Plastic Changes Of A-type Potassium Channels Underlie Acquired Epileptogenesis

Although epilepsy can be idiopathic, over half of epilepsy cases are initiated by neurological insults and are called acquired epilepsy (AE). Epileptogenesis is the process by which injuries such as status epilepticus, stroke, or traumatic brain injury produces short- and long-term plastic changes in the neurons, resulting in spontaneous recurrent seizures (SRS) in previously normal brain tissue. These plastic changes have been recognized as the most important determinants of neuronal excitability, and thereby contribute to the development of AE. Selective and coordinate changes in certain voltage- and transmitter-gated ion channels have been found to be involved in this process.Voltage-gated potassium channels (Kv) are important regulators of electrical signaling in the brain and act to down-regulate the excitability of individual neurons. Alterations in potassium channels could potentially contribute to hyperexcitability and a predisposition toward seizure activity. A-type voltage-gated potassium channels recently attracted special attention because they contribute to controlling the amplitude of back-propagating action potentials and the neuronal action potential firing properties. Kv4.2 subunit is one of the key components underlying A-type current (IA). Changes in the properties of these physiologically important IA by the transcriptional and posttranscriptional regulation of Kv4.2 could lead to the dysregulation of neuronal excitability, which is a hallmark of epilepsy. Evidence has accumulated that Kv4.2 is an important determinant of neuronal excitability and is implicated in epilepsy. However, thus far, limited information is available on the dynamic regulation of Kv4.2 and its underlying mechanisms. Recent studies have emphasized the importance of voltage-dependent potassium channel interacting proteins (KChIPs) in regulating the expression levels and physiological properties of Kv4 channels. KChIPs are calcium binding proteins that belong to the family of EF-hand neuronal calcium sensor (NCS) proteins. KChIPs appear to transduce Ca2+ signals to actively altering neuronal membrane excitability by modulating the properties of Kv4. It is implied that the intracellular Ca2+ concentration ([Ca2+]i) may influence Kv4 channels activities through the KChIP pathway. KChIP1 is prominently expressed in the brain and is tightly associated with Kv4.2. Currently, information regarding a possible involvement of KChIPs in epilepsy is insubstantial. There are not enough evidences to support a role for Kv4.2 channels in pathogenesis of epilepsy either. We were particularly curious about the association of Kv4.2 with KChIP1 and their involvement in short- or long-term plasticity in epileptogenesis. Thus, the present study was undertaken to examine the distribution of KChIP1 and its colocalization with GABAergic neuron and Kv4.2. We also used lithium-pilocarpine induced epilepsy model to reveal the possible plastic changes in Kv4.2 and KChIP1 expression and the intracellular calcium homeostasis during epileptogenesis.METHODSBrains of adult SD rats were processed according to the common immunoreaction procedure and stained with DAB to detect the distribution of KChIP1 in brain. Other sections of normal rat brains were processed for double-labeling to detect the presence of Kv4.2/KChIP1 or GABA/KChIP1. The labeled sections were observed by laser scan confocal microscopy. For the establishment of acquired epilepsy, rats were administrated with LiCl and pilocarpine to induce SE. Tissues of hippocampus and cerebral cortex were sampled at different time points after seizures. We used the techniques of Western blot and immunohistochemistry to detect the plastic changes of Kv4.2 and KChIP1. The expression levels of Kv4.2 and KChIP1 were quantified by measuring the relative optical density or cell counting. Rats that failed to exhibit seizures after pilocarpine injection were also investigated. For the intracellular calcium imaging, we used confocal microscopy to detect the 4-aminopyridine (4-AP) induced calcium elevation in the slices after SE. Statistical analysis was using two-tailed paired t-test. The accepted level of significance was 0.05.RESULTKChIP1 was found to be abundantly expressed in the adult SD rat brain, including cerebral cortex, hippocampus, thalamus, and cerebellum. KChIP1 preferentially locates in GABAergic interneurons in hippocampus and cerebral cortex. The co-existing proportion in hippocampus is around 60%. We also found KChIP1 and Kv4.2 were coexpressed in the neurons within cerebral cortex and hippocampus. KChIP1-positive signals were found mainly in the membrane of somata and proximal dendrites, while Kv4.2 mostly distributed on the membrane of distal dendrites.We used lithium-pilocarpine to induce SE and found a remarkable cell loss after the prolonged SE. Using immunohistochemistry, we found the increase in Kv4.2 and KChIP1 expression right after SE, primarily in CA1 and CA3 subfields of hippocampus, but the decrease during chronic spontaneous recurrent seizures. The results of immunoblot showed that the expression levels of Kv4.2 and KChIP1 in hippocampus were elevated after the prolonged SE. Evidently, Kv4.2 expression was increased significantly at 3h, 6h, 24h, 48h after seizure. The decrease in Kv4.2 expression at 50d after seizure had no statistical difference at a whole-hippocampus level. Similarly, KChIP1 showed statistically significant increases at 3h, 6h after seizure, but no difference at 48h, 50d. The increasing peak time of KChIP1 was earlier than that of Kv4.2. The controls and those rats that failed to exhibit seizures after PILO injection showed no significant change.We compared the difference in 4-AP-induced intracellular calcium ([Ca2+]i) elevation between the slices undergone SE and the control brain slices. The results showed that the [Ca2+]i elevation induced by the Kv4 channel blocker 4-AP was aggravated and prolonged in the model slice after SE. The functional relevance of these changes in Ca2+ homeostasis and Kv4.2 and KChIP1 expression may be associated with intrinsic neuronal excitability regulation and epileptogenesis.CONCLUSIONThe present study shows that the expression of Kv4 and KChIP1 is increased at the early latency stage of the Li-Pilo epilepsy model, which is possibly a sort of self-protection mechanism following prolonged seizures. KChIP1 increases before Kv4 does, suggesting that KChIP1 modulates Kv4.2 expression. The descending trend in the Kv4 and KChIP1 levels during the chronic phase of the model, particularly the decrease in the number of KChIP1-positive neurons in the hippocampus, indicates a decompensation that might contribute to hyperexcitability in chronic epilepsy. Disturbed homeostasis of [Ca2+]i following SE has been found in the model, and this was elicited by the Kv4 channel blocker 4-AP; it suggests a close relationship between potassium channel function and calcium signaling. It is possible that altered Ca2+ dynamics trigger a series of pathological changes and contribute to epileptogenesis.