Opticalcin 1-based t26 site mutated cell-penetrating peptides and applications

Abstract

The application provides a T26 site mutation based on opicalcin 1 penetrating peptide and application. With OpiCal as a template, the T26 residue is replaced with basic amino acids arginine (R) and lysine (K) through site-directed mutagenesis to construct mutants T26R and T26K. The physicochemical properties, cell penetration efficiency and RyR binding energy of the mutants and OpiCal are systematically compared by comprehensively using molecular docking and dynamics simulation, fluorescence labeling and calcium imaging technologies. The results show that T26R with increased positive charge density shows significantly enhanced cell penetration efficiency compared with OpiCal, and has no obvious cytotoxicity in the concentration range of 10-60 µM; at the same time, T26R still retains the specific binding ability to RyRs, realizes the bidirectional functional regulation of the cell penetrating peptide and RyR targeting molecule. The application reveals that a single amino acid residue T26 can bidirectionally and independently regulate the intracellular delivery efficiency and target binding affinity of scorpion venom peptide, and provides a theoretical basis and experimental support for rationally designing a new type of polypeptide with high efficient penetration and precise targeting ability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides a modified membrane-penetrating peptide derived from scorpion venom, specifically a membrane-penetrating peptide based on the T26 site mutation of opicalcin1 and its application. Background Technology

[0002] Ryline receptors (RyRs) are the largest known ion channel proteins. Under normal physiological conditions, their mechanism of action involves mediating the release of Ca from the sarcoplasmic reticulum (SR). 2+ RyRs play a crucial role in the excitation-contraction coupling process of skeletal muscle. Dysfunction of RyRs typically stems from alterations in the physiological function of calcium release from the SR (synaptic systolic receptor), which are triggered by various cytoplasmic signaling molecules through phosphorylation modification or direct binding to channel proteins, such as calmodulin (CaM) and Fkbp12.6, which significantly affect channel gating properties. RyR dysfunction can also lead to various diseases. For example, specific amino acid mutations in the transmembrane region of RyR1 are key factors in serious diseases such as malignant hyperthermia (MH) and central axonal disease (CCD); while mutations in the N-terminus, C-terminus, and central domain of RyR2 can lead to catecholamine-sensitive polymorphic ventricular tachycardia (CPVT). However, clinical management of diseases caused by RyR dysfunction still primarily relies on prevention, and effective therapeutic drugs are lacking.

[0003] In recent years, Calcin family peptides derived from scorpion venom have demonstrated therapeutic potential due to their unique properties. Previous studies have shown that Calcin peptides not only penetrate cell membranes efficiently but also exhibit high affinity for ryne-based receptors (RyRs), making them promising candidate molecules for treating RyR dysfunction-related diseases. Notably, among the more than ten reported Calcin family members, Opicalcin1 (OpiCa1), isolated and identified from African scorpion venom, exhibits the highest activity, with an affinity for RyRs of 0.3 kDa. In contrast, its homolog Opicalcin2 (OpiCa2) shows approximately ten times lower affinity for RyRs than Opicalcin1. These two peptides differ only in the 26th residue of their amino acid sequences, and this single amino acid variation is what leads to the significant decrease in binding affinity.

[0004] Currently, our understanding of the role of T26 is limited to ligand-receptor binding. Whether and how this residue affects the cell penetration ability of Calcin-like peptides—a property equally crucial for their intracellular drug action—remains unexplored. Furthermore, whether rational single-site mutagenesis of this hotspot site can decouple or bidirectionally regulate these two functions of penetration and binding has not yet been investigated. Filling this knowledge gap could establish a new paradigm for designing precisely tailored peptide therapies. Summary of the Invention

[0005] This invention addresses the aforementioned problems by first proposing the hypothesis that the T26 residue acts as a bidirectional molecular switch simultaneously regulating OpiCa1 membrane permeability and RyR targeting specificity. To verify this hypothesis, T26 was systematically replaced with the basic amino acids arginine (R) and lysine (K) to increase positive charge and potentially enhance cell entry ability. By integrating computer molecular docking / kinetic simulations and in vitro experiments on H9C2 cardiomyocytes, the physicochemical properties, cytotoxicity, cellular uptake efficiency, and effects on RyR-mediated Ca2+ targeting were comprehensively evaluated. 2+ The release of functions has an impact.

[0006] The results showed that the mutant T26R, produced by replacing the neutral hydrophilic threonine with a positively charged basic amino acid (preferably arginine), significantly increased the net positive charge of the peptide at physiological pH, thereby directly enhancing the electrostatic interaction between the peptide and the negatively charged cell membrane. This resulted in T26R exhibiting significantly superior cell penetration efficiency compared to OpiCa1 and OpiCa2, with no significant cytotoxicity in the 10–60 µM concentration range. Molecular docking and molecular dynamics simulations showed that the binding free energy of T26R to RyR2 was significantly higher than that of OpiCa1. Although T26R possessed the strongest cell penetration ability, it induced Ca26R in H9C2 cardiomyocytes. 2+ The release amount was lower than that of OpiCa1, which further confirms the decoupling of cell penetration activity and targeting activity at the functional level.

[0007] This invention confirms that T26 is the structural basis for the high-affinity binding of OpiCa1 and RyRs, and also discovers that the mutant peptide T26R successfully decouples the two functions of the peptide—cell membrane penetration efficiency and target binding activity. This discovery provides a new strategy for designing peptide drugs with both efficient delivery and precise targeting capabilities.

[0008] Based on the above research, the technical solution to be protected by this invention is as follows: In a first aspect, the present invention provides a transmembrane peptide based on a T26 mutation of opicalcin1, wherein the threonine residue at the T26 site of the opicalcin1 protein is replaced with a positively charged basic amino acid.

[0009] Preferably, the basic amino acid is selected from arginine or lysine, and more preferably arginine.

[0010] Furthermore, the membrane-penetrating peptide based on the T26 mutation of opicalcin1 was synthesized into a polypeptide using the Fmoc solid-phase synthesis method, with the following steps: (1) Synthesized using 2-chlorotriphenylmethyl chloride resin with a degree of substitution of 1.1 mmol / g, the resin was first swollen in DMF for 50–70 minutes, then the first Fmoc-protected C-terminal amino acid (containing 10 molar excess of DIEA) was attached to the resin and capped with methanol; subsequently, deprotection was performed in two steps using 20% ​​piperidine / DMF solution. The product was washed after detection with Kaiser reagent.

