Downregulation of Dendritic HCN Channel Gating in Epilepsy Is Mediated by Altered Phosphorylation Signaling

Abstract

The onset of spontaneous seizures in the pilocarpine model of epilepsy causes a hyperpolarized shift in the voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated current (I h ) in CA1 hippocampal pyramidal neuron dendrites, contributing to neuronal hyperexcitability and possibly to epileptogenesis. However, the specific mechanisms by which spontaneous seizures cause downregulation of HCN channel gating are yet unknown. We asked whether the seizure-dependent downregulation of HCN channel gating was due to altered phosphorylation signaling mediated by the phosphatase calcineurin (CaN) or the kinase p38 mitogen-activated protein kinase (p38 MAPK). We first found that CaN inhibition upregulated HCN channel gating and reduced neuronal excitability under normal conditions, showing that CaN is a strong modulator of HCN channels. We then found that an in vitro model of seizures (1 h in 0 Mg 2ϩ and 50 M bicuculline at 35-37°C) reproduced the HCN channel gating change seen in vivo. Pharmacological inhibition of CaN or activation of p38 MAPK partially reversed the in vitro seizure-induced hyperpolarized shift in HCN channel gating, and the shift was fully reversed by the combination of CaN inhibition and p38 MAPK activation. We then demonstrated enhanced CaN activity as well as reduced p38 MAPK activity in vivo in the CA1 hippocampal area of chronically epileptic animals. Pharmacological reversal of these phosphorylation changes restored HCN channel gating downregulation and neuronal hyperexcitability in epileptic tissue to control levels. Together, these results suggest that alteration of two different phosphorylation pathways in epilepsy contributes to the downregulation of HCN channel gating, which consequently produces neuronal hyperexcitability and thus may be a target for novel antiepileptic therapies.

Differential expression of HCN subunits alters voltage-dependent gating of h-channels in CA1 pyramidal neurons from dorsal and ventral hippocampus

“…Therefore, differences in the ratio of HCN1 to HCN2 expression levels represent a plausible candidate mechanism for the functional augmentation of the h-conductance gradient (assuming that differences in subunit expression translate into differences in h-channel subunit composition). However, the influence of secondary factors such as interaction with ␤-subunits (such as TRIP8b), posttranslational modification (i.e., calcineurin and p38 MAPK), and interaction with chemical ligands (cAMP and PIP 2 ) cannot be ruled out on the basis of our data (Chen et al 2001;Jung et al 2010;Lewis et al 2009;Pian et al 2006;Poolos et al 2006;Santoro et al 2009;Ulens and Tytgat 2001;Zolles et al 2006Zolles et al , 2009.…”

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

“…The pathogenesis of epilepsy or epileptogenesis is complex and has not been clearly defined, but it generally involves an imbalance between excitatory and inhibitory neurotransmission in multiple brain structures (11). Changes in the expression, localization, and function of a number of ion channels, including Nav1.2 (12,13), occur in the period following the initial acute seizures and may contribute to the resultant epileptogenesis, and at least some of these are mediated through altered PTMs (14). Nav channels, including Nav1.2, are also mutated in several forms of inherited epilepsy (15).…”

Traumatic brain injury causes a Tacrolimus-sensitive increase in non-convulsive seizures in a rat model of post-traumatic epilepsy

“…One possibility is that a pathological increase in calcineurin activity leads to the dysregulation of hyperpolarization-activated cation (HCN) channels [10]. Calcineurin has been shown to down regulate gating of HCN channels, resulting in neuronal hyper excitability [10], and a reduction in HCN channel expression in rat cortex is associated with epileptogenesis after status epilepticus [38,39].…”

“…Calcium-sensitive enzymes which regulate neuronal excitability may thus play a critical role in epileptogenesis. Indeed, animal models of acquired epilepsy have implicated a calcium-sensitive phosphatase, calcineurin [9][10][11][12]. Recent research by our lab suggests that calcineurin may also be involved in the epileptogenesis after TBI.…”

“…In a study of lateral fluid percussion TBI in rats, a well-characterized model of post-traumatic epilepsy [13,14], we identified an acute post-injury increase in calcineurin activity [15]. This increase could facilitate epileptogenesis through the dysregulation of hyperpolarizing cyclic nucleotide-gated (HCN) channels [10] or voltage-gated potassium channels (K V 2.1) [16]. An increase in calcineurin activity may also alter excitatory synaptic circuits by destabilizing dendritic spines [17][18][19][20][21][22], potentially creating opportunities for maladaptive plasticity [23].…”