| Two pore domain potassium channels(K2P)are a new type of potassium ion channels discovered in the 1990s.They are different from traditional potassium ion channels.It has four transmembrane segments(M1-M4)and two domains(P1,P2),namely the 4TMS/2P structure.TREK-1 is distributed in various tissues of the human body,more in the central nervous system,and widely distributed in the brains of rats and mice.With the development of science and technology and the advancement of experimental equipment,people’s understanding of two pore domain potassium channels is becoming more and more profound.Many studies have shown that TREK-1 is associated with central nervous system diseases such as cerebral ischemia,stroke,and depression.Fluoxetine,a commonly used clinical treatment for depression,can significantly inhibit TREK-1 current and is often used as a tool medicine.Some commonly used clinical volatile anesthetics such as halothane and isoflurane can strongly activate TREK-1,and can play a neuroprotective role in cerebral ischemia models.The general anesthetics ketamine,chloroform,etomidate all have obvious activation effects on TREK-1,but the specific targets and mechanisms are not clear.More and more scientific studies show that TREK-1 is a channel closely related to human diseases,and the research on TREK-1 is more worthy of our in-depth and persistent.This thesis mainly used patch clamp technique to observe the effect and mechanism of ketamine and etomidate on TREK-1.The results are as follows.Section 1:Electrophysiological study of ketamineKetamine is an intravenous anesthetic and is widely used clinically.The experimental results show that ketamine 3,10,30,100 μM has obvious activation effect on TREK-1 channel,the activation rates are 27.2 ± 5.5%(n=4),52.1±16.5%(n=5),60.5 ± 23.2%(n=5),79.3%± 19.5%(n = 6),ketamine can activate TREK-1 in a dose-dependent manner.Using intracellular microdialysis to observe ketamine 10,30,100 μM can also activate TREK-1 in a dose-dependent manner,with activation rates of 18.6 ± 9.2%(n=4),25.4 ± 11.7%(n=5),43.5±20.9%(n=4),it is speculated that the site where TREK-1 is activated by ketamine also exists in the cell.Temperature is also one of the regulating factors of the two pore domain potassium channel TREK-1.Using a thermostat to heat,it was observed that the activation rate of TREK-1 by ketamine 10 μM at 30℃ was 66.77 ± 24.7%(n=4)significantly higher the activation rate of TREK-1 by ketamine 10 μM at 25℃ is 38.5 ± 29.2%(n=3),and the activation of TREK-1 by ketamine and temperature can play a synergistic role.Chloroform is a tool medicine often used in laboratories,and previous results in our laboratory indicate that chloroforrm has a certain activation effect on TREK-1.The research results of this paper show that chloroform 0.05,0.1,and 0.2 mM can strongly activate TREK-1.The rates were 176.46± 33.7%(n=5),206.5 ± 60.4%(n=5),215.2 ± 48.2%(n=4),and the activation effect of chloroform was stronger than that of ketamine.Ketamine is a kind of intravenous anesthetic,which is widely used in clinic because of its good anesthetic effect.The experimental results show that ketamine can activate TREK-1,and the activation of TREK-1 increases with the increase of ketamine dose.Chloroform is a kind of solvent,which is also used as an intravenous anesthetic in medicine,but because of its large side effects,it is basically not used clinically.But it still occupies a certain position in the laboratory.The previous results in this laboratory indicate that chloroform has a certain activation effect on TREK-1 in this paper,0.05、0.1、0.2 mM chloroform was used to observe its effect on TREK-1.And it was found that the activation of TREK-1 is enhanced with the increase of chloroform concentration,and the activation effect of chloroform is stronger than that of ketamine.May consider chloroform as a positive tool for screening TREK-1 activator.Observed by electrode osmotic administration method:Ketamine can also play an activation role in the cell,and the target of ketamine action exists inside and outside the cell.The activation of TREK-1 by ketamine was significantly enhanced when the temperature was increased.Ketamine and temperature could play a synergistic role in the activation of TREK-1.The experimental results show that ketamine can activate TREK-1,and can exert TREK-1 activation effect both inside and outside cells.In addition,ketamine can also cooperate with TREK-l’s regulatory factor,temperature,to activate TREK-1.Chloroform also has a dose-dependent activation effect on TREK-1,and the activation effect of chloroform 0.05 mM on TREK-1 is stronger than that of ketamine 100 μM on