超极化激活的环核苷酸门控通道1参与七氟烷诱导的顺行性遗忘作用的机制研究

Elucidating the role of HCN1 ion channels in sevoflurane-induced anterograde amnesia

目的旨在探讨超极化激活的环核苷酸门控通道1(hyperpolarization-activated cyclic nucleotide-gated cation channel 1,HCN1)在七氟烷诱导的顺行性遗忘中的作用机制。方法采用雄性SPF级C57BL/6J野生型小鼠(WT组,n=60)和HCN1基因全身敲除(HCN1-/-)小鼠(HCN1-/-组,n=60)。将两组小鼠分别暴露于不同浓度的七氟烷(0%、0.1%、0.2%、0.4%、0.6%和0.8%)下,每个浓度10只小鼠。随后进行条件性恐惧实验,计算两组小鼠七氟烷抑制条件性恐惧记忆的半数效应浓度(median effective concentration,用EC50表示)。采用全细胞膜片钳技术记录0.125mmol/L七氟烷浓度灌流液对转染HCN1的人胚肾293细胞(HEK293-HCN1)上HCN1电流的影响,比较七氟烷处理前(Control组)和处理后(Sevoflurane组)HCN1电流的变化。结果与-/-WT组小鼠相比,HCN1组小鼠在七氟烷抑制场景恐惧记忆和声音恐惧记忆中的EC50值升高,差异有统计学意义(场景恐惧记忆:0.18%比0.26%,P<0.001;声音恐惧记忆:0.47%比0.49%,P<0.001)。全细胞电生理记录显示,与基础值相比,0.125mmol/L七氟烷抑制HEK293-HCN1细胞在﹣90~﹣140mV范围内的电流幅度(P值均<0.05),并增加了半最大激活电压(V1/2a)值[(﹣108±2.9)mV比(﹣100.1±3.0)mV,(P<0.001)]。结论HCN1电流的抑制可能参与了七氟烷诱导的顺行性遗忘作用。

Objective To investigate whether hyperpolarization-activated cyclic nucleotide-gated cation channel 1(HCN1)contributes to sevoflurane-induced anterograde amnesia.Method Wild-type and HCN1 knockout(HCN1-/-)C57BL/6J mice underwent contextual and cued fear conditioning under varying sevoflurane concentrations(0%,0.1%,0.2%,0.4%,0.6%,and 0.8%).Median effective concentration(EC50)values for sevoflurane inhibition of fear memory were calculated.The effect of sevoflurane(0.125 mmol/L)on HCN1 in human embryonic kidney 293 cells transfected with HCN1(HEK293-HCN1)was recorded using whole-cell patch clamp technique.The changes in HCN1 before(control group)and after(sevoflurane group)sevoflurane treatment were compared.Result The EC50 for sevoflurane inhibition of contextual-/-fear memory was significantly higher in HCN1 mice than in wild-type mice(0.26%vs.0.18%,-/-P<0.001).The EC50 for cued fear memory inhibition was also higher in HCN1 mice(0.49%vs.0.47%,P<0.001),indicating HCN1 knockout attenuated sevoflurane-induced anterograde amnesia.0.125 mmol/L sevoflurane inhibited HEK293-HCN1 cell current amplitude at﹣90 mV to﹣140 mV(all P<0.05)and shifted V1/2a from(﹣108±2.9)mV to(﹣100.1±3.0)mV(P<0.001),demonstrating an inhibitory effect on HCN1 currents.Conclusion Inhibition of HCN1 channel currents may contribute to the anterograde amnesic effects of sevoflurane.