Milescu M , Vobecky J , Roh SH , Kim SH , Jung HJ , Kim JI , Swartz KJ .

ZIA NS002945-13 NINDS NIH HHS , ZIA NS002945-13 Intramural NIH HHS

	Figure 1. NMR structures of hanatoxin and SGTx1 and inhibition of the Kv2.1 channel by hanatoxin. Stereo views of hanatoxin (A) and SGTx1 (B) NMR solution structures. Side chain colors are as follows: green, hydrophobic; blue, basic; red, acidic; pink, Ser/Thr; gray, other side chains and backbone atoms. Protein database accession codes are 1D1H for hanatoxin and 1LA4 for SGTx1. (C) Voltage-clamp recording from an oocyte expressing the Kv2.1 channel. Currents were elicited by depolarization to −10 mV (above) or +50 mV (below), in the absence (black) or presence of 4 μM hanatoxin (gray). The holding voltage was −100 mV, and the tail voltage was −80 mV. The dashed line indicates the level of zero current. Leak, background, and capacitive currents were subtracted after blocking the channel with agitoxin-2. (D) Voltage– activation relations in the absence (black) and presence of 4 μM hanatoxin (gray). Tail currents obtained following depolarizations were averaged for 0.2 ms beginning 2 ms after repolarization to −80 mV. Data points are the mean ± SEM (n = 3).
	Figure 2. Interaction of hanatoxin and SGTx1 with lipid vesicles. Fluorescence emission spectra of hanatoxin (A) and SGTx1 (B) in the absence (black) or presence of lipid vesicles composed of a 1:1 mix of POPC:POPG (blue). The lipid concentration was 2.4 mM. Spectrum of W30A mutant of SGTx1 in solution is shown for comparison. Stern-Volmer plots for acrylamide quenching of W30 fluorescence of hanatoxin (C) and SGTx1 (D) in solution (2 μM, black diamonds) and in the presence of lipid vesicles (0.9 mM, blue circles). Fluorescence intensity at 320 nm plotted as a function of available lipid concentration (60% of total lipids) for hanatoxin (E) or SGTx1 (F). Smooth curves correspond to partition functions with Kx = (4.8 ± 0.4) × 106 and F/F0max = 2.0 ± 0.05 for hanatoxin and Kx = (3.4 ± 0.6) × 106 and F/F0max = 1.6 ± 0.05 for SGTx1. All data were obtained using HEB solution (10 mM HEPES, 1 mM EDTA, pH 7). In all cases data points are the mean ± SEM (n = 3).
	Figure 3. Effect of lipid composition on partitioning of hanatoxin and SGTx1 into lipid membranes. Fluorescence emission spectra of hanatoxin (A) and SGTx1 (B) in the absence (black) or presence of lipid vesicles composed POPC (blue). The lipid concentration was 2.4 mM. Fluorescence intensity at 320 nm plotted as a function of available lipid concentration (solid symbols) for hanatoxin (C) and SGTx1 (D). Data for vesicles containing a 1:1 mix of POPC:POPG (open symbols and smooth curves, same as in Fig. 2, E and F) is shown for comparison. Stern-Volmer plots for acrylamide quenching of W30 fluorescence of hanatoxin (E) and SGTx1 (F) in solution (2 μM, black diamonds) and in the presence of POPC vesicles (0.9 mM, blue circles). Data points are the mean ± SEM (n = 3). (G and H) Reversed-phase HPLC profiles of SGTx1 present in the supernatant after ultracentrifugation in the absence (black) or after addition of 9 mM lipid vesicles (blue). Traces were normalized to the maximum in the absence of lipid vesicles. All data were obtained using HEB solution (10 mM HEPES, 1 mM EDTA, pH 7).
