Chen YH et al. (2010), Homologue structure of the SLAC1 anion channel ...

XB-ART-46653

Nature 2010 Oct 28;4677319:1074-80. doi: 10.1038/nature09487.

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Homologue structure of the SLAC1 anion channel for closing stomata in leaves.

Chen YH, Hu L, Punta M, Bruni R, Hillerich B, Kloss B, Rost B, Love J, Siegelbaum SA, Hendrickson WA.

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The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 Å resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

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	Figure 2. Crystal structure of HiTehA and homology model of AtSLAC1a, Electron density distribution from the HiTehA crystal structure at 1.2Å resolution. The map has (2Fo-Fc) coefficients based on the superimposed model. Contours are at 2.5σ. b, Ribbon diagram of the HiTehA trimer. Each protomer is colored spectrally from blue at its N-terminus to red at its C-terminus. c, DelPhi49 electrostatic potential at the extracellular surface. Electronegative and electropositive potential are colored in degrees of red and blue saturation, respectively. d, Electrostatic potential49 at the intracellular surface. e, Ribbon diagram of an HiTehA protomer viewed from outside the membrane. The ribbon is colored spectrally as in 2b. f, Ribbon diagram of an HiTehA protomer viewed from within the membrane from below the view of e. g, Surface of a homology model of AtSLAC1, viewed as in f, and colored by sequence variability48. h, Surface of AtSLAC1 as in g, but colored by electrostatic potential49.
	Figure 3. Putative structure of the SLAC1 conductance porea, Cross-section through the homology model of AtSLAC1. The model is viewed as in 2i, with the electrostatic potential49 shown on the external surface of the molecular envelope. The side chain of Phe450 is shown as a stick model (red) on the backbone ribbon colored yellow. b, Cross-section as in a, but colored by surface conservation48 as in 2h. c, Pore-lining residues in the SLAC1 homology model. A cylinder model (left), spectrally colored as in 2b, provides a key for viewing the rolled-open structure (left) with pore-lining residues of AtSLAC1 shown on the TModd helices. d, Ribbon diagrams of HiTehA TM9 (left) and TM7 (right) viewed from within the conductance pore. The side chains of Pro207 and Phe262 are shown as well as a kink-stabilizing water HOH25 that is coordinated by the NH of Gly263 and by C=O groups of Gly202 and Ala259. Density contours are shown for the water molecule.
	Figure 4. Ionic conductance measurementsa, Typical microelectrode voltage-clamp current traces from oocytes injected with various channel cRNAs. Left column, oocytes injected with cRNAs encoding wild-type HiTehA channels (WT), or F262A, G15D/F262A or G15D mutants. Right two columns, oocytes injected with cRNAs for WT AtSLAC1, or F450A, G194D/F450A or G194D mutants, with or without co-injection of AtOST1. Dotted lines represent zero current levels. Extracellular solution contained 30mM CsCl. Schematic keys at the left place phenyl and/or aspartyl gates. b, Effects of gating residue mutations. Mean chloride currents, measured at −90 mV, are shown comparing WT HiTehA and its mutant series F262A,G,T,V,L with WT AtSLAC1 and its corresponding series F450A,G,T,V,L, both alone and co-expressed with AtOST1. Full I-V relations are shown in Fig. S5. c, Effect of gating residues on relative AtSLAC1 anion permeabilities. Relative permeabilities (P[X]/P[Cl]) for chloride, nitrate, sulfite and malate of WT, F450A and F450T SLAC1 channels were measured from the change in current reversal potential with Cl− or anion X− as the sole permeant anion in the bath solution (Methods, Table S6).
	Figure 5. Structural features at the SLAC1 homolog gatea,b, Cross-sections through the conductance pores of HiTehA F262A and HiTehA G15D. The view and presentations are as in 3a, except that helices are colored purple. c, Molecular basis for conformational strain in gating residue Phe262 of HiTehA. Helices TM1 (left), TM9 (center) and TM7 (right) are viewed from within the pore and presented as ribbon diagrams with selected side chains drawn in stick representation. The local low-energy conformation for the phenyl ring (χ2 = 90°) is shown in thin lines with short contacts indicated in dashed lines: Leu18 Cδ2 – Phe262 Cδ1, d = 2.4 Å; Val210 Cγ2 – Phe262 Cε2, d = 2.8 Å. d, Conformational shifts consequent to release of strain in gating residue Phe262. Cα backbone structures of F262A, F262V and F262L HiTehA were first superimposed onto WT HiTehA, and residues 258-266 were drawn in stereo as superimposed. All backbone atoms are shown for peptides 262±1, but only Cα atoms are shown otherwise. The WT backbone and phenyl group are green; other backbone are all magenta; side chains of Ala262, Val262 and Leu262 are cyan, blue and red, respectively; oxygen-directed bonds are red and nitrogen-directed bonds are blue. e, Typical microelectrode voltage-clamp current traces from oocytes injected with wild-type (WT) or F450L AtSLAC1 cRNA. Experimental conditions and displays are as in 4a.

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