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Sci Rep
2016 Jan 22;6:23894. doi: 10.1038/srep23894.
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Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels.
Salari A
,
Vega BS
,
Milescu LS
,
Milescu M
.
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Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b-S4 "paddle motif," which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3-S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning.
Figure 1. Paddle motifs in Cav3.1 voltage-sensor domains.(a) Sequence alignment of the paddle region of Kv2.1 and S3–S4 regions of the four Cav3.1 domains (DI–DIV)2. The conserved charged residues involved in voltage-sensing are shown in blue. Grey regions and arrows indicate the sequences that were swapped between the two channels to create paddle chimaeras. The numbers correspond to the amino acid residues within the parent channel. (b) Potassium currents for Cav3.1/Kv2.1 chimaeras. (c) Voltage-activation data and Boltzmann fits for Kv2.1 and Cav3.1/Kv2.1 paddle chimaeras. Conductance was determined from normalized tail currents. V1/2 values are (mV): 4.4 ± 0.9 (DI), 25.5 ± 0.7 (DII), −1.3 ± 1 (DIII), −87.1 ± 1 (DIV), and −5.8 ± 1 (Kv2.1). (d) Time constants (τ) of activation and deactivation determined from single exponential fits at each voltage. Data shown in (b–d) were obtained with the following voltage-step protocol: holding voltage −100 mV (Kv2.1 and DI-DIII) or −120 mV (DIV); test pulse duration 500 ms; tail voltage −60 mV (Kv2.1, DI and DIII), −10 mV (DII), or −120 mV (DIV). I/Imax (c) is the normalized tail current amplitude. In all panels, data points are mean ± SEM (n = 6).
Figure 2. ProTx-II binding sites on Cav3.1 voltage-sensors.(a) Voltage-activation data and Boltzmann fits in the absence (black) and presence (red) of 1.33 μM ProTx-II for Kv2.1 and Cav3.1/Kv2.1 chimaeras. I/Imax is the normalized tail current amplitude. In the presence of toxin, the V1/2 values are (mV): 17.7 ± 1.8 (DI), 38.8 ± 0.7 (DII), −75.4 ± 1.4 (DIV), 6.3 ± 1 (Kv2.1). A meaningful fit could not be obtained for DIII. (b) Alanine scan of DIII chimaera. Fraction of uninhibited current in the presence of 1.33 μM ProTx-II for alanine mutants (Fumut) normalized to DIII construct (Fucontrol). (c) Barium currents for wild type and D1372A Cav3.1 channels in the absence (left) and presence (right) of 1.33 μM ProTx-II. Current elicited at −40 mV is highlighted in green. (d) Voltage-activation relationships for wild type (solid circles) and D1372A Cav3.1 (open circles) in the absence (black) and presence (red) of 1.33 μM ProTx-II. Curves are Boltzmann fits with V1/2 values of −41 ± 0.4 mV and −31 ± 0.3 mV for wild type and D1372A channels, respectively, in the absence of toxin; and −34 ± 1 mV and −25 ± 1 mV, in the presence of toxin. (e) Fraction of uninhibited current of wild type (solid circles) and D1372 (open circles) Cav3.1 channels in the presence of two ProTx-II concentrations. (f) Dose-response data and fit curves for wild type (solid circles) and D1372A (open circles) channels. Data were fit with both n and Kd as free parameters (solid lines), or with Kd as free parameter and n constrained to 1 (dashed lines), 2, or 3 (dotted lines). With two free parameters, the best fit values are Kd = 1.30 ± 0.6 μM and n = 2.11 ± 0.8 for wild type, and Kd = 1.44 ± 0.3 μM and n = 1.45 ± 0.25 for D1372A. Fu was calculated at −40 mV for the wild type and −30 mV for the mutant channel. In all panels, data points are mean ± SEM (n = 6).
