2397-Pos Board B416 The Selectivity of MscS is Determined by the Cytoplasmic Domain Charles D. Cox 1 , Anthony K. Campbell 1 , Kenneth T. Wann 1 , Boris Martinac 2 . 1 Cardiff University, Cardiff, United Kingdom, 2 Victor Chang Cardiac Research Institute, Sydney, Australia. The mechanosensitive channel of small conductance (MscS) is a heptameric pressure sensitive channel expressed in the inner membrane of E. coli. This channel possesses three transmembrane helices and a large water-filled cyto- plasmic cage which comprises more than 50% of the total protein. This cyto- plasmic domain is conserved throughout the MscS channel family and in MscS has been shown to be a dynamic structure with an active role in channel gating. It has also been suggested that the cytoplasmic domain determines the weak anion selectivity exhibited by MscS. In order to address this question this study reconstituted wild type MscS and single residue mutants of the cytoplas- mic vestibule in azolectin liposomes (protein:lipid 1:10000). Patch clamp re- cordings of these channels were then performed in the presence of asymmetric solutions of KCl (600/200 mM) and BaCl 2 (50/200 mM). The MscS mutants studied were R184E, R185E, E187R and E227A. Both E187R and E227A mutants show reduced selectivity characterised by a lower anion- cation permeability ratio. These residues are likely to determine selectivity by binding cations. From these data it is clear that charged residues in the cy- toplasmic domain of MscS determine its selectivity. This is interesting because unlike K þ , Na þ and Ca 2þ channels the selectivity of MscS is not determined by residues in the pore region but residues situated in the large water-filled cyto- plasmic domain. 2398-Pos Board B417 The Right-Side-Out Orientation of MscS in Liposomal Membranes Takeshi Nomura 1 , Masahiro Sokabe 2 , Boris Martinac 1,3 . 1 Victor Chang Cardiac Research Institute, Sydney, Australia, 2 Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3 St. Vincent’s Clinical School, The University of New South Wales, Sydney, Australia. The bacterial mechanosensitive channel MscS plays a crucial role in the protec- tion of bacterial cells against hypo-osmotic shock. MscS functional character- istics have extensively been studied in both giant spheroplasts and liposomes. Despite many studies of MscS reconstituted into liposomes the channel orien- tation in liposomal membranes is still unknown. We examined the orientation of MscS in liposomes by patch-clamp and confocal microscopy. using its pre- viously determined electrophysiological and pharmacological properties we were able to determine that in liposomes MscS retains the right-side-out orien- tation as in giant spheroplasts based on the following evidence: (i) I-V curves recorded in both spheroplast and liposome preparations exhibited strong out- ward rectification at both negative and positive pipette pressures. (ii) MscS ac- tivation ratio in liposome patches at positive relative to negative pipette voltages and vice versa showed positive correlation at both positive and nega- tive pipette pressures similar to MscS in inside-out excised spheroplast patches. (iii) MscS exhibited a voltage-dependent hysteresis upon application of saw- tooth pressure ramps in both spheroplasts and liposomes. In both spheroplasts and liposomes the hysteresis was more pronounced upon positive pipette volt- ages compared to negative voltages. (iv) 2.5% of 2,2,2-trifluoroethanol (TFE) caused MscS inactivation in liposome patches when added to the cytoplasmic side of MscS, whereas addition of TFE to the periplasmic side did not inactivate the channel, although it caused a shift of the channel activation towards lower pipette pressures. We obtained a similar result when applying TFE to MscS in spheroplast patches. In conclusion, our findings strongly indicate that the cyto- plasmic domain of MscS in liposome membrane patches faces the bath solution as in spheroplast patches. Consequently, upon liposome reconstitution MscS channels preserve their right-side-out orientation comparable to what was pre- viously reported for the MscL channels. Supported by the NH & MRC. 2399-Pos Board B418 Modulation of the G22E MscL Mutant Channel Gating by Lipid Bilayer Constituents and Gadolinium Andrew R. Battle 1 , Evgeny Petrov 2 , Boris Martinac 2,3 . 