CN117069820B - Voltage-gated Nav1.4 mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a voltage-gated Nav1.4 mutant and application thereof. The mutant is obtained by mutating a wild rat voltage-gated Nav1.4 amino acid sequence, wherein the mutation is that the 378 th tyrosine of the wild rat voltage-gated sodium channel Nav1.4 amino acid sequence is subjected to site-directed mutation to form phenylalanine; the amino acid sequence of the wild rat voltage-gated sodium channel Nav1.4 is shown as SEQ ID NO. 1. Compared with the body, the mutant of the invention shows that the voltage activation characteristic drifts towards the depolarization direction, the voltage deactivation characteristic drifts towards the hyperpolarization direction on the electrophysiological characteristic, is consistent with the electrophysiological characteristic of the typical HOKPP disease, and can provide a receptor model for preparing the medicine for treating the hypokalemia type periodic paralysis.

Description

A voltage-gated Nav1.4 mutant and its application

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a voltage-gated Nav1.4 mutant and an application thereof.

Background Art

Voltage-gated sodium (Nav) channels are key structures for generating excitability in cell membranes and are responsible for the initiation and propagation of action potentials in excitable cells. Dysfunction of Nav channels can cause a variety of diseases. Currently, more than 1,000 disease-related gene mutations have been found in human Nav channels.

Nav1.4 is highly expressed in skeletal muscle, accounting for more than 90% of Nav channels in adult muscle tissue. It is key to regulating the initiation and proliferation of action potentials in skeletal muscle contraction. Mutations in the gene SCN4A encoding the α subunit of Nav1.4 are associated with a variety of hereditary channel diseases, including periodic paralysis hyperkalemic (HYPP), periodic paralysis hypokalemia (HOKPP2), sodium channel myotonia (SCM), potassium-aggravated myotonia and congenital myasthenic syndrome (CMS16). More than 70 disease-related mutation sites have been found in human Nav1.4. For example, the mutation of Gln at position 270 on S5 of DI on the α subunit of Nav1.4 to Lys can cause congenital paramyotonia, the replacement of Thr at position 704 on S5 of DII by Met can cause hyperkalemic periodic paralysis, and the mutation of Cys at position 1209 on PD of DIII to Phe can cause myasthenic syndrome.

Nav1.4-related diseases are complex and diverse. Although more and more mutation sites of Nav1.4-related diseases have been discovered and analyzed, there are few reports overall, and further research is needed on the pathogenic mechanism of the disease. In addition, there is still a lack of effective treatment for diseases related to Nav1.4 gene mutations. Therefore, it is necessary to construct an expression model of Nav1.4 mutants in vitro, which is of great significance for revealing the structural and functional relationship of Nav1.4, clarifying the molecular mechanism of disease occurrence, and drug screening and development.

Summary of the invention

In view of this, the object of the present invention is to provide a voltage-gated Nav1.4 mutant and application thereof.

The present invention provides the following technical solutions:

A voltage-gated Nav1.4 mutant, wherein the mutant is derived from a mutation of the amino acid sequence of a wild-type rat voltage-gated sodium channel Nav1.4, wherein the mutation is a site-directed mutation of the tyrosine at position 378 of the amino acid sequence of the wild-type rat voltage-gated sodium channel Nav1.4 to phenylalanine; the amino acid sequence of the wild-type rat voltage-gated sodium channel Nav1.4 is shown in SEQ ID NO.1.

The present invention provides a gene encoding the mutant.

Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

The invention provides a recombinant vector carrying the gene.

The present invention provides a recombinant strain of the gene.

The present invention also provides use of the voltage-gated Nav1.4 mutant in preparing a drug for treating hypokalemic periodic paralysis.

The beneficial effects of the present invention are:

The mutant of the present invention is derived from the wild-type rat voltage-gated sodium channel Nav1.4 amino acid sequence (as shown in SEQID NO.1), and the tyrosine at position 378 of the wild-type rat voltage-gated sodium channel Nav1.4 amino acid sequence is site-directedly mutated to phenylalanine. The experimental results show that the mutant exhibits a voltage activation characteristic drifting toward the depolarization direction and a voltage inactivation characteristic drifting toward the hyperpolarization direction in terms of electrophysiological characteristics compared with the original body, which is consistent with the electrophysiological characteristics of typical HOKPP2 diseases, and can provide a receptor model for the preparation of drugs for treating hypokalemic periodic paralysis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a gel electrophoresis of the PCR product of mutation 378, where M is a marker and 1 is a sample;

FIG2 is an activation curve diagram of the wild-type and 378-position mutant voltage-gated sodium channel Nav1.4;

FIG. 3 is a graph showing the inactivation curves of the wild-type and 378-position mutant voltage-gated sodium channel Nav1.4.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

The invention provides a voltage-gated sodium channel Nav1.4 mutant, wherein the mutant is derived from a wild-type rat voltage-gated sodium channel Nav1.4 amino acid sequence mutation, wherein the tyrosine at position 378 of the wild-type rat voltage-gated sodium channel Nav1.4 amino acid sequence is site-directedly mutated to phenylalanine.