[0011] (2) Subsequently, Fmoc-protected amino acids, HBTU and DIEA were added for condensation reaction. After detection and washing with negative Kaiser reagent, the deprotection, detection, washing and condensation steps were carried out in sequence from C-terminus to N-terminus until the Fmoc protecting group of the last amino acid was removed.

[0012] (3) After washing and drying, the product was treated with a cutting solution containing 95% TFA to lyse the product; the lysate was dried with nitrogen, washed with ether and evaporated to dryness; then disulfide bonds were formed by DMSO oxidation, and Ellman's reagent was used to monitor until the oxidation was complete.

[0013] (4) The crude product was dissolved in H2O / acetonitrile. The elution time of the target peak was determined by HPLC analysis. The preparation was carried out using a C18 reversed-phase chromatography system. The detection conditions were as follows: detection wavelength 220 nm, flow rate 15 mL / min, injection volume 20 mL, column temperature 25℃, mobile phase: 0.1% TFA aqueous solution and 0.1% TFA acetonitrile solution. A small amount of the component was collected for mass spectrometry confirmation and purity detection. After passing the test, the product was freeze-dried.

[0014] In a second aspect, the present invention provides the application of the membrane-penetrating peptide based on the T26 mutation of opicalcin1 described above in the preparation of a reagent targeting the ryne receptor (RyRs) binding.

[0015] In a third aspect, the present invention provides a binding agent targeting the reniform receptor, comprising the membrane-penetrating peptide based on the T26 mutation of opicalcin1 described above, or using it as the sole active ingredient.

[0016] In a fourth aspect, the present invention provides the application of a membrane-penetrating peptide based on a T26 mutation in opicalcin1 in the preparation of a therapeutic drug for RyR dysfunction-related diseases.

[0017] In a fifth aspect, the present invention provides the application of the above-mentioned transmembrane peptide based on the T26 mutation of opicalcin1 in the preparation of a vector that simultaneously possesses transport and target binding functions, wherein the target is preferably RyR2.

[0018] Preferably, the active ingredient of the carrier includes a membrane-penetrating peptide, or a membrane-penetrating peptide is the sole active ingredient.

[0019] Furthermore, the carrier has the functions of drug delivery, nuclear localization sequence delivery, probe delivery, or nanoparticle delivery.

[0020] Because cell-penetrating peptides can enter cells without causing any damage and can be successfully used as carriers for siRNA, nucleotides, macromolecular drugs, proteins, etc., they have been widely used in the field of novel drug carriers.

[0021] In addition, in cell imaging, cell-penetrating peptides (CPPs) are used to promote internalization by capturing labeled peptide probes at target sites. The high efficiency of CPPs makes them a promising tool in the imaging field. In nuclear localization, to improve nuclear localization efficiency, CPPs with nuclear localization sequences (NLSS) can be directly or indirectly linked to DNA or gene vectors to facilitate nuclear localization.

[0022] In addition, due to the pH gradient between the tumor environment and the physiological environment, cell-penetrating peptides can exert a controlling effect in the tumor microenvironment by linking with nanoparticles, such as through acid degradation cross-linking, removal of the protective layer, or enzymatic degradation. When cell-penetrating peptides are linked with drug-containing nanoparticles, the therapeutic efficiency of the nanoparticles is improved and the drug toxicity is reduced.

[0023] In a sixth aspect, the present invention provides a transport and target binding functional carrier, using the cell-penetrating peptides described above as active ingredients.

[0024] The role and effect of invention This study used OpiCa1 as a template and constructed mutants T26R and T26K by replacing the T26 residue with the basic amino acids arginine (R) and lysine (K) through site-directed mutagenesis. Using molecular docking and kinetic simulation, fluorescent labeling, and calcium imaging techniques, the physicochemical properties, cell penetration efficiency, and RyR binding energy of the mutants and OpiCa1 were systematically compared. The results showed that T26R with increased positive charge density exhibited significantly enhanced cell penetration efficiency compared to OpiCa1, and showed no significant cytotoxicity in the concentration range of 10–60 µM. Simultaneously, T26R retained its specific binding ability to RyRs, achieving bidirectional functional regulation as both a cell-penetrating peptide and a RyR-targeting molecule. This study preliminarily reveals that a single amino acid residue, T26, can bidirectionally and independently regulate the intracellular delivery efficiency and target binding affinity of scorpion venom peptides, providing a theoretical basis and experimental support for the rational design of novel peptides with both high penetration efficiency and precise targeting capabilities. Attached Figure Description

[0025] Figure 1The physicochemical properties and membrane penetration potential of the peptides are shown. (A) Sequence alignment of OpiCa1, OpiCa2, T26K, and T26R (* indicates mutation sites; C3-C17, C10-C21, and C16-C17 are three disulfide bonds). (B) Biochemical and biophysical properties of OpiCa1, OpiCa2, T26K, and T26R, including pI, Mw, membrane penetration efficiency, electrostatic potential surface, and GRAVY. (C) pH-related net charge and positive charge count analysis of OpiCa1, OpiCa2, T26K, and T26R. (The horizontal axis represents the pH range from 0 to 13.35, and the vertical axis represents the total net charge of the peptide at the corresponding pH.) (D) Hydrophobic plot of key residues of OpiCa1, OpiCa2, T26K, and T26R (Blue: hydrophobic; red: hydrophilic, the horizontal axis represents the types of amino acids present in the four peptides, and the vertical axis represents the hydrophilicity of each amino acid. Positive values ​​indicate hydrophilicity, and negative values ​​indicate hydrophobicity).

[0026] Figure 2 The diagram shows the interaction analysis of OpiCa1, OpiCa2, T26R with RyR. (A) Schematic diagram of the binding of Calcin family members with RyR; (B) Free energy binding of OpiCa1, OpiCa2, and T26R with RyR1 and RyR2 (x-axis represents RyR acceptor type, y-axis represents ΔiG kcal / mol); (C) Detailed interaction analysis of OpiCa1, OpiCa2, and T26R with RyR1 and RyR2 (inner page: intermolecular hydrogen bonds in the interaction); (D) Number of interactions (hydrogen bonds, salt bridges) between OpiCa1, OpiCa2, and T26R with RyR1 and RyR2, respectively.

