Abstracts

h-channels (hyperpolarization-activated cation channels; I[sub]h[/sub]; HCN) have been hypothesized to play a role in epileptogenesis due to recent evidence that they are downregulated by seizures, are upregulated by several antiepileptic drugs, and produce epilepsy when genetically deleted in an animal model. Little is known about possible intracellular modulators of h-channel function apart from the well described role of cAMP. We sought to identify novel protein kinase modulators of h-channels. Putative kinase modulators of rat HCN1 and HCN2 were identified using the Scansite software tool to search the SWISS-PROT amino acid sequence database for kinase consensus phosphorylation motifs. We then tested the highest rank kinase candidate against I[sub]h[/sub] in rat CA1 hippocampal pyramidal and [quot]pyramidal-like[quot] principal (PLP) neurons using cell-attached patch clamp recordings in rat hippocampal slices. Kinase inhibitors were applied through the recording pipette and via bath pre-incubation for at least 20 mins. Whole-cell current clamp recordings were used to verify kinase inhibitor actions on hippocampal neuron passive membrane properties. Use of the Scansite tool produced a rank-ordered list of 15 putative kinase modulators of either HCN1 or HCN2. The highest ranked kinase was p38 MAPK, a member of the mitogen-activated protein kinase family which also includes ERK and JNK. Application of a specific cell-permeable inhibitor of p38, SB203580 (10 uM), produced an [sim]20 mV hyperpolarizing shift in I[sub]h[/sub] voltage-dependent activation recorded in pyramidal neuron dendrites (V[sub]1/2[/sub] in control = -86 [plusmn] 3.5 mV [mean [plusmn] SEM], n=12; V[sub]1/2[/sub] in SB = -107 [plusmn] 4.7 mV, n=8, p=.002). This shift caused a hyperpolarization in pyramidal neuron resting potential (RP in control = -60 [plusmn] 3.5 mV, n=7; RP in SB = -71 [plusmn] 3.5, n=5, p=.05). Measurement of passive membrane properties using whole-cell recordings in PLP neurons and delivery of drug through the pipette only showed an increase in input resistance (IR) 30 min after the start of recording of [sim]19%, consistent with downregulation of I[sub]h[/sub] (change in IR from start of recording in control
= -7.4 [plusmn] 2.4%, n=3; in SB= 11.8% [plusmn] 1.9%, n=4, p=.001). These results demonstrate that blockade of p38 MAPK activity produces a significant downregulation of I[sub]h[/sub] in hippocampal pyramidal neurons, with concomitant hyperpolarization of resting potential and increased input resistance. There has been little prior evidence that p38 MAPK modulates voltage-gated ion channels, but these results suggest that p38 MAPK may be a candidate mediator of recently described seizure-induced alterations in h-channel activity. (Supported by NINDS and the Epilepsy Foundation.)