparations reproducibly generated the homohexamer as a putative basic self-assembly unit (Figure 2). However, it is unlikely that these larger and smaller structures observed using EM represent hexameric and monomeric MG23, respectively. This is because the MG23 monomer at 23 kDa, composed of 210 amino acids, is too small for EM observation (for reference, see the images of 150 kDa mAbs in Figure 3B). We therefore propose that the crescent-shaped particle is a hexameric mini- mum structural subunit of MG23, and that multiple subunits subsequently constitute a larger assembly such as the bowl- shaped structure. With the available data, we could not determine the exact number of crescent-shaped subunits that would make up the full particle, but biochemical and structural analysis points to a number composed of two to six subunits. Fitting of more than six crescent-shaped subunits to the bowl-shaped structure seemed impossible. We present a docking of six crescent-shaped subunits as the most plausible model (Figure S4 of the Support- ing Information). The 3D map of the crescent-shaped subunit (Figure 5B) is contoured at an isosurface containing a volume corresponding to 138 kDa (23 kDa 6), and the bowl-shaped particle (Figure 4B) at 828 kDa (138 kDa 6) using Imagic V,19 assuming a hexagonal assembly of the crescent-shaped subunit. However, this simulation could not exclude other possibilities of subunit construction in the bowl-shaped particle. MG23 is expressed in various cell types and localized in the SR/ ER and nuclear membranes.11 Our bilayer experiments demon- strate that recombinant MG23 prepared using DDM or NP-40 as a detergent forms cation channels displaying little diﬀerence in permeability between Kþ and Ca2þ. MG23 gating behavior is very unusual; the simultaneous openings (and therefore apparent 2628 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632