In the present invention, the amino acid sequence of the wild-type rat voltage-gated sodium channel Nav1.4 is shown in SEQ ID NO.1,

In the present invention, the nucleotide sequence of the voltage-gated Nav1.4 mutant is shown in SEQ ID NO.2.

Example 1 Preparation of Nav1.4 single-site mutants

(1) Obtaining the mutant Y378F gene

The voltage-gated Nav1.4 was subjected to a single-point mutation. The recombinant plasmid of the wild-type rat voltage-gated sodium channel Nav1.4 was used as a template. The PCR technique was used to introduce a single mutation at position 378 of the Nav1.4 amino acid sequence. The primers were:

Y378F-IR:TGGTGTAGCC AAA GTTGGGGT (the underlined base is the mutant, as shown in SEQ ID NO. 3).

Y378F-IF: ACCCCAAC TTT GGCTACACCA (the underlined base is the mutant, as shown in SEQ ID NO.4).

PCR reaction system: The PCR mutation system in the present invention preferably includes: 6 μL of 16 ng/μL wild-type Nav1.4 DNA, 1 μL each of mutant primers IF and IR (1 μM), 25 μL of Q5 High-Fidelity DNA Polymerase, and ddH2O supplementation system to 50 μL.

PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at 60°C for 10 s, extension at 68°C for 3 min, 25 cycles; extension at 68°C for 5 min; and storage at 4°C.

(2) Perform agarose gel electrophoresis on the PCR product to determine whether the mutant gene is successfully constructed

Accurately weigh 0.3g agarose, heat and dissolve it with 30mL 1×TAE Buffer, add 2.5μL 4S Red Plus nucleic acid stain when cooled to 60℃, shake well and slowly pour it into the gel tank with the sample comb inserted. After it solidifies, put the gel and the support plate into the electrophoresis tank, with one end of the sample well close to the negative pole of the electrophoresis tank, and add 1×TAE buffer to cover the gel. Unplug the sample comb and add 7μL DNA Marker to the first well for comparison of the size of the target gene. Add 2μL DNA loading buffer to the sample, mix well and add the treated sample from the second well, start the electrophoresis instrument, and set the program to 90V for 30min. Analyze the gel after electrophoresis with a gel imager and save the picture.

The standard for successful construction of mutant genes: the brightest part of the target band is compared with the marker 10000bp band, and if they are close, the construction is successful.

As can be seen from Figure 1, the mutant Y378F gene was successfully constructed.

Example 2 Expression and purification of mutants

(1) Transformation of PCR products

After obtaining the PCR product, the obtained PCR product was digested with Dpn I enzyme to obtain a mutant recombinant vector. In this embodiment, the digestion temperature was 37° C. and the time was 1 hour.

Take 10 μL of PCR product, add it to 100 μL of competent cell suspension in ice bath, let it stand on ice for 30 minutes, heat shock the transformation product at 42℃ for 90 seconds, and quickly cool it on ice for 2 minutes. Add 900 μL of liquid LB medium without antibiotics to the EP tube, culture at 37℃, 200r/min for 60 minutes, centrifuge at 5000r/min for 3 minutes, discard 900 μL of supernatant, suspend the remaining bacterial liquid and apply it to the plate, and after the bacterial liquid is completely absorbed by the culture medium, culture it at 37℃ overnight for about 14 hours to obtain the recombinant strain.

(2) Extraction and restriction digestion of mutant plasmid

The obtained recombinant strain was expanded and cultured in LB liquid culture medium, and the plasmid was extracted from the bacterial liquid using the kit Fast PurePlasmidMini Kit. The specific experimental steps were carried out according to the instructions.

The extracted plasmid will be divided into a portion for sequencing, and the rest can be digested with the corresponding restriction endonuclease Not I to form a linearized plasmid. In the present invention, the enzyme digestion reaction system preferably includes: 15 μg of mutant DNA, 20 μL of 10×H Buffer, 20 μL of 0.1% BSA, 10 μL of restriction endonuclease Not I, and 200 μL of ddH ₂ O supplement system. The reaction is carried out in a constant temperature water bath at 37°C for 2 hours. After the enzyme digestion reaction is completed, the product needs to be purified. The process uses the kit MiniBESTDNA Fragment Purification KitVer.4.0 and is performed according to the instructions.