[0027] Figure 3Molecular dynamics simulations of OpiCa1, OpiCa2, T26R, RyR1, and RyR2 are shown: (A) Comparison of the initial and final conformational RMSFs of OpiCa1, OpiCa2, T26R with those of RyR1 and RyR2. The S6 helices of OpiCa1, OpiCa2, T26R, and RyR1 are different colors at the start of the simulation (OpiCa1: blue; OpiCa2: red; T26R: purple), while gray indicates the position of all complexes after 100 nanoseconds of simulation. (Displacement from the initial position to the final position is indicated by arrows in the simulation). (B) SASA fluctuation curves of OpiCa1, OpiCa2, T26R, RyR2, and RyR1 during a 300 nanosecond simulation. The x-axis represents time in nanoseconds (ns), and the y-axis represents SASA (in square nanometers). The yellow curve corresponds to OpiCa1-RyR2, the purple curve corresponds to OpiCa2-RyR2, and the green curve corresponds to T26R-RyR2. (C) RMSD fluctuation curves of OpiCa1, OpiCa2, T26R, RyR2, and RyR1 during a 300-nanosecond simulation. The x-axis represents time (ns), and the y-axis represents RMSD (nm). Yellow represents OpiCa1-RyR2, purple represents OpiCa1-RyR2, and green represents T26R-RyR2. (D) The number of hydrogen bonds formed during the 300-nanosecond simulation. The x-axis represents the interval of hydrogen bond number, and the y-axis represents the relative frequency of each interval. The orange, purple, and green bars correspond to OpiCa1-RyR2, OpiCa2-RyR2, and T26R-RyR2, respectively. (E) OpiCa1, OpiCa2, T26R, and RyR2 docked in Gibbs energy. Under 300 nanosecond simulation and projection, both complex models reached their most stable states. The x-axis represents RMSD (nm), and the y-axis represents Rg (nm). The color scale represents the relative Gibbs free energy (kcal / mol), with the blue / cyan region representing the most stable conformation. (F) Solvent accessible surface area (SASA) fluctuation curves during the 300 nanosecond simulation. The x-axis represents time (nanoseconds), and the y-axis represents SASA (square nanometers). The blue, pink, and gray curves correspond to OpiCa1-RyR1, OpiCa2-RyR1, and T26R-RyR1, respectively. (G) RMSD fluctuation curves during the 300 nanosecond simulation. The x-axis represents time (ns), and the y-axis represents RMSD (nm). The blue, pink, and gray curves correspond to OpiCa1-RyR1, OpiCa2-RyR1, and T26R-RyR1, respectively. (H) Number of hydrogen bonds formed during the 300 nanosecond simulation. The x-axis represents the range of hydrogen bond numbers, and the y-axis represents the relative frequency of each range.The blue, pink, and gray bars correspond to OpiCa1-RyR1, OpiCa2-RyR1, and T26R-RyR1, respectively. (I) Gibbs free energy landscape plots of the OpiCa1, OpiCa2, T26R, and RyR1 complexes during a 300-nanosecond simulation, and projections of the two complex models in their most stable states. The x-axis represents RMSD (nm), and the y-axis represents Rg (nm). The color scale indicates the relative Gibbs free energy (kcal / mol).

[0028] Figure 4 The effects of the T26 mutation on membrane penetration efficiency were shown: (AC) Cytotoxicity analysis (n=6, cell viability of H9C2 cells treated with different concentrations of OpiCa1, OpiCa2, and T26R); (DE) Statistical analysis of peptide membrane penetration using fluorescence microscopy (30 μM, 2 hours; H9C2 cardiomyocyte cytoskeleton stained red with phalloidin, cell nuclei stained blue with DAPI, and single-stained portions of the three peptide drugs labeled with FITC green fluorescence under a 200× silver microscope) (****P<0.0001) (n=3); (F) Quantitative analysis of membrane penetration efficiency of OpiCa1, OpiCa2, and T26R using fluorescent microparticle readers at 0 μM, 10 μM, 30 μM, and 60 μM (n=6; X-axis: FITC-labeled CPP concentration (μM), Y-axis: intracellular FITC). Fluorescence excitation; gray: control; pink: OpiCa2; blue: OpiCa1; yellow: T26R); (GH) flow cytometry statistics (n=6; gray: control; pink: OpiCa2; blue: OpiCa1; yellow: T26R).

[0029] Figure 5 The effects of OpiCa1, OpiCa2, and T26R on intracellular Ca2+ in H9C2 cells were demonstrated. 2+ Effects of levels: (A) Representative fluorescence microscopy images showing intracellular Ca2+ levels in H9C2 cells after incubation with OpiCa1, OpiCa2, and T26R at 30 μM for 30 min. 2+ Changes (n=3). (B) H9C2 cells Ca after treatment with OpiCa1, OpiCa2 and T26R at 30 μM. 2+ Statistical analysis of fluorescence value changes (*P<0.01) (n=3). (C) OpiCa1, OpiCa2 and T26R were incubated at concentrations of 0 μM, 10 μM, 30 μM and 60 μM for 0.5 h, and then intracellular Ca in H9C2 cells was quantitatively analyzed using a fluorescence spectrometer. 2+ Level. (*P<0.05, **P<0.01, ****P<0.0001) (n=6). (D) Flow cytometry plotting intracellular Ca2+ Peak values ​​were plotted at 0, 30, 60, and 120 minutes after treatment with 30 μM peptide. (E) Quantitative analysis of intracellular Ca2+ by flow cytometry after treatment with 30 μM peptide. 2+ The levels were tested at 0 minutes, 30 minutes, 60 minutes, and 120 minutes respectively. Detailed Implementation

[0030] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0031] I. Materials and Methods 1.1. Physicochemical properties and molding efficiency The amino acid sequences of OpiCa1, OpiCa2, and their mutant peptides (T26R and T26K) were analyzed using the Allpeptide peptide calculator (https: / / www.allpeptide.com / ). The amino acid sequences of the four peptides were entered into the prediction box, and the disulfide bond pairing information formed by three natural folds (C3-C17, C10-C21, C16-C32) was simultaneously imported for analysis. The system-generated analysis report included parameters such as the peptide's molecular formula, molecular weight, number of amino acids, isoelectric point (pI), net charge at pH=7.0, and the arithmetic mean of average hydrophilicity and hydrophobicity. Subsequently, the cell penetration characteristics of the four peptides were predicted using the CellPPD online tool (https: / / webs.iiitd.edu.in / raghava / cellppd) to verify the differences in their cell penetration properties.