Figure 3. Tarantula toxins effects on Cav3.1 and Cav3.1/Kv2.1 chimaeras.(a) Sequence alignment of ProTx-II and three tarantula gating-modifier toxins. Regions of amino acid differences (sites 1–3) are highlighted in grey. Acidic residues are shown in red and hydrophobic residues in green. (b) Relative affinity of toxins for Kv2.1 and Cav3.1/Kv2.1 chimaeras. Data points are mean ± SEM (n = 6–9). (c) Alanine scan of DIII chimaera. Fraction of uninhibited current in the presence of ProTx-II, PaTx-1, GsAF-I, and GsAF-II for alanine mutants (Fumut) normalized to DIII construct (Fucontrol). Data points are mean ± SEM (n = 3–6). (d) Voltage-activation data and Boltzmann fits for wild type (solid circles) and D1372A (open circles) Cav3.1 channels, in the absence (black, same data as in Fig. 2d) and presence (red) of PaTx-1, GsAF-I, and GsAF-II. V1/2 values are −35 ± 0.7 mV, −34 ± 0.6 mV, and −38 ± 0.5 mV for the wild type channel, and −31 ± 0.6 mV, −25 ± 0.8 mV, and −25 ± 0.8 mV for the D1372A channel. Toxin concentration was 1.33 μM. Data points are mean ± SEM (n = 6).
Figure 4. Interaction of tarantula toxins with lipid vesicles.(a) Intrinsic tryptophan fluorescence emission spectra of toxins in the absence (black) and presence of lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC; pink) or a 1:1 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2oleoyl-sn-glycero-3-[phosphor-rac-(1-glycerol) (PC:PG; blue). The lipid concentration was 1.5 mM. (b) Fluorescence intensity at 320 nm plotted vs. available lipid concentration (60% of total lipids). Curves are partition function fits with Kx = 10 ± 3 × 106 and Flipids/Fsolution = 1.65 ± 0.02 for ProTx-II; Kx = 6.1 ± 2 × 106 and Flipids/Fsolution = 1.32 ± 0.02 for PaTx-I; Kx = 9.8 ± 3 × 105 and Flipids/Fsolution = 1.53 ± 0.05 for GsAF-II, Kx = 7.6 ± 2 × 106 and Flipids/Fsolution = 1.27 ± 0.02 for GsAF-I. In all panels, data points are mean ± SEM (n = 3).
Figure 5. Comparison of related toxins on Cav3.1 inhibition and membrane partitioning strength.(a) Cav3.1 inhibition vs. strength of membrane partitioning. (b) ProTx-II, PaTx-1, GsAF-I, and GsAF-II structures superimposed based on their backbone fold. Side chains of residues within sites 1–3 are colored red (acidic), green (hydrophobic), purple (serine), and yellow (glycine and alanine). (c) Toxin surface profiles, colored based on the normalized Eisenberg hydrophobicity scale64. The most hydrophobic residues are in red, following a color gradient to the most hydrophilic residues in white.
Alabi,
Portability of paddle motif function and pharmacology in voltage sensors.
2007, Pubmed,
Xenbase
Alabi,
Portability of paddle motif function and pharmacology in voltage sensors.
2007,
Pubmed
,
Xenbase
Beschiaschvili,
Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes.
1990,
Pubmed
Bladen,
Block of T-type calcium channels by protoxins I and II.
2014,
Pubmed
Bosmans,
Deconstructing voltage sensor function and pharmacology in sodium channels.
2008,
Pubmed
,
Xenbase
Bosmans,
Palmitoylation influences the function and pharmacology of sodium channels.
2011,
Pubmed
,
Xenbase
Campos,
beta-Scorpion toxin modifies gating transitions in all four voltage sensors of the sodium channel.
2007,
Pubmed
,
Xenbase
Campos,
Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels.
2008,
Pubmed
,
Xenbase
Capes,
Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.
2013,
Pubmed
,
Xenbase
Catterall,
Voltage-gated calcium channels.
