Modulation of h-channels in hippocampal pyramidal neurons by p38 mitogen-activated protein kinase

Abstract

Hyperpolarization-activated cyclic nucleotide-gated ion channels (h-channels; I(h); HCN) modulate intrinsic excitability in hippocampal and neocortical pyramidal neurons, among others. Whereas I(h) mediated by the HCN2 isoform is regulated by cAMP, there is little known about kinase modulation of I(h), especially for the HCN1 isoform predominant in pyramidal neurons. We used a computational method to identify a novel kinase modulator of h-channels, p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK in hippocampal pyramidal neurons caused a approximately 25 mV hyperpolarization of I(h) voltage-dependent activation. This downregulation of I(h) produced hyperpolarization of resting potential, along with increased input resistance and temporal summation of excitatory inputs. Activation of p38 MAPK caused a approximately 11 mV depolarizing shift in I(h) activation, along with depolarized resting potential, and decreased input resistance and temporal summation. Inhibition of related MAPKs, ERK1/2 (extracellular signal-related kinase 1/2) and JNK (c-Jun N-terminal kinase), produced no effect on I(h). These results show that p38 MAPK is a strong modulator of h-channel biophysical properties and may deserve additional exploration as a link between altered I(h) and pathological conditions such as epilepsy.

Figure 1.

Characteristics of I_h in pyramidal and PLP neurons. A, Biocytin-filled image of a typical CA1 hippocampal pyramidal neuron and accompanying traces showing I_h from a dendritic recording. Voltage commands are shown above each trace. The dendritic recording distance was 180 μm. B, Biocytin-filled image of a PLP neuron and sample I_h traces from a somatic recording. PLP neurons showed a similar morphology to CA1 pyramidal neurons, and I_h from somatic PLP recordings demonstrated similar kinetics to that from pyramidal dendrite recordings. C, Voltage-dependent activation of I_h was similar in pyramidal dendrites (circles) and PLP somas (triangles). D, I_h activation time constants were similar for pyramidal dendrites (black bars) and PLP somas (gray bars). Error bars indicate SEM.

Figure 2.

Blockade of p38 MAPK caused a hyperpolarizing shift in I_h activation in both pyramidal dendrites and PLP somas. A, Application of SB203580 (10 μm), a selective inhibitor of p38 MAPK, by bath preincubation and in the recording pipette caused an ∼25 mV hyperpolarizing shift in I_h half-activation voltage (V_1/2) in pyramidal dendrites (triangles) compared with control (circles). B, Under similar recording conditions to those in A, blockade of p38 MAPK by SB202190 (20 μm), another p38 MAPK inhibitor, caused a hyperpolarizing shift in V_1/2 of similar magnitude in PLP somas to that seen in pyramidal dendrites. C, Preincubation and pipette delivery of U0126 (20 μm), an inhibitor of ERK1/2, had no effect on I_h activation in pyramidal dendrites (triangles) compared with control (circles). D, Preincubation and pipette delivery of SB202474 (10 μm; crosses), an inactive analog of SB203580, and SP600125 (10 μm; triangles), an inhibitor of JNK, caused no significant change in I_h activation in PLP somas compared with control (circles). E, Application of a membrane permeable form of cAMP, 8-Br-cAMP (100 μm; triangles), caused an insignificant depolarizing shift in I_h activation in pyramidal dendrites compared with control (circles). F, Application of anisomycin (20 μm; triangles), an activator of p38 MAPK, caused an ∼11 mV depolarizing shift in I_h activation in PLP somas compared with control (circles). Error bars indicate SEM.

Figure 3.