[0032] 1.2. Polypeptide Synthesis The peptide was synthesized using the Fmoc solid-phase synthesis method with a 1.1 mmol / g degree of substitution (DOC) of 2-chlorotriphenylmethyl chloride resin. First, the resin was swollen in DMF for 60 minutes. Then, the first Fmoc-protected C-terminal amino acid (containing a 10-fold molar excess of DIEA) was attached to the resin, followed by end-capping with methanol. Next, deprotection was performed in two steps using a 20% piperidine / DMF solution. After detection with a positive Kaiser's reagent, the product was washed. Subsequently, the Fmoc-protected amino acid, HBTU, and DIEA were added for a condensation reaction. After detection with a negative Kaiser's reagent and washing, the deprotection, detection, washing, and condensation steps were repeated sequentially from the C-terminus to the N-terminus until the Fmoc protecting group of the last amino acid was removed. After washing and drying, the product was cleaved for 180 minutes with a cleavage buffer containing 95% TFA. The lysate was dried under nitrogen, washed with diethyl ether, and evaporated to dryness. Disulfide bonds were then formed using DMSO oxidation, monitored with Ellman's reagent until complete oxidation. For purification, the crude product was dissolved in H₂O / acetonitrile, and the elution time of the target peak was determined by HPLC analysis. Preparation was performed using a C18 reversed-phase chromatography system (detection wavelength 220 nm, flow rate 15 mL / min, injection volume 20 mL, column temperature 25°C, mobile phase: 0.1% TFA aqueous solution and 0.1% TFA acetonitrile solution). A small amount of the fraction was collected for mass spectrometry confirmation and purity determination. After passing the test, the product was freeze-dried. Finally, a small amount of the final product was subjected to MS molecular weight identification and HPLC purity verification. The qualified white powder peptide was sealed and stored at -20°C.

[0033] 1.3. Cytotoxicity Detection H9C2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and passaged in a 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments. Cells were then cultured at 5 × 10⁻⁶ cells / year. 3 In a 96-well plate, the seeding density was 5 × 10⁶ cells / well. 3 Cells were cultured in 100 μL of culture medium per well for 24 h until cell adhesion was achieved. The supernatant was discarded, and the cells were washed with PBS buffer. Six replicates were then added to each well containing final concentrations of 10 μM, 30 μM, 60 μM, and 100 μM of OpiCa1, OpiCa2, and the mutant peptide T26 R (purity ≥95%). A blank control group was also included, containing only serum-free culture medium. All cells were incubated for 2 h. After incubation, 100 μL of CCK-8 reagent was added to each well, and incubation continued for 30 min. The absorbance (OD) at 450 nm was measured using a microplate reader.

[0034] 1.4 Verification of membrane penetration efficiency 1.4.1 Cell penetration efficiency of peptides at different concentrations H9C2 cells cultured in serum-containing medium were digested, counted, and then seeded evenly into 96-well plates. The plates were incubated at 37°C with 5% CO2 for 24 hours. Cell morphology was confirmed to be good after observation under an inverted microscope, and the cell count reached 5 × 10⁶ cells per well. 3 For cell incubation, the old culture medium in the wells was aspirated and discarded, and the cells were washed once with DMEM. Subsequently, FITC-labeled OpiCa1, OpiCa2, and T26R were added at concentrations of 0 μM (control), 10 μM, 30 μM, and 60 μM, respectively. A negative control group was also established, with only culture medium added. After co-incubation for 2 hours (n=6), the supernatant was discarded, and the cells were washed three times with 0.05%–0.1% Tween-20 TBST to remove free peptides from the cell surface. Intracellular fluorescence intensity was then detected using a microplate fluorescence analyzer.

[0035] 2.4.2 Cell penetration efficiency of different peptides at different time points H9C2 cells were cultured in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. After confirming good growth by observing cell morphology under an inverted microscope, the cell count was determined to be 5 × 10⁻⁶ cells / well. 5 For each well, the old culture medium was aspirated and discarded, and the cells were then washed once with DMEM. Subsequently, three FITC-labeled peptides were added to the wells at a concentration of 30 μm at 30 min, 60 min, and 120 min, with three replicates per group. The cells were washed three times with TBST to remove free peptides from the cell surface, then digested with trypsin and resuspended in PBS. The average fluorescence intensity was detected by flow cytometry to quantify membrane penetration efficiency.

[0036] 2.4.3 Cardiac cell Ca 2+ Changes in concentration Cells were cultured in 6-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. Cell morphology was observed using an inverted microscope to verify good cell growth and a cell count of 5 × 10⁶ cells / well. 5 After each well, the old culture medium was aspirated and discarded, and the cells were then washed once with DMEM. Three FITC-labeled peptides at a concentration of 30 μM were then added, and the cells were incubated for 120 min, with three replicates per group. Cells were washed three times with TBST to remove free peptides from the cell surface, and then the 6-well plates were placed under a fluorescence microscope. The fluorescence intensity was then quantitatively analyzed using ImageJ software. The relative intracellular Ca2+... 2+The concentration calculation formula is as follows: ΔF / F0 = (F - background) / F0 (F represents the measured fluorescence intensity, F background represents the background fluorescence intensity, and F0 is the baseline fluorescence intensity).

[0037] 1.5. Obtaining Receptor and Ligand Models The 3D structures of RyR1 (PDB ID: 7T65) and RyR2 (PDB ID: 7U9Z) were obtained from the RCSB protein database (https: / / www.rcsb.org / ). Based on previous studies that clearly demonstrated the binding of OpiCa1 to the S6 helix of RyR, we precisely defined the docking receptor range as the segments constituting the S6 helix in RyR1 (residue numbers: 4901-4968) and RyR2 (residue numbers: 4836-4896). The structures of OpiCa1, OpiCa2, and T26R mutants were modeled using homology on the SWISS-MODEL server, with Impercalcin as the template. The final model was performed in GROMACS 2022.4 using the steepest descent algorithm to minimize energy within 5000 steps to remove stereo collisions and obtain a stable conformation.

[0038] 1.6. Molecular docking and molecular dynamics simulation 1.6.1. Molecular docking Molecular docking was performed on the GRAMM-X docking server (https: / / gramm.compbio.ku.edu / ). After docking, the PDBePISA protein interface analysis tool (https: / / www.ebi.ac.uk / pdbe / pisa / ) was used to systematically calculate the binding free energy and analyze the interaction interfaces of all generated complex conformations; this tool can assess the contribution of hydrogen bonds and salt bridge interactions formed in the complexes to the binding free energy. Based on this, we ranked the conformations according to their binding free energy values ​​and selected the representative model with the lowest energy and the most reasonable conformation for subsequent detailed analysis. PyMOL molecular visualization software was used for three-dimensional visualization analysis of the complex structures, visually identifying and verifying the specific hydrogen bond network and key salt bridge connections formed at the acceptor-ligand interface. This allowed us to elucidate the molecular basis of their interactions, ensuring that our analytical conclusions are both statistically significant and structurally rational.

