mittx  (Alomone Labs)

Journal: Frontiers in Physiology

Article Title: Piezo buffers mechanical stress via modulation of intracellular Ca2+ handling in the Drosophila heart

doi: 10.3389/fphys.2022.1003999

Figure Lengend Snippet: Pharmacological inhibition of Piezo causes heart arrest under mechanical stress. (A–C) Quantification of LDIs with GsMTx-4 applied exogenously (A, 5mM; n = 12), or when expressed from a transgenic construct using Piezo-Gal4 (B) or Tin-Gal4 (C) , under conditions of ambient and negative (−482 mmHg) pressure. Each data point represents an individual heart preparation; the y -axis shows additive time in LDI over a 60 s period for each individual preparation. p -values, unpaired t-test. (D) Schematic drawing of the GsMTx-4 transgenic constructs used in the study: secreted ( GsMTx4-FL ); non-secreted ( GsMTx4-AP ); SR-targeted ( GsMTx4-SR ). UAS = Upstream Activation Sequence; ss = signal sequence; KDEL = ER/SR translocation signal; red arrow = mature peptide cleavage site.

Article Snippet: For pharmacological inhibition of Piezo 5µM GsMTx4 (Alomone Labs) was added to HBSS.

Techniques: Inhibition, Transgenic Assay, Construct, Activation Assay, Sequencing, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

doi: 10.1016/j.jbc.2022.102326

Figure Lengend Snippet: The synthetic peptides (type II) derived from loop2 of GsMTx4 sustains the essential inhibitory effect of GsMTx4 on the stretch-activated BK channel. A , sequences for peptides designed from loop2 of GsMTx4 ( e.g. , Pept 02, Pept 03, and Pept 04). The insets on the right show the surface images of Pept 02 and Pept 03. Hydrophobic residues (Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Tyr, and Val) are in green , positive residues (Arg and Lys) are in blue , and negative residues (Asp and Glu) are in red . B , the sample traces showing the inhibition effect of Pept 02 on the mechanosensitive SAKcaC. The rights show the corresponding histogram events of channels open (O) and closed (C), which were fitted with the Gaussian function. C , time courses of normalized open probability ( P o / P o(control) ) for the synthetic Pept 02, Pept 03, and Pept 04 during peptide diffusion to the cell membranes. The control (no peptide) and the effect of Pept 01 were shown for comparison. P o was normalized to the control level ( P o / P o(control) ) following the excised inside–out patch configuration (before peptide diffusion to the cell membrane). D , the same as in B , but for the inhibition effect of Pept 03. E , bars represent the inhibition rates (τ) for Pept 02, Pept 03, and Pept 04 on the SAKcaC. The result for the full length of GsMTx4 was shown for comparison. Taus were obtained with the standard single-exponential function in D . F , the inhibited effects (inhibited [%]) for Pept 02, Pept 03, and Pept 04 on SAKCaC. The effect of GsMTx4 was shown for comparison. The cartoons on the left of B and C represent the peptide (Pept 02 in B and Pept 03 in C ) backfilled in the pipette with tension ( P m ) automatically formed upon membrane deformation following the excised inside–out patch-clamp configuration. Time points were measured from the onset of backfilling for peptides in the extracellular side of the cell membrane (see the Experimental procedures section). Peptide concentrations used were 5 μM. Membrane potentials ( V m ) were held at −80 mV. ∗ p

Article Snippet: The peptide GsMTx4 was purchased from Alomone Labs.

Techniques: Derivative Assay, Inhibition, Diffusion-based Assay, Transferring, Patch Clamp

Journal: The Journal of Biological Chemistry

Article Title: Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

doi: 10.1016/j.jbc.2022.102326

Figure Lengend Snippet: The synthetic Pept 02 inhibits SAKcaC activity in a dose-dependent manner. A and B , the sample traces showing the dose-dependent inhibition effects for GsMTx4 ( A ) and Pept 02 ( B ) on the SAKcaC with 0 nM (control, upper panel ), 50 nM ( middle panel ), and 100 nM ( lower panel ) peptides applied from the extracellular side of the cell membrane. The corresponding total histogram events of channel open (O) and closed (C) states were presented on the right . Membrane potential ( V m ) was held at −50 mV. Traces were obtained 25 min following the backfilling. C , dose–response curves for Pept 02 and GsMTx4 at −50 mV. The solid lines are fit to Hill equation with disassociation constants ( K d ) summarized in G . Note, Y -axis represents the normalized P o ( P o(Pept) / P o(maxi) ). Hill cofactors were 1.8 ± 0.52 for Pept 02 and 1.6 ± 0.47 for GsMTx4. D , statistical comparison of the K d between Pept 02 and GsMTx4 determined from the Hill equation fits in C . Data points at each concentration represent three to eight determinations. E , P o – V relationships for the effects of Pept 02 and GsMTx4 on the SAKcaC. The peptide concentrations used were 50 nM. The solid lines are fits to the standard Boltzmann function: P o = P o(max) /[1 + exp(−( V m − V 1⁄2 )/ K )], where V 1⁄2 represents the voltage required for half of the maximum channel opening and K represents the slope factor. The V 1⁄2 and K −1 obtained were −91.5 ± 1.36 mV and 25.1 ±1.16 for control (no peptide), −23.7 ± 1.06 mV and 22.9 ± 1.11 for Pept 02, and −47.2 ± 0.97 mV and 24.5 ± 1.63 for GsMTx4. F , statistical comparison of △ V 1/2 shifted by Pept 02 ( red ) versus GsMTx4 ( gray ). V 1/2 was obtained from E . △ V 1/2 = V 1/2(Pept) − V 1/2(Control) . n = 6 to 9 per group. ∗∗ p

