PKA Mediated Aβ_25-35 -Induced Inhibition Of K⁺ Channels (I A ) And Antagonism Of Humanin

Alzheimer's disease (AD) is a primary irreversible neurodegenerative disorder characterized by the presence of extensive extracellular amyloid plaques, intracellular neurofibrillary tangles and neuronal death in cerebral cortex and hippocampus, along with progressive impairment of learning, memory and unrelenting cognitive decline.β-amyloid (Aβ) is a polypeptide of 39-43 amino acids and major protein component of senile plaques. In addition, it is generally accepted that Aβ_25-35, a shorter fragment of Aβpeptide, exerts a similar effect as that of the full molecule in different experimental models and thus widely used for exploring the neurotoxicity of Aβ. There are many doctrines related to the etiology and pathogenesis of AD, but has not been determined. Several lines of evidence indicates that an abnormal accumulation of Aβis the leading cause and pathological characteristic of AD. Therefore, it is critical to study the mechanisms of Aβ-induced neurotoxicity and find a neuroprotective agence for inhibiting the Aβ's toxicity.There are study confirmed that voltage-dependent potassium channels are widely distributed in the central nervous system, primarily by influencing the shape of action potential, regulating action potential frequency, adjust the resting membrane potential of nerve function and other activities, including learning and memory.In hippocampal neurons, there are two voltage-gated potassium channels, namely the transient outward potassium current (I_A) of fast activation and fast inactivation and the delayed rectifier potassium current (I_K) of slow activation and hardly inactivation. Potassium channels are involved with aging-related damages in cognitive functions and play important roles in the process of learning and memory. I_A current is the main component of outward currents in the early repolarization of action potential. It has important roles in the regulation of neuron discharge frequency, action potential generation and discharge patterns. Large number of experiments show that the dynamics characteristics of transient outward potassium current are regulated by phosphorylation and the opening probability of I_A channels in hippocampal pyramidal cells was significantly reduced after activation of protein kinase A (PKA). PKA-CREB pathway plays an important role in memory formation. In Alzheimer's disease (AD) there are PKA-CREB pathway dysfunction. Aβcan lead to PKA-CREB signaling pathway dysfunction in low concentrations, causing hippocampal CA1 LTP (long-term potentiation) closely related with learning and memory was inhibited. Preliminary studies in our laboratory has observed that Aβcan significantly inhibit voltage-gated potassium channels (including I_A and I_K), Thus we speculate that Aβ_25-35 inhibition I_A current may be related to activation of PKA pathway or PKA may mediate the Aβ_25-35 inhibition I_A current.Humanin (HN) is a newly identified neuroprotective peptide that specifically inhibit familial Alzheimer's disease (FAD) gene mutations and neuronal cell death caused by Aβdeposition. Humanin suppressed Aβ_25-35 induced inhibition of K+ currents by antagonizing Aβ_25-35 induced phosphorylation of PKC and PKC-mediated inhibition of I_K current, and no effect on I_A currents. Thus,whether humanin suppressed Aβ_25-35 induced inhibition of K+ currents by antagonizing Aβ_25-35 induced phosphorylation of PKA and PKA-mediated inhibition of I_A current? Based on the above suggest, we observed the roles of PKA in the course of the Aβ-induced potassium channels changes and HN antagonism using the whole cell patch clamp technique. The mechanism of Aβ-induced potassium channel dysfunction and the protective effect of HN are clarified.Methods:1. Isolation of hippocampal neurons select hippocampal neurons of SD rats aged 7-10 days, isolated CA1 area into cell suspension, added to the Petri dish. After cells adherence, add 2ml clean extracellular fluid. recorded with patch clamp under the inverted microscope.2. The experimental group: Control group, Aβgroup (5μmol / L), HN group (5μmol / L), 8-brom-camp group (5μmol / L, 50μmol / L, 500μmol / L), H- 89 (10μmol / L) + Aβgroup (5μmol / L) .3. Electrophysiological measurement The whole-cell patch clamp mode use voltage clamp recording mode. Total K+ currents stimulated with 200 ms depolarizing pulse from -40 mV to +70 mV in 10 mV steps following a hyperpolarizing prepulse of 150 ms to -110 mV. I_K stimulated with similar protocol as total K+ currents, except for a 150 ms prepulse to -50 mV. Then I_A was translated by the formula. The amplitude of total K+ currents, I_A was measured at the peak of the current, and the amplitude of I_K was measured at 158 ms of the current. In the same hippocampal neurons, compare changes in potassium currents caused by different methods of administration.4. Data analysis All datas were expressed as mean±SEM. Statistical analysis of the data of patch clamp was performed by Analysis of Repeated Measure and one-way ANOVA using an SPSS program (SPSS 13.0) to study the relationship between the different variables. p<0.05 was considered to indicate statistically significance.Results:1. Aβ_25-35 ( 5μmol / L) inhibited the voltage-dependent total potassium currents and I_A current.Under the +70 mV depolarizing pulse, applied Aβ_25-35 alone the total potassium currents amplitude in the control group from 3421.70±409.98pA reduced to 2022.60±442.68pA, current amplitude compared with the control group decreased 59.11±12.94% (n = 5, p <0.05); I_A current from the control group 2196.77±170.56pA decreased to 1340.61±259.71pA, current amplitude compared with the control group decreased 61.03±11.82% (n = 5, p <0.05).2. PKA inhibitor H-89 (10μmol / L) can antagonize Aβinduced inhibition of I_A. Apply Aβ_25-35 and H-89, compared with the control group, the relative currents amplitude of total K+ current and I_A current were 104.78±10.19%(n=5)和119.68±22.87%(n=5). Apply Aβ_25-35 alone the relative values of the two currents were 59.11±12.94% (n=5), 61.03±11.82 % (n=5).They were statistically significant (p<0.05);3. Different concentrations PKA agonist 8-brom-camp (5μmol / L, 50μmol / L, 500μmol / L) can inhibit transient outward potassium current (I_A) in dose dependent. Compared with the control group, the relative currents amplitude of I_A were 87.18±15.33% (n=5, p >0.05), 59.91±5.11% (n=5, p<0.05), 46.59±2.56% (n=5, p<0.05). The delayed rectifier potassium current (I_K) slightly increased;4. HN ( 5μmol / L) can antagonize the inhibitory effect of Aβ_25-35;HN antagonize PKA agonist 8-brom-camp (50μmol / L) induced inhibition of I_A currents. Given HN (5μmol / L) after Aβ_25-35 total potassium currents and I_A current amplitude were increased to 2952.00±272.01pA and 1928.63±172.68pA (n = 5, p <0.05), compared with Aβ_25-35, have statistically significant.After 8-brom-camp HN (5μmol / L) was applied, relative current amplitude of I_A increased from 59.91±5.11% to 78.68±10.00% (n=5).Conclusions:1. Acute administration of Aβ_25-35 (5μmol / L) inhibited voltage-dependent total potassium currents and I_A currents .2. PKA mediated the inhibition effects of Aβon potassium channels;Aβinhibition of I_A by activation of PKA pathway may be one of the mechanisms of its toxicity.3. HN antagonize the Aβ_25-35 inhibition of I_A and HN antagonize PKA agonist induced inhibition of I_A. Inhibition of Aβinduced PKA activation and PKA-mediated Aβneurotoxicity may be one of the mechanisms of HN against Aβinduced neurotoxicity.