[0039] 1.6.2. Molecular Dynamics Membrane Simulation The complex structure with the optimal binding mode from previous molecular docking results was selected as the initial conformation. The topology and coordinate files compatible with the AMBER force field were constructed using the CHARMM-GUI (https: / / charmm-gui.org / ) online platform. Subsequently, GROMACS was used to perform 300-nanosecond molecular dynamics simulations on the selected complex system, outputting the simulated trajectory. Several key physical parameters were calculated and analyzed to evaluate the dynamic behavior and stability of the system, including the root mean square deviation (RMSD) characterizing the overall structural drift, the number and occupancy of hydrogen bonds (HB) used to quantify specific intermolecular interactions, and the solvent-accessible surface area (SASA) reflecting the degree of hydrophobic burial on the protein surface. By comparing the parameters of OpiCa1, OpiCa2, and T26R docking with RyR, the structural differences and dynamic change rules exhibited by them during receptor binding were revealed.

[0040] 1.7. Ca in cardiomyocytes 2+ Determination of content 1.7.1. Measurement using a fluorescent microplate reader The density after digestion and counting is 5 × 10⁻⁶ per well. 3 H9C2 cells were seeded into black 96-well plates at a moderate density and cultured at 37°C with 5% CO2 for 24 hours. The culture medium was discarded, and the cells were gently washed once with DMEM, followed by the addition of 100 μL of peptide solutions containing 0, 10, 30, and 60 μL of OpiCa1, OpiCa2, or T26R, and cultured continuously for 30 minutes. After incubation, the cells were washed once with DMEM, and 100 μL of Fluo-4 AM working solution was added to each well, and incubated in the dark for 30 minutes. Subsequently, the cells were cultured with Ca-free... 2+ Cells were washed three times with PBS, and the fluorescence intensity of each well was detected using a fluorescent microplate reader to reflect intracellular Ca2+. 2+ level.

[0041] 1.7.2. Flow cytometry analysis of intracellular Ca 2+ Dynamic changes H9C2 cells were seeded into 6-well plates and cultured for 24 hours until the cell count reached 5 × 10⁶ cells / well. 5 After each well was filled, the culture medium was discarded, and the cells were washed with PBS, followed by treatment with 30 μM of each peptide at 0, 30, 60, and 120 minutes. After treatment, the cells were washed once with DMEM and incubated in the dark for 30 minutes with Fluo-4 AM. The cells were then treated with Ca... 2+ The probe was washed three times with free PBS to remove it, then digested with trypsin and resuspended. Intracellular Ca2+ was quantitatively analyzed at different time points by flow cytometry to detect mean fluorescence intensity.2+ Dynamic changes in the horizontal plane.

[0042] 1.8. Data Processing and Statistical Results Analysis All experimental data were derived from at least three independent biological replicates, with three technical replicates for each assay. Sample size (n) is indicated in the corresponding legend. Outliers were removed using the 3σ rule; background signal was subtracted for fluorescence and spectroscopic assays to ensure accuracy. Results are expressed as mean ± standard error of mean (SEM). Multiple comparisons were performed using one-way ANOVA and Tukey post-hoc tests after validating data normality (Shapiro-Wilk test) and variance homogeneity (Lvene test). Significance was defined as P < 0.05 and labeled as P < 0.05, P < 0.01, and *P < 0.001. All data analysis, statistical tests, and plotting were performed using GraphPad Prism 9.0. Molecular docking and dynamic simulation data (combining free energy, RMSD, SASA, hydrogen bond number, and Gibbs free energy maps) were analyzed using AutoDock Vina, GROMACS 2021, and PyMOL 2.5 built-in modules to calculate the mean difference and standard deviation of 100 nanosecond trajectories.

[0043] 2. Results 2.1 Investigation of the physicochemical properties and membrane penetration potential of peptides Both OpiCa1 and OpiCa2 are derived from the venom of the African scorpion *Pandinus imperator*. They are full-length peptides of 33 amino acids, differing by only a single amino acid at position 26: OpiCa1 has a threonine residue (T26), while OpiCa2 has an alanine residue (A26). Figure 1 A). Regarding amino acid distribution, compared to the classic cell-penetrating peptide TAT, OpiCa1 and OpiCa2 exhibit sequence characteristics characteristic of cell-penetrating peptides, containing a large number of basic amino acids. Using OpiCa1 as a template, we mutated the T26 residue to the basic amino acids arginine (T26R) and lysine (T26K), respectively. This mutation resulted in a change in molecular weight and increased the isoelectric point of the mutant peptide (…). Figure 1 B). Furthermore, cell-penetrating peptide (CPP) predictions showed that its membrane penetration efficiency was higher than that of OpiCa1 and OpiCa2, but lower than that of classic cell-penetrating peptides, which is closely related to the ratio of basic amino acids (B). Figure 1 B). All peptides carry a strong positive charge at low pH values, and the charge gradually decreases as pH increases, turning negative at high pH values. This indicates that they are all basic peptides with an isoelectric point greater than 7, and carry a positive charge at the physiological pH of 7.4. Figure 1(BC). Among them, the T26R mutant has the highest isoelectric point and the largest positive charge density in physiological environments, indicating that it has a stronger binding affinity for negatively charged molecules and greater tolerance to alkaline microenvironments (1B). The net charge of these four peptides gradually decreases with increasing pH, while maintaining a strong positive charge (approximately 15-20) at physiological pH (≈7.4). This sustained positive charge provides a key electrostatic driving force for the peptides to interact with and penetrate negatively charged cell membranes. The charge distribution of OpiCa1 and OpiCa2 is almost identical across the entire pH range, indicating highly consistent electrostatic properties. In contrast, T26R and T26K deviate slightly from the OpiCa1 peptide, maintaining a higher net positive charge under alkaline conditions, indicating enhanced tolerance to alkaline microenvironments (see BC). Figure 1 C). These four peptides exhibit highly similar distribution patterns of hydrophilic and hydrophobic residues, with hydrophobic residues (blue bars) being the dominant component, accompanied by a limited number of strongly hydrophilic residues (red bars), forming a typical amphiphilic structure. The hydrophobic domain serves as the structural basis for peptide insertion into the hydrophobic core of the cell membrane, while the hydrophilic domain maintains sufficient solubility in the aqueous environment, preventing abnormal peptide aggregation. Figure 1 D).

