Background:Voltage-gated sodium channels are the targets of many commonly used antiepileptic drugs.NaV1.6,encoded by Scn8a,increased in chronic mesial temporal epilepsy animal models and co-localized with Ankyrin-G,en...Background:Voltage-gated sodium channels are the targets of many commonly used antiepileptic drugs.NaV1.6,encoded by Scn8a,increased in chronic mesial temporal epilepsy animal models and co-localized with Ankyrin-G,encoded by Ank3.We hypothesized that inhibition of Ank3 transcription by siRNA decrease the expression of NaV1.6.Results:We characterized expression of the target genes in hippocampal neuron HT22 cells by Real time-PCR.The melt peak in the resolution curve of Scn1a,Scn8a and Ank3 were all unique.Ank3 transcription was interfered and the relative Ank3 mRNA levels of the three interfered groups compared to GAPDH were 0.89±0.13,0.52±0.07 and 0.26±0.05 while that of the negative control group was 1.01±0.08(P<0.05).When Ank3 transcription was inhibited by siRNA,the relative mRNA levels of Scn8a decreased in the three groups(0.91±0.09,0.33±0.06 and 0.25±0.05),compared to the negative control group(1.10±0.09).Tested by Western blotting,protein levels of ankyrinG and Nav1.6 decreased after ank3-siRNA.Ankyrin-G in negative control group,group1,group2 and group1+2 were 0.813±0.051,0.744±0.041,0.477±0.055 and 0.351±0.190 respectively(P<0.01)while Nav1.6 were 0.934±0.036,0.867±0.078,0.498±0.070 and 0.586±0.180(P<0.01).The quantity analysis of immunofluorescence showed significant decrease of ankyrin-G and Nav1.6(Student’s test,P=0.046 and 0.016 respectively).Conclusion:We therefore concluded that in HT22 cells the expression of Nav1.6 was down-regulated by Ank3 RNA interference.展开更多
The axon initial segment(AIS)region is crucial for action potential initiation due to the presence of high-density AIS protein voltage-gated sodium channels(Nav).Nav channels comprise several serine residues responsib...The axon initial segment(AIS)region is crucial for action potential initiation due to the presence of high-density AIS protein voltage-gated sodium channels(Nav).Nav channels comprise several serine residues responsible for the recruitment of Nav channels into the structure of AIS through interactions with ankyrin-G(AnkG).In this study,a series of computational experiments are performed to understand the role of AIS proteins casein kinase 2 and AnkG on Nav channel recruitment into the AIS.The computational simulation results using Virtual cell software indicate that Nav channels with all serine sites available for phosphorylation bind to AnkG with strong affinity.At the low initial concentration of AnkG and casein kinase 2,the concentration of Nav channels reduces significantly,suggesting the importance of casein kinase 2 and AnkG in the recruitment of Nav channels.展开更多
Neurons grow multiple axons after treatment with glycogen synthase kinase-3（GSK-3） inhibitors. However,whether they are electrically active is not known. Here, we examined the role of multiple axons as electrophysio...Neurons grow multiple axons after treatment with glycogen synthase kinase-3（GSK-3） inhibitors. However,whether they are electrically active is not known. Here, we examined the role of multiple axons as electrophysiological components during neuronal firing. Combining pharmacological, immunofluorescence, and electrophysiological methods, we found that more neurons had multiple axon initial segments（AISs） after inhibition of GSK-3 activity with SB415286. The multiple AISs induced by GSK-3 inhibition were enriched with voltage-gated sodium channels. The depolarization rate of the multiple-AIS neurons was increased, but their action potential threshold and halfwidth were normal. By calculating derivatives of the actionpotential rising phase, an extra d2 V/dt2 peak from the extra AIS was distinguished; this indicated that the extra AIS fired ahead of the soma and increased the rate of depolarization.Our study demonstrates that the multiple axons induced by GSK-3 inhibition have AIS structures that are electrically active, and provides insight for axon and AIS studies.展开更多
基金the National Nature Science foundation(81000554)Guangdong Nature Science foundation(2018A030313345)the Science and Technology Foundation of Guangdong Province(2008B030301058).
文摘Background:Voltage-gated sodium channels are the targets of many commonly used antiepileptic drugs.NaV1.6,encoded by Scn8a,increased in chronic mesial temporal epilepsy animal models and co-localized with Ankyrin-G,encoded by Ank3.We hypothesized that inhibition of Ank3 transcription by siRNA decrease the expression of NaV1.6.Results:We characterized expression of the target genes in hippocampal neuron HT22 cells by Real time-PCR.The melt peak in the resolution curve of Scn1a,Scn8a and Ank3 were all unique.Ank3 transcription was interfered and the relative Ank3 mRNA levels of the three interfered groups compared to GAPDH were 0.89±0.13,0.52±0.07 and 0.26±0.05 while that of the negative control group was 1.01±0.08(P<0.05).When Ank3 transcription was inhibited by siRNA,the relative mRNA levels of Scn8a decreased in the three groups(0.91±0.09,0.33±0.06 and 0.25±0.05),compared to the negative control group(1.10±0.09).Tested by Western blotting,protein levels of ankyrinG and Nav1.6 decreased after ank3-siRNA.Ankyrin-G in negative control group,group1,group2 and group1+2 were 0.813±0.051,0.744±0.041,0.477±0.055 and 0.351±0.190 respectively(P<0.01)while Nav1.6 were 0.934±0.036,0.867±0.078,0.498±0.070 and 0.586±0.180(P<0.01).The quantity analysis of immunofluorescence showed significant decrease of ankyrin-G and Nav1.6(Student’s test,P=0.046 and 0.016 respectively).Conclusion:We therefore concluded that in HT22 cells the expression of Nav1.6 was down-regulated by Ank3 RNA interference.
文摘The axon initial segment(AIS)region is crucial for action potential initiation due to the presence of high-density AIS protein voltage-gated sodium channels(Nav).Nav channels comprise several serine residues responsible for the recruitment of Nav channels into the structure of AIS through interactions with ankyrin-G(AnkG).In this study,a series of computational experiments are performed to understand the role of AIS proteins casein kinase 2 and AnkG on Nav channel recruitment into the AIS.The computational simulation results using Virtual cell software indicate that Nav channels with all serine sites available for phosphorylation bind to AnkG with strong affinity.At the low initial concentration of AnkG and casein kinase 2,the concentration of Nav channels reduces significantly,suggesting the importance of casein kinase 2 and AnkG in the recruitment of Nav channels.
基金supported by the Fund for Distinguished Young Scholars of National Natural Science Foundation of China (81425009)National Natural Science Foundation of China (31630028 and 91632305)a Financial Grant from the China Postdoctoral Science Foundation (2013M540015)
文摘Neurons grow multiple axons after treatment with glycogen synthase kinase-3（GSK-3） inhibitors. However,whether they are electrically active is not known. Here, we examined the role of multiple axons as electrophysiological components during neuronal firing. Combining pharmacological, immunofluorescence, and electrophysiological methods, we found that more neurons had multiple axon initial segments（AISs） after inhibition of GSK-3 activity with SB415286. The multiple AISs induced by GSK-3 inhibition were enriched with voltage-gated sodium channels. The depolarization rate of the multiple-AIS neurons was increased, but their action potential threshold and halfwidth were normal. By calculating derivatives of the actionpotential rising phase, an extra d2 V/dt2 peak from the extra AIS was distinguished; this indicated that the extra AIS fired ahead of the soma and increased the rate of depolarization.Our study demonstrates that the multiple axons induced by GSK-3 inhibition have AIS structures that are electrically active, and provides insight for axon and AIS studies.