	Figure 4. Effects of solution composition on partitioning of hanatoxin and SGTx1 into lipid membranes. Fluorescence intensity at 320 nm plotted as a function of available anionic lipid concentration for hanatoxin (A) and SGTx1 (B). Smooth curves are the fits of a partition function to the data as follows: diamonds for 100 mM KCl data: Kx = (5.3 ± 0.03) × 105 and F/F0max = 2.0 ± 0.02 for hanatoxin, and Kx = (1.1 ± 0.001) × 105 and F/F0max = 1.7 ± 0.03 for SGTx1; triangles for 100 mM K+, 1 mM free Ca2+ data: Kx = (4.8 ± 0.01) × 104 and F/F0max = 2.1 ± 0.1 for hanatoxin, and Kx = (1.9 ± 0.01) × 104 and F/F0max = 2.0 ± 0.5 for SGTx1; closed circles for 100 mM K+, 1 mM Ca2+, 0.3 mM Mg2+, pH 7.6 (PB). Data from Fig 2 (E and F) for HEB solution are shown for comparison using open circles. Stern-Volmer plots for acrylamide quenching of W30 fluorescence of hanatoxin (C and E) and SGTx1 (D and F) in physiological buffer (PB) (2 μM, black diamonds) and in the presence of either anionic (C and D) or neutral (E and F) lipid vesicles (0.9 mM, blue circles). In all cases data points are the mean ± SEM (n = 3). (G) Reversed-phase HPLC profiles of hanatoxin, SGTx, and agitoxin-2 present in the supernatant in the absence (black) or in the presence of 300 oocytes (blue) resuspended in physiological buffer (PB). The calculated fraction partitioned (fP = ([Toxintotal] − [Toxinfree])/[Toxintotal]) is 0.31 ± 0.05 for hanatoxin, 0.25 ± 0.04 for SGTx1, and 0.01 ± 0.01 for agitoxin-2. Data are the mean ± SEM (n = 3). Traces were normalized to the maximum absorbance in the absence of oocytes.
	Figure 5. Effect of SGTx1 mutations on partitioning into membranes. (A) Toxins fluorescence spectra in the absence (black) or presence of anionic lipid vesicles (blue). The lipid concentration was 0.4 mM. (B) Fluorescence intensity at 320 nm for WT, D24A, and R3A plotted as a function of available lipid concentration (60%). Data points are the mean ± SEM (n = 3). Smooth curves are fits of a partition function to the data with the following parameters: Kx = (1.0 ± 0.03) × 107 and F/F0max = 2.0 ± 0.02 for D24A (open triangles), and Kx = (7.0 + 0.1) × 105 and F/F0max = 1.4 ± 0.03 for R3A (open circles). Data for WT SGTx is shown for comparison (solid circles, same as in Fig. 2 F). (C) Kx values for partitioning of WT and mutant SGTx1 into anionic lipids. (D) Perturbations in partitioning into anionic membranes mapped onto the SGTx1 NMR solution structure, shown as a surface rendering with a probe radius of 1 à . Side-chain colors are as follows: light gray for |ΔΔGP| < 1 kcal/mol, pink for |ΔΔGP| = 1–1.5 kcal/mol, red for |ΔΔGP| > 1.5 kcal/mol, and purple for ΔΔGP < −1 kcal/mol. Backbone and all other residues are colored dark gray. Structure in the right panel was rotated 180° about the indicated axis. Changes in free energy were calculated as: ΔΔGP = −RTln(Kxmut/KxWT). The change in Kx value for W30A relative to WT (ΔΔGP ∼1 kcal mol−1) was estimated from depletion experiments. (E) Reversed-phase HPLC profiles of WT and mutant SGTx1 toxins present in the supernatant in the absence (black) or in the presence of 300 X. laevis oocytes (blue). Traces were normalized to the maximum in the absence of oocytes. (F) Fraction of toxin partitioned into oocytes membranes. Data are the mean ± SEM (n = 3). The dotted line corresponds to the value for wild type SGTx1. (G) Comparison between the effect of mutations on partitioning into model (gray, same as in C and D) and native membranes (black).
	Figure 6. Spectral characteristics of SGTx1 peptides. (A) λmax for WT and SGTx1 mutants free in solution (open bar) or in the presence of 2.4 mM anionic liposomes (blue for λmax values within 2 nm of that observed for WT and purple for all others). (B) Spectral characteristics of membrane-bound toxins mapped onto the NMR solution structure of SGTx1. Side-chain colors for the stereo pairs are as follows: blue for WT-like spectra, purple for red-shifted maxima, and green for W30. All other side chains and backbone are gray.
	Figure 7. Comparison of the biochemical and functional behavior of SGTx1 enantiomers. (A) Reversed-phase HPLC profiles of L-SGTx (gray) and D-SGTx (red) injected either separately or together (dotted black trace). (B) Circular dichroism spectra of L-SGTx (gray symbols) and D-SGTx (red symbols). (C) Fluorescence intensity at 320 nm plotted as a function of available lipid concentration (60%) for a 1:1 mix of POPC: POPG. Smooth red curve is the fit to the D-SGTx data and corresponds to a partition function with Kx = (1.2 ± 0.2) × 106 and F/F0max = 1.7 ± 0.03. Data for L-SGTx is shown for comparison (gray, same as in Fig. 2 F). (D) Voltage-clamp recording from an oocyte expressing the Kv2.1 channel. Currents were elicited by depolarization to −10 mV in the absence (black) or presence of 8 μM L-SGTx1 (gray) or 8 μM D-SGTx1 (red). The holding voltage was −100 mV, and the tail voltage was −50 mV. The light gray line indicates the level of zero current. Leak, background, and capacitive currents were subtracted after blocking the channel with agitoxin-2. Voltage–activation relations in the absence (black) and presence of 8 μM L-SGTx1 (gray) (E) or 8 μM D-SGTx1 (red) (F). Tail currents obtained following depolarizations were averaged for 0.2 ms beginning 2 ms after repolarization to −50 mV. In all case data points are the mean ± SEM (n = 3).