[0044] 2.2 Structural analysis of the interaction between the peptide and RyR Molecular docking simulations show that OpiCa1, OpiCa2, T26R, and T26K all interact with the four S6 helices in the transmembrane region of the central channel domain in the RyR1 and RyR2 tetramers. Figure 2 A). However, binding energy analysis showed that, except for T26K, the binding energies of OpiCa1, OpiCa2, and T26R to RyR were negative, indicating a stable interaction between these peptides and RyR. Figure 2 B). In contrast, T26K exhibited a positive binding energy, indicating that it did not form a stable binding conformation; therefore, this mutant was excluded from further studies. Molecular binding analysis further revealed that the binding of OpiCa1, OpiCa2, and T26R to RyR was primarily mediated by HB interactions. Specifically, OpiCa1 and RyR2 formed 21 HBs, including two pairs of key residues, T26 and E4878 and E4882 of RyR2, which together promoted complex stability. In contrast, A26 in OpiCa2 did not participate in hydrogen bond formation. Instead, K30 formed a hydrogen bond with E4882 in RyR2, while H6 formed a hydrogen bond with E4878 in RyR2. In this binding mode, the T26R mutant formed a hydrogen bond between R26 and L4859. When bound to RyR1, OpiCa1, OpiCa2, and T26R form only 8, 11, and 9 HBs, respectively, indicating potential differences in their binding modes with RyR2 (see [link to article]). Figure 2 C, D).

[0045] 2.3 Molecular dynamics simulations between peptides and RyRs Molecular dynamics simulations elucidated the stability and interaction dynamics of the peptide-RyR complex. Comparative analysis of the simulated trajectories showed that binding to OpiCa1, OpiCa2, or T26R induces an asymmetric conformational shift in the RyR tetramer structure. Figure 3 This is consistent with the symmetric regulation pattern during ligand-induced channel opening. Analysis of the solvent accessible surface area (SASA) of these complexes showed that all systems reached equilibrium after the initial relaxation period (approximately 10–50 nanoseconds). 300-nanosecond molecular dynamics (MD) simulations showed that the solvent accessible surface area (SASA) of all peptide-RyR complexes gradually decreased and stabilized after 100 nanoseconds, indicating that the binding interfaces of each system reached a steady state during the simulation (see [link to simulation]). Figure 3 B, 3F). For the RyR1-constrained system, the root mean square deviation (RMSD) of all complexes increases within the first 100 nanoseconds, then plateaus. T26R-RyR1 exhibits a slightly higher RMSD in the subsequent 200 nanoseconds than the other two groups, indicating lower overall binding stability (see B, 3F). Figure 3 C). Intermolecular HBs analysis showed that OpiCa2-RyR1 had the most stable HB-binding state and a significantly higher probability of high HB counts, while T26R-RyR1 exhibited the weakest HBs interaction, confirming that the T26R single-point mutation impaired the peptide's specific HB-binding ability to RyR1 (see [link to article]). Figure 3 D). Gibbs Energy Landscape (GEL) analysis shows that OpiCa2-RyR1 has the deepest low-energy well, with the largest free energy span of the three groups, reaching 0.0–20.0; its low-energy region is highly concentrated and the energy gradient is steep, giving it the best conformational stability. Although T26R-RyR1 has the same free energy span, it exhibits a wider distribution and a slower gradient in the low-energy region, corresponding to a significantly shallower actual well depth. OpiCa1-RyR1 has the smallest free energy span (0.0–17.5), with a dispersed low-energy region and the shallowest low-energy well (see...). Figure 3 E). For systems entering the RyR2 direction, the RMSD of all complexes increased within the first 50 nanoseconds, followed by a plateau. The RMSD of OpiCa2-RyR2 increased significantly within the subsequent 250 nanoseconds, indicating its lower binding stability with RyR2 (see E). Figure 3 G). HBs analysis showed that OpiCa1-RyR2 had the most stable HB binding and a significantly higher incidence of high-quantity HBs, while T26R-RyR2 could hardly form stable intermolecular HBs, confirming that the T26R mutation failed to optimize the peptide's specific HB binding ability to RyR2 (see G). Figure 3 GEL analysis showed that OpiCa1-RyR2 had the deepest low-energy well, with the largest free energy range of 0.0–20.0; its low-energy region was highly concentrated and the energy gradient was steep, giving it the best conformational stability. Although T26R-RyR2 had the same free energy range, it exhibited a widely distributed low-energy region, a gentle gradient, and a significantly shallower well depth. OpiCa2-RyR2 had the smallest free energy range (0.0–17.5), with a dispersed low-energy region and the shallowest low-energy well (H). Figure 3 I). Overall, the OpiCa1-RyR2 complex exhibited the best binding stability across all testing systems. Quantitative analysis of binding kinetic parameters (RMSD, SASA, HBs, and ΔG_bind) is summarized in Table S6, providing a numerical basis for the observed stability differences among the complexes.

[0046] 2.4 Verification of the penetration efficiency of the T26 residue-regulated peptide on the H9C2 cell membrane After treating H9C2 cells with OpiCa1, OpiCa2, and T26R at concentrations of 10, 30, 60, and 100 μM, cytotoxicity assays showed that none of the three peptides at these concentrations induced significant cytotoxicity in H9C2 cells (Figures 4A-C). Membrane penetration efficiency assays at peptide concentrations of 10, 30, and 60 μM showed that T26R had significantly higher membrane penetration efficiency than OpiCa1 and OpiCa2 at all three tested concentrations (Figure 4D). After treating H9C2 cells with 30 μM FITC-labeled OpiCa1, OpiCa2, and T26R for 2 hours, the intracellular fluorescence intensity of the T26R group was significantly higher than that of the OpiCa1 and OpiCa2 groups (Figures 4E-F). Flow cytometry results showed that after treatment of H9C2 cells with FITC-labeled peptides for 30 min, 60 min, and 120 min, the T26R group exhibited the highest cell membrane penetration efficiency at each time point (Figure 4G-H). These results indicate that the T26R single-point mutation can significantly enhance the cell membrane penetration efficiency of peptides into H9C2 cardiomyocytes without producing significant cytotoxicity within a concentration range of 10–100 μM.

