Biochemistry Figure 7. Distinguishing gating characteristics of MG23. (A) Coordi- nated gating of recombinant MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH7.2) at a holding potential of 20 mV. C indicates the zero current level (all channels closed), and O indicates the current level of a single MG23 channel opening; the dashed lines aid the visualization of these levels across the trace. An asterisk denotes a single MG23 channel opening. The arrowheads indicate where coordinated gating of multiple MG23 channels occurs, and the bar shows a long coordinated gating event. (B) Typical example of the coordinated MG23 channel gating in solutions where trans Ca2þ was the only permeant ion (250 mM Tris/HEPES, cis; 65 mM Ca2þ, trans) at a holding potential of 10 mV. The dashed lines between the top and bottom traces indicate the portion of the trace that has been reproduced below on an expanded time scale to visualize the individual channel openings more clearly. (C) Noise analysis illustrates the voltage dependence of MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH 7.2). As the holding potential became increasingly negative, Kþ current across the bilayer became more elevated. MG23, when reconstituted into liposomes, retains the same orientation as native MG23 in SR vesicles, it is most probable that the cytosolic side also faces into the cis chamber. Thus, in a cellular environment, given the voltage dependence of MG23 (Figure 7), it would be expected that during a Ca2þ release event, the ER/SR would become negative relative to the cytosol and this would reduce the Po of the MG23 channel, although it would not close completely. If voltage was the only controlling inﬂuence on Po, MG23 would tend to be more open during reﬁlling of the ER/SR stores or just before a Ca2þ release event. MG23 may therefore contribute to the spontaneous Ca2þ leak that is known to occur from intracellular stores in many cell types.33-35 In ARTICLE cardiac cells, this is termed diastolic SR Ca2þ leak, and there is growing evidence that this is increased in disease states such as heart failure and leads to an increased likelihood of fatal cardiac arrhythmias.35-37 Indeed, in cardiac cells, it has been suggested that there must be another route for the release of Ca2þ from the SR during diastole apart from ryanodine receptors;38 further investigation is required to determine whether MG23 is that pathway. On the other hand, the voltage dependence and sporadic gating behavior of MG23 suggest that it may play a more minor role in balancing charge asymmetry during a large and rapid Ca2þ release event (such as during a cardiac Ca2þ transient) than the recently characterized TRIC channels that are activated to high Po values as negative charge develops in the SR.9 The full magnitude of MG23’s contribution to counterion currents, however, will not be realized until we have an improved understanding of whether the function of MG23 can be regulated by soluble ligands or by other mechanisms such as phosphoryla- tion and nitrosylation. There is recent evidence that MG23 is involved in the process that leads to cell death because apoptotic signaling is attenuated in mutant thymocytes prepared from MG23-knockout mice.12 It is accepted that changes in the Ca2þ content and leak of intracellular stores can aﬀect the sensitivity to apoptotic death,39 but the molecular identity of the leak pathway(s) has not been identiﬁed. Our data, demonstrating that MG23 is permeable to monovalent and divalent cations, suggest that MG23 could fulﬁll that role. It is clear that much work is required to link the unique structural features of MG23 to its equally unique behavior as an ion channel. For example, does the transient assembly or break- down of the bowl-shaped MG23 particle underlie the sporadic activity of multiple MG23 channels opening simultaneously, or does the production of MG23 bowls and their appearance in the ER membrane lead to the destruction of ionic gradients across the ER and immediate cell death? What is the role of MG23 in nuclear membranes because it is clearly present there in high levels? Again, to answer this question, we require a much more detailed understanding of the ion conducting properties of MG23 and of how MG23 channel gating can be regulated. Investigating the phenotype of the knockout mouse will also be vital in identifying the key physiological and pathophysiolo- gical roles of the MG23 protein. ’ ASSOCIATED CONTENT bS Supporting Information. Detailed methodology (Sup- porting Experimental Procedures), puriﬁcation and cross-linking of native MG23 (Figure S1), continuous sections through the bowl-shaped MG23 particle (Figure S2), Euler angle distribution and FSC function in the 3D reconstruction of MG23 particles (Figure S3), docking model of the crescent-shaped subunits into the bowl-shaped particle (Figure S4), MG23 is not permeable to Tris and HEPES (Figure S5), and MG23 PCa2þ/PKþ data (Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *H.T.: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan; telephone, þ81-75-753- 4572; fax, þ81-75-753-4605; e-mail, takeshim@pharm.kyoto-u. ac.jp. C.S.: Biomedical Research Institute and Biomedicinal 2630 dx.doi.org/10.1021/bi1019447 |Biochemistry 2011, 50, 2623–2632