Biochemistry ARTICLE Figure 5. Reconstruction of the smaller-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations; fourth row, projections of the 3D reconstruction. (B) Dimensions of the smaller-sized crescent- shaped molecule are 14 nm (height), 11 nm (wide side length), and 6 nm (narrow side length). The massive bowl-shaped particle in Figure 3 is considered to be an assembly of multiples of this small subunit. The scale bars are 10 nm. coordination) of seemingly random numbers of MG23 channels were a consistent feature of the gating of this channel (Figure 7). As described above, particle image analysis implies that the crescent-shaped subunits are unstably assembled into the bowl- shaped particle that is equipped with a putative central pore. Although we can only speculate about how MG23 conducts ions, the unusual gating features could be caused by the proposed instability of the bowl-shaped particle. For example, MG23 may operate as a single conduction pathway with a central pore in the bowl-shaped assembly. During the disintegration and re-forma- tion of the assembly, the proposed channel pore might display multiple conductance states and could pass huge currents super- ﬁcially observed as simultaneous gating events. Alternatively, it may be possible that the crescent-shaped subunits form the unitary ion channel and can gate independently in the bilayer. In response to the structural transitions of the bowl-shaped assembly, the resulting conformational changes might aﬀect ionic conduction through each crescent-shaped channel or could induce the simultaneous openings of the multiple-channel components. On the basis of our reconstructed 3D particle images, it is not possible to locate the ion conduction pathway of the MG23 channel. However, if MG23 is to follow the structural conventions of ion channels with known X-ray crystal structures, the apparent thick- ness at the transmembrane region of the crescent-shaped subunit (Figure S2 of the Supporting Information) suggests that it may be too narrow to form a unitary conduction pathway, hence favoring the idea of a single central pore in the bowl-shaped particle. Figure 6. Single-channel behavior of MG23. (A) Representative single- channel recordings of native MG23 from rabbit skeletal muscle in solutions of symmetrical 260 mM KCl and 20 mM HEPES (pH 7.2). The holding potentials are indicated, and O and C represent the open and closed channel levels, respectively. (B) Single-channel current- voltage relationship of recombinant MG23 in symmetrical 260 mM KCl solutions (n = 3). (C) Single-channel current-voltage relationship of recombinant MG23 in symmetrical 260 mM K-PIPES solutions (n = 5). (D) Channel gating of recombinant MG23 with Ca2þ as the permeant ion. The bathing solutions contained 250 mM HEPES, 80 mM Tris (pH 7.2), and 15 μM free Ca2þ in the cis chamber and 250 mM glutamic acid and 10 mM HEPES (pH 7.2) with Ca(OH)2 (free Ca2þ concentration of 65 mM) in the trans chamber. The holding potential was 0 mV. Channel openings at holding potentials more positive than 10 mV could not be resolved. (E) Current-voltage relationship of MG23 under the conditions described in panel D (n = 5). The data represent means ( the standard deviation. A novel ER/SR cation channel such as MG23, exhibiting no obvious selectivity for monovalent over divalent cations, would be expected to contribute signiﬁcantly to the regulation of cellular Ca2þ homeostasis. There are many aspects of intracel- lular Ca2þ release that are not fully understood, and characteriz- ing the biophysical properties of SR proteins such as MG23 will be important in resolving these issues. For example, electrogenic release of Ca2þ from and reuptake of Ca2þ into the ER/ SR require synchronized counterion movements to balance the buildup of charge asymmetry across the ER/SR mem- brane,5,6,30,31 yet the molecular machinery responsible for the proposed counterion ﬂuxes remains undeﬁned. MG23 could play some part in that process. The puriﬁed MG23 channel displays a consistent voltage dependence of activation, indicating that it incorporates into the bilayer in a ﬁxed orientation. SR vesicles also form in a ﬁxed orientation13 and are incorporated into artiﬁcial membranes in a ﬁxed orientation such that the cytosolic side of SR ion channels always faces into the cis chamber.32 Because our biochemical data (Figures 1-4) indicate that puriﬁed 2629 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632