[0047] 2.5 Differential Regulation of Intracellular Ca by OpiCa1, OpiCa2, and T26R in H9C2 Cardiomyocytes 2+ steady state After incubating H9C2 cells with 30 μM OpiCa1, OpiCa2, and T26R for 30 min, fluorescence microscopy imaging showed significant differences in Fluo-4 fluorescence signals among the treatment groups. Figure 5A). Quantitative fluorescence intensity analysis showed that the intracellular Fluo-4 fluorescence intensity in the OpiCa1 treatment group was significantly higher than that in the OpiCa2 group and the T26R group ( Figure 5 B). H9C2 cells were further incubated for 30 min with OpiCa1, OpiCa2, and T26R at concentration gradients of 10, 30, and 60 μM, respectively. Fluo-4 quantitative PCR results showed that intracellular Ca2+ in all peptide-treated groups was significantly reduced. 2+ The levels of intracellular Ca2+ were significantly increased; and at the same test concentration, the OpiCa1 treatment group induced a significant increase in intracellular Ca2+. 2+ The fluorescence intensity was significantly higher than that of the OpiCa2 group and the T26R group ( Figure 5 C). Further targeting of intracellular Ca after treatment with 30 μM peptide. 2+ Signal temporal gradient analysis and flow cytometry results showed that intracellular Ca2+ levels were higher in all peptide-treated groups. 2+ The fluorescence signals all showed a transient upward trend, reaching a peak at 30 min after treatment and gradually decaying within 120 min. Figure 5 D); The fluorescence signal time progression curve is completely consistent with this dynamic change trend ( Figure 5 E). The above results confirm that OpiCa1 induces intracellular Ca2+ in H9C2 cells. 2+ The release capacity was significantly stronger than that of OpiCa2 and T26R. A single-point mutation in T26R could significantly downregulate peptide-mediated intracellular Ca2+. 2+ Release effect.

[0048] 3. Discussion The Calcin family represents a unique class of bioactive molecules with dual functions: intrinsic cell penetration and highly specific targeting of RyR. Within this family, OpiCa1 and OpiCa2 are the most active members, differing by only one amino acid at position 26, yet exhibiting a 10-fold difference in binding affinity. This divergence highlights the T26 site as a key focus for understanding the structural basis of receptor-ligand interactions. This study employed site-directed mutagenesis to investigate whether this residue regulates membrane translocation and target binding. Our approach is based on the established principle that sufficient basic residue density and a balanced hydrophobicity (30%–50%) are core determinants of CPP efficiency. Our structural basis is supported by the fact that the S6 helix of the RyR pore domain mediates channel gating, and previous cryogenic EM studies have localized IpCa to this specific region. We further elucidated how T26 substitution alters these interactions by utilizing molecular docking, a robust computational tool, to predict binding postures and affinities. Unlike traditional CPP-based delivery systems, which often suffer from poor target specificity or inherent cytotoxicity, cadherin peptides offer the unique advantages of high receptor selectivity and low toxicity. Our results show that T26 acts as a regulatory hub, differentially modulating the balance between membrane permeability and RyR binding. Specifically, T26R mutations indicate that enhancing the net positive charge at this site can significantly improve cellular uptake, even if it leads to a functional trade-off in binding affinity. This finding provides a strategic framework for the rational design of next-generation peptide therapies that require both high delivery efficiency and precise intracellular targeting.

[0049] To investigate the role of the T26 residue, we designed two mutants with basic amino acid substitutions: T26K and T26R. Systematic analysis of their physicochemical properties showed that the T26R mutation significantly increased the peptide's net positive charge and Ip, resulting in a higher predicted membrane penetration rate than T26K. Both mutants, as well as the wild-type peptide, met the established criteria for CPPs, including well-separated amphipathic domains and stable disulfide bonds. Experimental results confirmed that the charge at the T26 site is a key determinant of cellular uptake; replacing neutral threonine with positively charged arginine (T26R) directly enhanced electrostatic interactions with the negatively charged cell membrane. Therefore, T26R showed significantly higher penetration efficiency than OpiCa1 and OpiCa2 in both fluorescent microplate and flow cytometry analyses. Notably, our results showed that the cell penetration rate of the T26R mutant was related to the basic amino acid substitution. 2+ There is a clear inverse correlation between release. Although T26R is superior in membrane translocation efficiency, its induced Ca2+ release... 2+ Mobilization levels were significantly lower than OpiCa1. This functional decoupling strongly suggests that the observed Ca... 2+The fluctuations are mediated by specific RyR interactions, rather than nonspecific membrane disruption, as the latter typically increases proportionally to peptide uptake. Furthermore, secondary Ca... 2+ Oscillations typically indicate impaired membrane integrity or metabolic stress. However, our CCK-8 assay for Ca... 2+ No significant cytotoxicity was observed at imaging concentrations (10–60 μm), confirming that these signals represent physiological responses in healthy cells, rather than passive Ca2+. 2+ Leakage. In summary, these findings validate that the membrane permeability of OpiCa1 can be effectively enhanced through single-residue charge engineering at the T26 site, while its target binding affinity is determined by unique structural requirements.

[0050] Interestingly, the enhanced cell penetration efficiency of the T26R mutant did not translate into a corresponding increase in its targeting activity. To elucidate the structural basis of this observation, we compared the binding stability of OpiCa1, OpiCa2, and T26R with RyR1 and RyR2 using molecular docking and 300-nanosecond MD simulations. T26K was excluded from these advanced analyses due to its poor membrane permeability and unfavorable binding free energy, failing to form a stable peptide-protein complex. Through multidimensional evaluation, combining RMSD, SASA, intermolecular hydrogen bonding ability, and ΔG_bind, we demonstrated that the OpiCa1-RyR2 complex exhibited the highest binding affinity and structural stability across all testing systems. At the atomic level, the T26 residues in OpiCa1 form key HBs, with the E4878 and E4882 residues of RyR2 providing strong anchoring within the binding pocket. In contrast, OpiCa2 lacks these specific interactions, and the T26R bond positional variation results in a less ideal binding conformation. These structural findings are highly consistent with our functional data: although T26R reached the highest intracellular concentration, it induced the release of Ca2+ in H9C2 cardiomyocytes. 2+ The difference was significantly lower than that of OpiCa1. This divergence confirms that the T26 site acts as a key determinant, differentially regulating the relationship between intracellular delivery and target binding. The partial decoupling of these two functions at the T26 site highlights the inherent complexity of simultaneously optimizing the delivery efficiency and biopotency of scorpion venom-derived peptides.

[0051] In summary, the most important finding of this study is that the T26 residue acts as a key site, regulating both the membrane permeability and RyR binding affinity of OpiCa1. Our results indicate that rational mutagenesis at this single site can be used to differentially tune different functional modules within a single molecular scaffold. Furthermore, comparison with the homologous peptide OpiCa2 reveals the high structural sensitivity of this site; even subtle changes in the polarity or side chain size between neutral residues are sufficient to induce significant changes in binding stability, reflecting the subtle functional evolution of the calcinin family. Our comprehensive approach, combining 300-nanosecond molecular dynamics simulations with in vitro cell assays, shows that while increasing the net positive charge (T26R) significantly enhances membrane translocation, it simultaneously alters the specific interactions required for optimal RyR binding. This partial decoupling of delivery efficiency from bioefficiency facilitates peptide uptake and Ca26 binding. 2+ The observed inverse correlation between releases provides a structural explanation. While the T26 site does not alter the fundamental binding position of RyR, its regulatory role in the anchoring peptide is crucial. Taken together, this work provides key experimental evidence and a conceptual framework for developing efficient, targeted CPP drug delivery systems. This research also lays a solid foundation for future studies on RyR channel gating kinetics and granulation reticulum calcium processing, ultimately guiding the balanced delivery and targeting performance optimization of calcification-derived therapeutics.