1 School of Pharmacy, Griffith University, Australia, 2 Victor Chang Cardiac Institute, Darlinghurst, Australia, 3 St Vincent’s Clinical School, The University of New South Wales, Sydney, Australia. Bacteria respond to hypoosmotic changes through the mechanosensitive (MS) channels of Large (MscL) and Small (MscS) conductance, MscS responds first to pressure, i.e. bilayer tension changes, followed by MscL 1 . The lipid environ- ment, lyso lipids and cholesterol have been shown to significantly influence the ratio of the opening of MscL to MscS 2,3 . Furthermore, introduction of the highly negatively charged cardiolipin to both azolectin and POPE/POPC lipid membranes causes rapid gating of MscS 4 . Here we report an expanded study using the spontaneously active G22E MscL mutant which, although spontane- ously active, is still mechanosensitive. Addition of sub-millimolar amounts of the metal ion Gadolinium(III) reversibly inhibits spontaneous channel activity, but upon application of pressure, the channel exhibits mechanosensitivity sim- ilar to the wild-type MscL. Our results are consistent with the previous studies showing that Gd(III) inhibits MscL mechanosensitivity by binding to the lipid bilayer 5,6 1 Martinac B, Curr Top Membr. 2007 58, 25 2 Battle AR, Petrov, E, Pal P, Martinac B. Febs Lett2009,583, 487 3 Nomura T, Cranfield CG, Deplazes E, Owen DM, Macmillian A, Battle AR, Constantine M, Sokabe M, Martinac B. PNAS2012, 109, 8770 4 Battle AR, Nomura T, Martinac B, Biophys J 2011, 100 S1, 278 5 Ermakov YA, Kamaraju K, Sengupta K, Sukharev, S. Biophys J, 2010, 98, 1018 6 Petrov E, Martinac B. E. Biophys. J., 2007, 36, 95 Supported by the National Health & Medical Research Council of Australia. 2400-Pos Board B419 A Novel Approach to follow Helical Movements of an Ion Channel in Real-Time Duygu Yilmaz 1 , Anna Dimitrova 1 , Martin Walko 2 , Armagan Kocer 1 . 1 University of Groningen, Groningen, Netherlands, 2 Pavol Jozef Safarik University, Kosice, Slovakia. Mechanosensitive channel of large conductance (MscL) is one of the best- studied mechanosensitive channels in bacteria (Sukharev et al., 1994, Blount et al., 2007). It acts as a safety valve in response to hyperosmotic shock. High-resolution structure of Mycobacterium tuberculosis MscL revealed that it forms a homopentamer with two transmembrane helices per subunit. Al- though in nature the channel opens in response to tension, breaking the hydro- phobic interactions at its pore region leads to the spontaneous opening of the channel. Here, by using this principle, we modified the hydrophobic gate of the channel with designed chemical switches and we gained external control over its activation. We followed the resulting structural changes on the protein by following the Electron Paramagnetic Resonance signal from a spin label on different positions at the pore forming helices. We developed a method in which we could control the number of switches and EPR spin labels per pen- tamer. By this approach, we start following the gradual activation of the chan- nel in real time. 2401-Pos Board B420 MscL as a Triggered Nanovalve: New Modifications to Improve Design Irene R. Iscla, Robin Wray, Christina Eaton, Juandell Parker, Paul Blount. U.T. Southwestern Med. Ctr., Dallas, TX, USA. MscL is a small homopentameric bacterial protein that has, among other char- acteristics, an incredible pore size greater than 30A ˚ and the ability to gate in response to mechanical tension in the membrane. Because of its amenability, E.coli MscL has been the most studied mechanosensitive channel, serving as a paradigm of how a protein can sense and transduce mechanical force. Early on in the study of the channel a critical domain for MscL gating was revealed by forward genetic experiments screening for mutations that led to a gain-of- function (slowed- or no-growth) phenotypes: mutations at residue G22, within the pore, led to severe gain-of-function phenotypes. This residue is thought to form part of a ‘‘hydrophobic lock’’ that stabilizes the closed state of the chan- nel. If this hydrophobic lock is disrupted by the insertion of a charge, the tran- sition energy barrier for MscL gating is destabilized and the channel gates even in the absence of membrane tension. using this observation, researchers have successfully changed the modality of MscL to be sensitive to stimuli such as light and pH simply by chemically modifying the G22 site within the MscL channel. Due to its ability to be triggered by different stimuli and the large pore size, MscL has been proposed as a triggered nanovalve for its use in nano- devices such as a liposome drug delivery system. Here, by utilizing in vivo, flux and patch clamp assays, we characterize other neighboring residue that also form part of the hydrophobic lock and we show that the G22 site may not be the best choice for all modifications that change channel modality. 2402-Pos Board B421 Mechanosensor and Gate is Tightly Coupled in the Bacterial Mechanosen- sitive Channel MscL Yasuyuki Sawada 1 , Takeshi Nomura 2 , Masahiro Sokabe 1,3 . 1 Nagoya University Graduate School of Medicine, Nagoya, Japan, 2 Victor Chang Cardiac Research Institute, New South Wales, Australia, 3 FIRST research center for innovative naobiodevice, Nagoya University, Nagoya, Japan. Tuesday, February 5, 2013 469a