(3) Preparation of mutant RNA

In vitro transcription was performed using the in vitro transcription kit mMESSAGE mMACHINETM T7 Kit, and the purified linearized plasmid DNA was used as a template for in vitro transcription to prepare the mutant cRNA. The in vitro transcription reaction system was as follows: 1.5 μg of mutant linearized DNA, 2 μL of 10×Reaction Buffer, 10 μL of 2×NTP/CAP, 1 μL of GTP, 2 μL of Enzyme Mix, and 20 μL of RNase-free Water supplement system. The reaction was carried out in a constant temperature water bath at 37°C for 4 hours. After the in vitro transcription reaction was completed, the transcription product was purified using the Purification of Transcription Reactions MEGAclear ^TM Kit, and the operation was carried out according to the instructions.

Example 3 Detection of mutant activity

(1) Microinjection of mutant RNA

Select sexually mature female African clawed frogs, place them on ice for anesthesia for 1 hour, dissect and obtain their oocytes, use 0.5g~1g/L collagenase for oscillation digestion and enzymatic hydrolysis, select frog eggs with distinct poles, smooth and rounded, and microinjection. The volume of injected cRNA is 30~50nL/cell, and the amount of cRNA injected into each oocyte is not less than 0.6ng. After injection, the oocytes are stored in an ND96 solution containing antibodies and incubated for 3~6 days at 17℃ and 34% humidity to detect the expression of wild-type and mutant Nav1.4.

(2) Activity detection

The activity of mutants was detected using a Warner 725D two-electrode voltage clamp system (amplifier, Warner 725D; digital-to-analog converter, Axon Digidata 1550B). To obtain the activation characteristics of the channel, the stimulation method was as follows: the oocytes were clamped at -80 mV, the depolarization voltage range was -80 mV to +60 mV, the step stimulation voltage was 5 mV, the duration was 2 s, and the current signal of the channel was recorded after P/N leak subtraction (N = 4). For the measurement of the half-inactivation voltage, the cells were clamped at -100 mV, and the cells were depolarized by the pre-stimulation potential of 5 mV steps from -70 mV to +20 mV and maintained for 300 ms, and then the current was recorded with a test voltage of -10 mV and maintained for 200 ms. The activation curve was obtained by fitting the normalized peak current using the Boltzmann equation, and the half-activated and half-inactivated voltages of the voltage-gated Na _v channel were calculated.

As shown in Figure 2 and Table 1, the half-activation voltage of the original channel is -26.07±0.89mV, and the mutant Y378F causes the voltage dependence of channel activation to drift toward the depolarization direction, and the half-activation voltage becomes -21.46±0.71mV, which drifts nearly 5mV toward the depolarization direction, indicating that the mutation affects the activation ability of the channel.

Table 1 Activation-inactivation characteristics of wild-type and mutant rNav1.4

Note: The data shown in the table are in the form of mean±SEM, V _1/2 : midpoint voltage; n: the number of oocytes tested in the experiment.

As shown in Figure 3 and Table 1, the half-inactivation voltage of the main body is -32.93±0.45mV, and the inactivation curves of the mutants Y378F and shifted toward the hyperpolarization direction compared with the main body. The inactivation V1/2 is -39.08±0.39mV, which drifted toward the hyperpolarization direction by nearly 6mV, indicating that the mutation enhanced the inactivation process of the channel.

Similar phenomena are also manifested in the electrophysiological characteristics of HOKPP2. The disease is mostly caused by mutations in the highly conserved Arg residue on S4 of DI-DIII of Nav1.4. However, in addition to the conservative Arg mutation on S4 of DI-DIII, only one mutation at another position, that is, the Pro at position 1158 on the S4-S5 linker of DIII was replaced by Ser, which also caused the disease. The establishment of the mutant expression model of the present invention is of great significance for revealing the structural and functional relationship of Nav1.4, clarifying the molecular mechanism of disease occurrence, and drug screening and development.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (5)

1. A voltage-gated Nav1.4 mutant, characterized in that the mutant is derived from the amino acid sequence mutation of the wild-type rat voltage-gated sodium channel Nav1.4, and the mutation is a site-directed mutation of the tyrosine at position 378 of the amino acid sequence of the wild-type rat voltage-gated sodium channel Nav1.4 to phenylalanine; the amino acid sequence of the wild-type rat voltage-gated sodium channel Nav1.4 is shown in SEQ ID NO.1. 2. A gene encoding the mutant according to claim 1. 3. The gene according to claim 2, characterized in that its nucleotide sequence is shown as SEQ ID NO.2. 4. A recombinant vector carrying the gene according to claim 2. 5. A recombinant strain comprising the gene according to claim 2.