[0052] In summary, our study identifies the T26 residue as a key site with a dissociative and differential regulatory role in the intracellular delivery efficiency and target-specific performance of OpiCa1. These findings not only advance the mechanistic understanding of the calcinin peptide family but also establish key design principles for the development of bifunctional peptide therapeutics. While our results indicate that precise mutagenesis can decouple and rebalance membrane penetration and targeting activity, we also acknowledge several limitations that warrant further investigation. First, although existing data support a partially functionally decoupled model, reverse phenotypic mutants (such as T26D or T26F) are still lacking. Furthermore, to fully map the regulatory landscape of this residue, further experimental characterization of a broader panel of mutants (including T26K / E) is needed, demonstrating that the absolute functional independence of these two properties has not yet been definitively established. Second, although our calculations and functional assays strongly suggest a difference in RyR binding affinity, direct biophysical measurements, such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) using purified RyR domains, are crucial for accurately quantifying the thermodynamic parameters of these interactions. Third, while flow cytometry provides a powerful quantification of cellular uptake, future studies utilizing confocal Z-stacking and endocytosis pathway inhibitors will further elucidate the specific mechanisms of transmembrane translocation. Finally, the in vivo delivery efficiency and therapeutic potential of these mutant peptides in disease models remain to be systematically elucidated. Overall, this work provides a structural framework and crucial experimental evidence for optimizing calcium acid-based delivery systems, laying the foundation for next-generation peptide drugs targeting RyR dysfunction-related diseases.

[0053] 4. Conclusion This invention systematically analyzes the functional role of a single residue, T26, in the scorpion venom-derived peptide OpiCa1. By combining computational modeling with cellular function assays, we demonstrate that mutations at the T26 site mediate partial functional decoupling of two key peptide properties: cell penetration efficiency and RyR targeting affinity. Specifically, the T26R mutation increases the peptide's positive charge density, leading to a significantly enhanced membrane translocation compared to OpiCa1 and OpiCa2. However, this increased penetration is accompanied by intracellular calcium... 2+The reduced release potency strongly suggests that the cellular response is mediated by specific RyR interactions, rather than nonspecific membrane disruption. Mechanistically, our 300-nanosecond MD simulations show that this decoupling stems from changes in the interfacial interaction network, particularly disruption of the native hydrogen bond network and reduced tightness of the binding interface. These findings establish the T26 residue as a key regulatory hub for differentially modulating the delivery and activity profile of calcified peptides. This work lays the foundation for structure-based engineering strategies that can selectively modulate cellular delivery and bioactivity to address RyR-related pathologies, such as cardiac arrhythmias. Future research will focus on mapping a more comprehensive structure-activity profile through broader amino acid substitutions (e.g., T26D, T26F) and further refining the therapeutic potential of these engineered peptides using direct biophysical methods to quantify binding thermodynamics.

[0054] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A transmembrane peptide based on a T26 mutation in opicalcin1, characterized in that, Replace the threonine residue at the T26 site of the opicalcin1 protein with a positively charged basic amino acid.

2. The membrane-penetrating peptide based on the T26 mutation of opicalcin1 according to claim 1, characterized in that, The basic amino acid is selected from arginine or lysine.

3. The membrane-penetrating peptide based on the T26 mutation of opicalcin1 according to claim 1 or 2, characterized in that, The peptides were synthesized using the Fmoc solid-phase synthesis method, and the steps are as follows: (1) Synthesized using 2-chlorotriphenylmethyl chloride resin with a degree of substitution of 1.1 mmol / g, the resin was first swollen in DMF for 50-70 minutes, then the first Fmoc-protected C-terminal amino acid of DIEA containing a 10-fold molar excess was attached to the resin and capped with methanol; subsequently, deprotection was performed in two steps using 20% ​​piperidine / DMF solution. The product was washed after detection with Kaiser reagent; (2) Subsequently, Fmoc-protected amino acids, HBTU and DIEA were added for condensation reaction. After detection and washing with negative Kaiser reagent, the deprotection, detection, washing and condensation steps were carried out in sequence from C-terminus to N-terminus until the Fmoc protecting group of the last amino acid was removed. (3) After washing and drying, the product was treated with a cutting solution containing 95% TFA to lyse the product; the lysate was dried with nitrogen, washed with ether and evaporated to dryness; then disulfide bonds were formed by DMSO oxidation, and Ellman's reagent was used to monitor until the oxidation was complete. (4) The crude product was dissolved in H2O / acetonitrile, and the elution time of the target peak was determined by HPLC analysis. The preparation was carried out using a C18 reversed-phase chromatography system. The detection conditions were as follows: detection wavelength 220 nm, flow rate 15 mL / min, injection volume 20 mL, column temperature 25℃, mobile phase: 0.1% TFA aqueous solution and 0.1% TFA acetonitrile solution. A small amount of the component was collected for mass spectrometry confirmation and purity testing. After passing the test, the product was freeze-dried.

4. The use of the membrane-penetrating peptide based on the T26 mutation of opicalcin1 as described in claim 1 or 2 in the preparation of a reagent targeting the rynyl alkaloid receptor (RyRs) binding.

5. A binding agent targeting the rennet receptor, characterized in that, This includes the transmembrane peptide based on the T26 site mutation of opicalcin1 as described in claim 1 or 2, or as the sole active ingredient.

6. The use of the membrane-penetrating peptide based on the T26 mutation of opicalcin1 as described in claim 1 or 2 in the preparation of a therapeutic drug for RyR dysfunction-related diseases.

7. The application of the membrane-penetrating peptide based on the T26 mutation of opicalcin1 as described in claim 1 or 2 in the preparation of a vector that simultaneously possesses transport and target binding functions.

8. The application according to claim 7, characterized in that: in, The active ingredient of the carrier includes a membrane-penetrating peptide, or a membrane-penetrating peptide is the sole active ingredient.

9. The application according to claim 7, characterized in that: in, The carrier has the functions of drug delivery, nuclear localization sequence delivery, probe delivery, or nanoparticle delivery.

10. A carrier integrating transport and target combination functions, characterized in that, The cell-penetrating peptide described in claim 1 or 2 is used as the active ingredient.

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