2011,
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
Chagot,
Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus.
2004,
Pubmed
Chanda,
Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.
2002,
Pubmed
,
Xenbase
Chuang,
Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin.
1998,
Pubmed
,
Xenbase
Cuello,
Molecular architecture of the KvAP voltage-dependent K+ channel in a lipid bilayer.
2004,
Pubmed
Diochot,
Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis.
1999,
Pubmed
Edgerton,
Evidence for multiple effects of ProTxII on activation gating in Na(V)1.5.
2008,
Pubmed
Edgerton,
Inhibition of the activation pathway of the T-type calcium channel Ca(V)3.1 by ProTxII.
2010,
Pubmed
Eisenberg,
Analysis of membrane and surface protein sequences with the hydrophobic moment plot.
1984,
Pubmed
Frech,
A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning.
1989,
Pubmed
,
Xenbase
Goldschen-Ohm,
Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel.
2013,
Pubmed
,
Xenbase
Hanck,
Site-3 toxins and cardiac sodium channels.
2007,
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
Kalia,
From foe to friend: using animal toxins to investigate ion channel function.
2015,
Pubmed
Ladokhin,
How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?
2000,
Pubmed
Lee,
Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels.
2003,
Pubmed
,
Xenbase
Lee,
Solution structure and functional characterization of SGTx1, a modifier of Kv2.1 channel gating.
2004,
Pubmed
,
Xenbase
Lee,
A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom.
2004,
Pubmed
Lee,
Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane.
2005,
Pubmed
Lee,
Solution structure of GxTX-1E, a high-affinity tarantula toxin interacting with voltage sensors in Kv2.1 potassium channels .
2010,
Pubmed
,
Xenbase
Lee,
Solution structure of kurtoxin: a gating modifier selective for Cav3 voltage-gated Ca(2+) channels.
2012,
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
Long,
Crystal structure of a mammalian voltage-dependent Shaker family K+ channel.
2005,
Pubmed
Long,
Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment.
2007,
Pubmed
Middleton,
Two tarantula peptides inhibit activation of multiple sodium channels.
2002,
Pubmed
Mihailescu,
Structural interactions of a voltage sensor toxin with lipid membranes.
2014,
Pubmed
Milescu,
Tarantula toxins interact with voltage sensors within lipid membranes.
2007,
Pubmed
,
Xenbase
Milescu,
Interactions between lipids and voltage sensor paddles detected with tarantula toxins.
2009,
Pubmed
Milescu,
Opening the shaker K+ channel with hanatoxin.
2013,
Pubmed
,
Xenbase
Pallaghy,
A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides.
1994,
Pubmed
Pantazis,
Functional heterogeneity of the four voltage sensors of a human L-type calcium channel.
2014,
Pubmed
Park,
Studies examining the relationship between the chemical structure of protoxin II and its activity on voltage gated sodium channels.
2014,
Pubmed
Perez-Reyes,
Molecular physiology of low-voltage-activated t-type calcium channels.
2003,
Pubmed
Perez-Reyes,
Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.
1998,
Pubmed
,
Xenbase
Phillips,
Voltage-sensor activation with a tarantula toxin as cargo.
2005,
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,
Localization of the voltage-sensor toxin receptor on KvAP.
2004,
Pubmed
Ruta,
Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel.
2005,
Pubmed
Schmidt,
Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.
2008,
Pubmed
,
Xenbase
Smith,
Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains.
2005,
Pubmed
Smith,
Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation.
2007,
Pubmed
Swartz,
Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites.
1997,
Pubmed
,
Xenbase
Swartz,
Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels.
1997,
Pubmed
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
Tilley,
Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.
2014,
Pubmed
Wang,
Molecular surface of tarantula toxins interacting with voltage sensors in K(v) channels.
2004,
Pubmed
,
Xenbase
Winterfield,
A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels.
2000,
Pubmed
Xiao,
The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation.
2010,
Pubmed