Article Snippet: The peptide GsMTx4 was purchased from Alomone Labs.

Techniques: Activity Assay, Inhibition, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

doi: 10.1016/j.jbc.2022.102326

Figure Lengend Snippet: Structural features for synthetic short peptides and the proposed gating modes for SAKcaC. A and B , the surface structures for type I (Pept 01, A ) and type II peptides (only Pept 02 are presented, B ). Left ( side views ), where Trp 1 forms the hydrophobic protrusion at the peptide head and the positive residues ( e.g. , Lys 2 /Arg 12 in Pept 01 and Lys 2 in Pept 02) form a positively charged protrusion directly following Trp1. Middle ( top views ); right ( bottom views ). The side views are presented based on the orientation of GsMTx4 with trypotophan (Trp) facing the intracellular side (from the outside to the inside) of the cell membrane, where Trp was assumed for GsMTx4 to be involved in peptide penetration into the cell membrane. C , the simplified peptide structural models showing the predominant features of both types of peptides and the supposed peptide action with the lipid bilayer (below). Note that both types of peptides contain a hydrophobic head (indicated as “H”) formed with Trp, a positively charged protrusion directly following H (indicated as “P”), and a peptide cap at the ending (indicated as “C”). D , MD simulation revealed that peptide has the ability to insert into the lipid bilayer with the hydrophobic head ( green ) down and induce membrane deformation. Snapshots were taken from a free simulation of type I (Pept 01) with a POPC bilayer membrane at the time points indicated. E , proposed gating modes modulated by membrane tension (MT) ( left ) and short peptide ( middle and right ) for SAKcaC: MT first pulls SAKcaC gate opening through the interaction element between STREX-lipid, where STREX domain in BK channel targets the plasma membrane by palmitoylation of two cysteine residues ( blue spheres ) ( 39 , 40 ); under hyperpolarized/resting conditions ( middle ): the negative charges accumulated in the inner monolayer could attract electrostatically the positively charged protrusion in peptides ( purple ) upon its portioning into the cell membrane, thus facilitating peptide moving down ( move inward ) along the electrochemical gradients. The peptide was placed at a deep position to interact with both inner and outer monolayers, where the inner cell membrane has been suggested to interact with STREX in BK channel ( blue spheres ) ( 39 , 40 ) and induce a stronger membrane deformation (also see inset above in the middle ), to close the channel gate firmly; Under depolarized state (right) , the positively charged protrusion in peptides ( purple ) was repelled/driven back ( move outward ) by membrane potential. Alternatively, the outward electrostatic forces may also prevent the absorption of peptides from the extracellular side into the lipid bilayer. Thus, the peptide was placed at a shallow position to interact with the outer monolayer (also see the inset above on the right ) and induced less membrane deformation. The schematic of the inset above ( left ) illustrates the topology of SAKcaC, where the STREX-exon located between RCK1 and RCK2 domains in the intracellular C terminus is attached to the plasma membrane by palmitoylation of the two cysteines (highlighted in green ) ( 39 ). The snapshots in the insets above ( middle and right ) represent the peptide (Pept 01)–POPC bilayer interaction under the hyperpolarized/resting ( middle ) versus depolarized ( right ) conditions (see the Experimental procedures section) ( 12 ). Note, that the positively charged protrusions ( blue ) interacted directly with the negatively charged carbonyl oxygen atoms in the monolayer in both modes. The gating mechanism modes for SAKcaC are primarily based on the spring model proposed for BK channel ( 50 , 58 ). BK, big potassium; MD, molecular dynamics; POPC, 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine; RCK, regulator of K + conductance; SAKcaC, stretch-activated big potassium channel.