	Figure 8. Comparison of the effects of SGTx1 mutations on membrane partitioning and apparent affinity. (A) Perturbations in toxin partitioning into model membranes (ΔΔGP) from Fig. 5, C and D, plotted against changes in apparent toxin affinity for Kv2.1 channels (ΔΔGO). Blue symbols indicate the values for residues proposed to be involved in direct toxin–channel interactions. ΔΔGO = −RTln(Kdmut/KdWT), where Kd is the apparent equilibrium dissociation constant determined from experiments in which aqueous toxin concentration is varied and the resulting fraction of unbound channels measured when activating the channel using weak depolarizations (Swartz and MacKinnon, 1997a). Apparent Kd values are from Wang et al. (2004). (B) Perturbations in toxin partitioning into native membranes (ΔΔGP) from Fig. 5 G plotted against changes in apparent toxin affinity for Kv2.1 channels (ΔΔGO). Data from A is shown for comparison (gray). (C) Perturbations in apparent Kd mapped onto the SGTx1 NMR solution structure, shown as a surface rendering with a probe radius of 1 à . Side-chain colors are as follows: light gray for |ΔΔGO| < 1 kcal/mol, pink for |ΔΔGO| = 1–1.5 kcal/mol, red for |ΔΔGO| > 1.5 kcal/mol, and purple for ΔΔGO < −1 kcal/mol. Backbone and all other residues are colored dark gray (Wang et al., 2004). (D) Residues proposed to participate in direct toxin–channel interaction are colored blue, all other residues studied are colored white, backbone and unstudied residues are colored dark gray. In the right panels the structures were rotated 180° about the indicated axis.
	Figure 9. Effect of sphingomyelinase D on inhibition of Kv2.1 channels by hanatoxin. (A) Time course for effects of hanatoxin (500 nM) on the Kv2.1 channel before (gray) and after 15 min treatment of oocytes with SMaseD (blue). I is tail current amplitude at −80 mV elicited following depolarizations to −10 mV for control (gray) or −50 mV after SMaseD treatment (blue), normalized to the value in the absence of toxin. Voltage–activation relations for Kv2.1 before (B) or after SMaseD treatment (C) in the absence (open symbols) or presence (solid symbols) of 100 nM hanatoxin. Tail currents obtained following depolarizations were averaged for 0.2 ms beginning 2 ms after repolarization to −80 mV. (D) Fraction of uninhibited current plotted against test voltage at three hanatoxin concentrations (50 nM, 100 nM, 4 μM) for untreated (top, gray) and SMaseD-treated (bottom, blue) oocytes. (E) Concentration dependence for inhibition of Kv2.1 channel. Fu, the fraction of unbound channels, was estimated from fractional inhibition at negative voltages (Swartz and MacKinnon, 1997a; Phillips et al., 2005). Smooth curves are fits of Fu = (1 − P)4 where P = [Toxin]/([Toxin] + Kd) with Kd = 298 ± 14 nM for unmodified membranes (gray), and Kd = 170 ± 10 nM after SMaseD treatment (blue). Leak, background, and capacitive currents were subtracted after blocking the channel with agitoxin-2. In all case data points are the mean ± SEM (n = 3).
	Figure 10. Interaction of tarantula toxins with voltage-sensor paddles within the membrane. Illustration of SGTx1 partitioning into the membrane and interacting with S3–S4 helices. Side chain colors for left SGTx1 structure are green for hydrophobic, blue for basic, red for acidic, pink for Ser/Thr, and gray for other side chains and backbone atoms. Right SGTx1 structure shows residues important for protein–protein interactions in light blue. In both cases G32-F34 have been removed for clarity. Backbone fold of the activated/open conformation of the Kv1.2 (protein database accession code2A79) shown with only one of four voltage-sensing domains. Side chains of the outer four S4 Arg residues are shown and both S1 and S2 helices have been deleted for clarity. The contribution of the back subunit to the pore domain has also been omitted for clarity. Purple spheres are positions of potassium ions within the ion conduction pathway.

Ahern, Stirring up controversy with a voltage sensor paddle. 2004, Pubmed

Ahern, Stirring up controversy with a voltage sensor paddle. 2004, Pubmed
Andersen, Ion channels as tools to monitor lipid bilayer-membrane protein interactions: gramicidin channels as molecular force transducers. 1999, Pubmed
Bemporad, Vstx1, a modifier of Kv channel gating, localizes to the interfacial region of lipid bilayers. 2006, Pubmed
Beschiaschvili, Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes. 1990, Pubmed
Burstein, Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms. 2001, Pubmed
Calderón, Lipid composition and phospholipid asymmetry of membranes from a Schwann cell line. 1997, Pubmed
Cestèle, Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II. 1998, Pubmed
Cestèle, Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin. 2006, Pubmed
Cohen, Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes. 2006, Pubmed
Cuello, Molecular architecture of the KvAP voltage-dependent K+ channel in a lipid bilayer. 2004, Pubmed
Doyle, The structure of the potassium channel: molecular basis of K+ conduction and selectivity. 1998, Pubmed
Garcia, Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom. 1994, Pubmed , Xenbase
Gill, Calculation of protein extinction coefficients from amino acid sequence data. 1989, Pubmed
Hill, Isolation and characterization of the Xenopus oocyte plasma membrane: a new method for studying activity of water and solute transporters. 2005, Pubmed , Xenbase
Jiang, X-ray structure of a voltage-dependent K+ channel. 2003, Pubmed
Jiang, The principle of gating charge movement in a voltage-dependent K+ channel. 2003, Pubmed
Jung, Solution structure and lipid membrane partitioning of VSTx1, an inhibitor of the KvAP potassium channel. 2005, Pubmed
Kubo, Primary structure and functional expression of a mouse inward rectifier potassium channel. 1993, Pubmed , Xenbase
Ladokhin, How to measure and analyze tryptophan fluorescence in membranes properly, and why bother? 2000, Pubmed
Lee, Solution structure and functional characterization of SGTx1, a modifier of Kv2.1 channel gating. 2004, Pubmed , Xenbase
Lee, Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels. 2003, Pubmed , Xenbase
Lee, Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane. 2005, Pubmed
Lee, A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom. 2004, Pubmed
Li-Smerin, Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca2+ and K+ channels. 1998, Pubmed , Xenbase
Li-Smerin, Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel. 2000, Pubmed , Xenbase
Li-Smerin, Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels. 2001, Pubmed
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Lu, Ion conduction pore is conserved among potassium channels. 2001, Pubmed
Lundbaek, Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers. 1994, Pubmed
Miller, The charybdotoxin family of K+ channel-blocking peptides. 1995, Pubmed
Nagle, Structure of lipid bilayers. 2000, Pubmed
Patton, Some precautions in using chelators to buffer metals in biological solutions. 2004, Pubmed
Phillips, Voltage-sensor activation with a tarantula toxin as cargo. 2005, Pubmed
Polozov, Studies of kinetics and equilibrium membrane binding of class A and class L model amphipathic peptides. 1998, Pubmed
Posokhov, Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding. 2007, Pubmed
Posokhov, Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers? 2007, Pubmed
Ramu, Enzymatic activation of voltage-gated potassium channels. 2006, Pubmed
Reshetnyak, Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins. 2001, Pubmed
Rogers, Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit. 1996, Pubmed
Ruta, Functional analysis of an archaebacterial voltage-dependent K+ channel. 2003, Pubmed
Ruta, Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. 2005, Pubmed
Schmidt, Phospholipids and the origin of cationic gating charges in voltage sensors. 2006, Pubmed
Simons, Lipid sorting in epithelial cells. 1988, Pubmed
Smith, Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains. 2005, Pubmed
Stith, Quantification of major classes of Xenopus phospholipids by high performance liquid chromatography with evaporative light scattering detection. 2000, Pubmed , Xenbase
Suchyna, Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers. 2004, Pubmed
Swartz, Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels. 1997, Pubmed
Swartz, An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. 1995, Pubmed
Swartz, Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites. 1997, Pubmed , Xenbase
Swartz, Tarantula toxins interacting with voltage sensors in potassium channels. 2007, Pubmed
Takahashi, Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins. 2000, Pubmed , Xenbase
Tombola, How far will you go to sense voltage? 2005, Pubmed
Wang, Molecular surface of tarantula toxins interacting with voltage sensors in K(v) channels. 2004, Pubmed , Xenbase
Wee, SGTx1, a Kv channel gating-modifier toxin, binds to the interfacial region of lipid bilayers. 2007, Pubmed
Winterfield, A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels. 2000, Pubmed