Relationship Between Kv1.1 Expression And Neuronal Excitability In A Mouse Model Of Fragile X Syndrome

Objective Fragile X syndrome(FXS),a common form of inherited mentalretardation,is characterized by hyperexcitability in neural network including intellectual disability and a high incidence of epileptic seizures.Functional deficiency of FMRP is the main causes of FXS.Abundant studies indicate that A-type potassium channels,known as key modulators of neuronal excitability,may play a crucial role in FXS.As a Shaker homologue potassium channel,Kv1.1 is an important key component of the A-type potassium channel and has been shown to be involved in the regulation of neuronal excitability.Its abnormal function has been evidenced to be associated with hyperexcitability of neural networks in various neurological disorders.We speculate that the expression and/or function of Kv1.1 channel may be altered,consequently affecting the Kv1.1 mediated A-type potassium currentduring the pathophysiological process of FXS.To test this hypothesis,we employed a genetic mouse model of FXS,Fmr1 gene knockout mice(Fmr1KO).The Fmr1 KO and WT mice at the age of 10 days(P10),30 days(P30)and 60 days(P60)were examined for the expression and distribution of Kv1.1 channel.In the brain slice,we used patch-clamp technique to record Kv1.1-mediated currents isolated by a Kv1.1 specific inhibitor DTX-? and investigated the changes of Kv1.1-mediated current and its electrophysiological kinetics.These studies will help us to understand the relationship between Kv1.1 channel and neuronal excitability in FXS.Methods 1.Conventional PCR technique was used to determine the genotype of experimental animals.2.Fmr1 KO FVB mice and WT controls at the age of P10,P30,and P60 were randomly selected for the experiments.3.Western blotting method was applied to measure the expression leve of Kv1.1 in the hippocampus of Fmr1 KO and WT mice.4.Immunohistochemistry was carried out to detect the expression and distribution of Kv1.1 in the region ofhippocampus.5.After preparation of acute brain slice,neurons in the region of CA3 were recorded with potassium currents by whole-cell patch clamp techinque.With application of Kv1.1 specific blocker DTX-?,specific Kv1.1-mediated currents were isolated to learn their density and kinetics.Current-clamp was used to evoke action potentials(AP)forcomparing firing ability of neurons.Results 1.Western blotting and immunohistochemistry results showed that the expression of Kv1.1 channel was increased with age in both Fmr1 KO and WT mice.In the Fmr1 KO hippocampus,the expression level of Kv1.1 was significantly lowerby around 50% than that in the age-matched WT mice for the P10 and P60 groups(p < 0.01),but no difference was found in the P30 age groups.Immunohistochemistry showed that Kv1.1 subunit was highly expressed in the strata radiatum and oriens of hippocampus,especially in CA3 region of both Fmr1 KO and WT mice.The topography of Kv1.1 distribution did not differ markedly between Fmr1 KO and WT mice.There were overall decreased intensities of Kv1.1 positive signals in the Fmr1 KO mice for each age group.2.Electrophysiological recording showed that theamplitude of total potassium current was decreased in the pyramidal neurons in the region of CA3 from Fmr1 KO mice at the age of P10.We used a Kv1.1-specificinhibitor DTX-? to create concentration-response curve.It showed that half maximal inhibitory concentration(IC50)in Fmr1 KO neurons was 1.65 n M,lower than that of WT controls(3.03 n M).By curve fitting,we calculated that total potassium amplitude in Fmr1 KO is equivalent to that in WT controls treated with 2.49 n M DTX-?.By using 100 n M DTX-?,the traces of Kv1.1-mediated current were obtained from the total potassium current by digital subtraction.We found that the amplitude of Kv1.1-mediated current evoked by depolarized voltages was significantly decreased in the Fmr1 KO neurons,with increased half activation voltage(V1/2)and reduced time constant of recovery from inactivation.3.By using current clamp,identical depolarizing current injections evoked more APs in Fmr1 KO neurons than in WT neurons(p < 0.01).WT neurons treated with 2.49 n M DTX-?also led to a prominent increase in the average number of evoked APs,similar to the increase in Fmr1 KO neurons.The results indicated that the increased APs in Fmr1 KO neurons may be ascribed to the reduction of Kv1.1-mediated current.Conclusions 1.The expression levels of Kv1.1 are increasing with development.In Fmr1 KO hippocampus,the developmental elevations of Kv1.1 expression were delayed and insufficient in the neonatal and adult periods.2.Total potassium currents,especially the Kv1.1-mediated components,were significantly decreased in the Fmr1 KO neurons,which might due to the insufficient expression of Kv1.1.In the Fmr1 KO neurons,abnormalties of Kv1.1-mediated currents include decreased amplitude,depolarization shift of activation voltage,and accelerated recovery from inactivation.3.The evidence that downregulation of Kv1.1-mediated current contributes to increased firing ability of neurons indicates that insufficient expression and impaired function of Kv1.1 channel maybe an important mechanism underlying hyperexcitability of FXS.