TREK-1.It may be considered that chloroform is used as a positive drug for screening TREK-1 activator within an appropriate concentration range.Section 2:Electrophysiological study of etomidateThe previous research in this laboratory showed that etomidate has an activation effect on TREK-1.Based on the previous research in this laboratory,this paper continues to observe the regulatory mechanism of etomidate on TREK-1.The experimental results show that etomidate 3,10,and 15 μM have obvious activation effect on the two pore domain potassium channel TREK-1,which can be activated to 115.0± 3.9%(n=5,P=0.06),142.1 ± 16.3%(n=6,*P<0.05),122.9 ± 3.8%(n=4,*P<0.05)and etomidate 20,25,30,100 μM has a certain inhibitory effect on TREK-1,which can be suppressed to 96.2%± 5.0%(n=5),90.3 ± 14.3%(n=7),87.8 ±14.6%(n=8),70.3 ± 8.8%(n=5.*P<0.05).The above results indicate that etomidate has a bidirectional regulation effect on TREK-1.Fit one phase exponential decay to the concentration and rate of change of etomidate in Graphpad Prism 5.0 software.According to the equation y=(y0-plateau)*exp(-k*x)+plateau(y0=136.50 plateau=-28.70 k=0.08)The inflection point of bidirectional regulation of etomidate is calculated to be 20.7 μM.Next,the effect of etomidate on the voltage-dependent potassium channel Kv2.1 was observed,and the I-V curve of etomidate on Kv2.1 was found:etomidate had a slight inhibitory effect on Kv2.1 at 10 μM,but the same there was no significant difference between the control groups;etomidate 30 μM had no effect on Kv2.1.In order to verify the bidirectional regulation of etomidate on TREK-1,the effects of etomidate 1 0 and 30μM on the TREK-1 Ⅰ-Ⅴ curve,resting membrane potential and ramp current were observed.The results show that etomidate 10μM can shift the TREK-1 Ⅰ-Ⅴ curve to the left,and etomidate 30 μM has no effect on the TREK-1 Ⅰ-Ⅴ curve.The membrane potential of the etomidate 10 μM group was-46.0± 1.2 mV,the membrane potential of the control group was-40.3 ± 2.3 mV,and the etomidate 10 μM shifted the TREK-1/CHO membrane potential in the direction of hyperpolarization.The membrane potential of the etomidate 30 μM group was-38.7±1.3 mV,the membrane potential of the control group was-47.7± 1.2 mV,and the etomidate 30 μM shifted the TREK-1/CHO membrane potential in the direction of depolarization.Ramp current stimulation results show that etomidate 10 μM can significantly shift the slope current curve to the left,and etomidate 30 μM general trend can shift the slope current curve to the right.Studies have shown that TREK-1 is affected by acidity,and the effect of different concentrations of etomidate on TREK-1 under different acidity conditions was observed.At 10 μM of etomidate,the activation of TREK-1 by etomidate when intracellular acidity was increased the effect is increased;when etomidate is 30 μM.increasing the intracellular acidity of etomidate has a stronger inhibitory effect on TREK-1.Observed the effect of chloroform on TREK-1 current under the condition of changing acidity.Chloroform 0.05,0.1,0.2 mM increased the activation of TREK-1 under increased intracellular acidity than under normal cell state,but increased extracellular acidity TREK-1 has no significant effect.The experimental results show that etomidate has a two-way regulation effect on TREK-1,using one pHase exponential decay pair to fit,according to the equation y=(y0-plateau)*exp(-k*x)+plateau(y0=136.50 plateau=-28.70 k=0.08),the inflection point of bidirectional regulation of etomidate is 20.7 μM.In order to verify the bidirectional regulation effect of etomidate on TREK-1,the effects of etomidate 10 and 30 pM on the TREK-1 Ⅰ-Ⅴ curve,resting membrane potential and slope current stimulation were observed.The results were consistent with the above results,indicating etomidate there is a bidirectional regulation effect on TREK-1.Acidity is one of the regulatory factors of TREK-1.The effects of etomidate 10 and 30 μM on TREK-1 were observed under the conditions of increasing extracellular acid and intracellular acidity.The results showed that the increase depends on changing the intracellular and external acidity.Imidate also has a bidirectional regulation effect on TREK-1.Observed the effect of chloroform on the current of TREK-1 under the condition of changing acidity.Chloroform 0.05,0.1,0.2 mM increased the activation of TREK-1 under the increase of intracellular acidity than under normal cell state,but increased the extracellular acidity chloroform 0.05,0.1,0.2 mM compared with normal cell state,increasing the extracellular acidity had no significant effect on TREK-1. |
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