Article Snippet: The peptide GsMTx4 was purchased from Alomone Labs.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

doi: 10.1016/j.jbc.2022.102326

Figure Lengend Snippet: Summary of the effects of type I, type II, and the mutant peptides on SAKcaC. A , comparisons of the inhibitory effects for type I, type II, and the mutant peptides on SAKcaC. The inhibitory effects were normalized to GsMTx4. Normalized inhibition (%) = [Inhibited (peptide) /Inhibited (GsMTx4) ] × 100. B , comparisons of diffusion rates for type I, type II, and the mutant peptides on SAKcaC. The diffusion rates were normalized to GsMTx4. Diffusion rates (%) = (Diffusion rate (peptide) /Diffusion rate (GsMTx4) ) × 100. (n = 4–11). p

Article Snippet: The peptide GsMTx4 was purchased from Alomone Labs.

Techniques: Mutagenesis, Inhibition, Diffusion-based Assay

Journal: The Journal of Biological Chemistry

Article Title: Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

doi: 10.1016/j.jbc.2022.102326

Figure Lengend Snippet: The synthetic peptide 01 (Pept 01), derived from loop2 and loop3 in neuropeptide GsMTx4, inhibits the stretch-activated BK (SAKca) channel (SAKcaC). A , sequence of Pept 01. B , schematic illustrates the topology of mechanosensitive BK channel (SAKcaC). The mechanosensory domain, STREX-exon, is located between RCK1 and RCK2 domains in BK C terminus and targets the plasma membrane via palmitoylation of two cysteine residues in the STREX-exon (highlighted in blue spheres ) ( 39 , 40 ). The gray spheres represent the two Ca 2+ -binding sides that are located in the RCK1 and RCK2 domains. C , typical current traces showing the inhibition effect of Pept 01 on mechanical-sensitive SAKcaC. The times above indicate the time points upon peptide backfilled in the pipette (see the Experimental procedures section). Membrane potential ( V m ) was held at −80 mV. The total histogram events of channels open (O 1 and O 2 ) and closed (C) were fitted by the Gaussian function on the right . D , time courses of normalized open probability ( P o / P o(control) ) for control (no peptide) and during Pept 01 diffusion to the cell membranes. The effect of the whole length of peptide GsMTx4 was shown for comparison. E , bars represent the inhibited effect (inhibited [%]) for Pept 01 on SAKcaC compared with GsMTx4. The inhibited (%) was obtained 20 min later following backfilling. F , the inhibition rates (τ) of Pept 01 on the SAKcaC compared with GsMTx4. τ were obtained with the standard single-exponential function fits in C . Membrane potentials ( V m ) were held at −80 mV. The insets beside A represent the 3D model of Pept 01 established from the known structure of GsMTx4 (Protein Data Bank code: 1TYK ). Time points were measured from the onset of backfilling for peptides in the extracellular side of the cell membrane (see the Experimental procedures section). Peptide concentrations used were 5 μM. The cartoons on the left in C represent the pipette backfilled with Pept 01 with tension ( P m ) automatically formed upon membrane deformation following the excised inside–out patch-clamp configuration. The arrows beside the traces represent the level of the channel closed. ∗∗ p

Article Snippet: The peptide GsMTx4 was purchased from Alomone Labs.

Techniques: Derivative Assay, Sequencing, Binding Assay, Inhibition, Transferring, Diffusion-based Assay, Patch Clamp

Journal: Cells

Article Title: The Role of AlphαSynuclein in Mouse Models of Acute, Inflammatory and Neuropathic Pain

doi: 10.3390/cells11121967

Figure Lengend Snippet: Acute cold nociception and related gene expression. ( A ) nociceptive threshold in the cold plate test. ( n = 11) ( B ) TRPM8 mRNA expression in the DRGs and the paws of wild type and Snca knock-out mice as assessed by RT-PCR analysis (relative gene expression of TRPM8 in relation to GAPDH). ( n = 3–4) ( C ) Western blot analysis of Pgp9.5 and TRPM8 in the paws of wild type and Snca knock-out mice; the blots show representative results and the diagrams the densitometric analysis of all samples ( n = 4). *** p

Article Snippet: The blots were then incubated overnight at 4 °C with primary antibodies against alphαSynuclein (BD Bioscience, Franklin Lanes, Evansville, IN, USA, 1:500), p-p38, p-38, p-p42/44, p42/44, (all Cell Signalling Technology, Boston, MA, USA, 1:250), COX-2 (Santa Cruz Biotechnology, Heidelberg, Germany, 1:500), CB1R, (Cayman, Biomol, Hamburg, Germany 1:500), DOR (Abcam, Cambridge, UK, 1:200), Pgp9.5 (Abcam, Cambridge, UK, 1:200) and TRPM8 (alamone labs, Jerusalem, Israel, 1:100) in blocking buffer.

Techniques: Expressing, Knock-Out, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot