Application of Neuregulin-1 (NRG1) in preparation of product to enhance Transient Receptor Potential Cation Channel Subfamily C Member 6 (TRPC6) channel activity
A neuregulin and channel technology, applied in the field of nervous system disease treatment, can solve problems such as NRG1TRPC6 channel function not seen
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Embodiment 1
[0038] This example provides the co-labeling of TRPC6 channel protein and ErbB4 receptor of NRG1 in the hippocampal region of the brain by immunofluorescence labeling.
[0039] 1. Experimental animal feeding:
[0040] SPF grade Erbb4-TD mice: male, aged 6 weeks. The animals are raised in an SPF grade animal center. The temperature in the breeding area is generally between 25°C and 28°C, and the humidity is controlled between 40% and 70%. The animal center is cleaned regularly, animal bedding is replaced, feed is added, and SPF-grade breeding-grade feed and drinking water are freely ingested.
[0041] 2. Drugs and main reagents:
[0042] Tamoxifen was purchased from Sigma-Aldrich Company (Shanghai, China).
[0043] 3. Experimental method and specific steps:
[0044] Erbb4-TD mice were obtained by crossing Erbb4-CreERT2 and Rosa::LSL-tdTomato mice (the neurons expressing ErbB4 receptors in Erbb4-TD mice will all express red fluorescent protein at the same time, so as to comp...
Embodiment 2
[0049] In this example, Western blotting and patch clamp electrophysiology were used to detect the effect of NRG1 on the nitrosylation level of TRPC6 channel protein and the promotion of GABA release before and after NEM intervention.
[0050] 1. Experimental animal feeding: the same as in Example 1.
[0051] 2. Drugs and main reagents: cesium chloride, HEPES, EGTA, ATP-Mg, GTP-Na, sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, calcium chloride, sodium bicarbonate, glucose , sucrose, and NEM were purchased from Sigma-Aldrich Company (China, Shanghai); recombinant human neuregulin 1 (recombinant human NRG1-β1, rNRG1-β1) (amino acid sequence: SHLVKCAEKE KTFCVNGGEC FMVKDLSNPS RYLCKCPNEFTGDRCQNYVM ASFYKHLGIE FMEAE) was purchased from ProSpec Company ( Israel, Rehovot); QX-314, TTX, CNQX, DL-AP5 were purchased from TOCRIS (Bristol, UK).
[0052] 3. Experimental method and specific steps:
[0053] (1) Tissue sample preparat...
Embodiment 3
[0067] This example provides an example of the effect of NRG1 on GABA release before and after blocking TRPC6 channels by patch clamp electrophysiology.
[0068] 1. Experimental animal feeding: the same as in Example 1.
[0069] 2. Drugs and main reagents: cesium chloride, HEPES, EGTA, ATP-Mg, GTP-Na, sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, calcium chloride, sodium bicarbonate, glucose , sucrose were purchased from Sigma-Aldrich Company (China, Shanghai); rNRG1-β1 was purchased from ProSpec Company (Israel, Rehovot); QX-314, TTX, CNQX, DL-AP5 were purchased from TOCRIS Company (UK, Bristol); SKF96365 was purchased from Merck Millipore (Billerica, USA); ML-7 was purchased from MCE (Monmouth Junction, USA).
[0070] 3. Experimental method and specific steps: Take adult male C57BL / 6 mice (ordinary mice) to conduct patch clamp experiments, and the experimental operation is the same as before (Example 2). Add TRPC c...
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