Effects of p38 MAPK blockade on I_h properties. A, Sample I_h traces from a pyramidal dendrite elicited by voltage commands from −30 to −150 mV under control conditions. Tail currents are shown at expanded time scale at right. Traces elicited by a −80 mV command are indicated by an arrowhead. Calibration: 5 pA (1.8 pA for tail currents), 250 ms (12 ms for tail currents). B, I_h traces from pyramidal dendrite using same protocol as in A after p38 MAPK inhibition by SB203580 (10 μm). Traces elicited by a −79 mV command are indicated by an arrowhead. Note reduction of I_h by p38 MAPK inhibition at physiological levels of hyperpolarization. Calibration: 10 pA (5.5 pA for tail currents), 250 ms (12 ms for tail currents). C, Average RP (in millivolts) after patch rupture after voltage-clamp experiments. Pyramidal dendrite (Dend) RP was significantly hyperpolarized after application of SB203580 (10 μm; ∗p < 0.01). PLP soma RP was not significantly hyperpolarized after application of SB202190 (20 μm). D, Maximal I_h density recorded at −150 mV in PLP somas showed no significant change after p38 MAPK inhibition by SB202190 (20 μm). Error bars indicate SEM.

Figure 4.

Downregulation of I_h by p38 MAPK inhibition increased IR. A, Sample current-clamp recordings in pyramidal dendrites from separate representative neurons in response to hyperpolarizing current injections of 100 and 200 pA under control conditions and after incubation in SB203580 (10 μm) shows that blockade of p38 MAPK increased IR. The arrowhead indicates depolarizing “sag” in voltage response associated with I_h activation. Resting potential was held at −65 mV by steady-state current injection for all measurements. Group data show that blockade of p38 MAPK by SB203580 caused an ∼72% increase in IR compared with the control population (∗p < 0.05). B, Similar increases in IR were seen during current-clamp recordings in PLP somas after incubation in SB203580. IR increased further when neurons were incubated in ZD7288 (10 μm), a blocker of h-channels, abolishing the depolarizing sag. When PLP neurons were incubated with SB203580 and ZD7288 together, there was no significant difference in IR compared with ZD7288 alone, demonstrating that the effects of SB203580 on IR required intact I_h (∗∗∗p < 0.001 compared with control by one-way ANOVA). Error bars indicate SEM.

Figure 5.

Downregulation of I_h by p38 MAPK inhibition increased TS. A, Representative current-clamp recordings in pyramidal dendrites in response to current injection of 20 Hz α-function waveforms under control conditions (thin trace, Ctrl), after incubation in SB203580 (10 μm; thick trace, SB), or superimposed (Both). TS was measured as the ratio of the peak amplitude of the fifth response to that of the first, and increased after p38 MAPK blockade. Resting potential was held at −65 mV by steady-state current injection for all measurements. Group data showed that incubation in SB203580 caused an ∼16% increase in TS in pyramidal dendrites (∗p < 0.05). B, Similar increases in TS were seen during current-clamp recordings in PLP somas after incubation in SB203580. Sample recordings from separate neurons are shown in response to 20 Hz α-function current injections. Responses in drug (thick traces) are superimposed over control response (thin trace). Group data show that TS increased ∼25% after incubation in SB203580. TS increased further when neurons were incubated in ZD7288 (10 μm). When PLP neurons were incubated with SB203580 and ZD7288 together, there was no significant difference in TS compared with ZD7288 alone, demonstrating that similar to the IR data, the effects of SB203580 on TS required intact I_h (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with control by one-way ANOVA). Error bars indicate SEM.

Figure 6.

Activated p38α MAPK subunits upregulate I_h. A, Representative current-clamp recordings from PLP neurons in response to 200 pA hyperpolarizing current injections measured initially (thin trace, 0 min) or after 15 min of recording in the same neuron (thick trace, 15 min). Resting potential was held at −65 mV by steady-state current injection for all measurements. Under control conditions and with inactivated p38α MAPK added to the pipette solution, IR had decreased minimally after 15 min of recording. With active p38α MAPK added to the pipette solution, IR decreased significantly after 15 min of recording. B, Measurement of TS in PLP neurons in response to 20 Hz α-function current injections and under the same conditions as in A showed that TS changed minimally after 15 min of recording in control and inactivated p38α MAPK conditions. Activated p38α MAPK caused a significant decrease in temporal summation after 15 min of recording. C, D, Group data showing decrease in IR and TS, respectively, after 15 min of recording with activated p38α MAPK compared with control and inactivated p38α MAPK (∗p < 0.05, ∗∗p < 0.01 compared with activated p38α MAPK by one-way ANOVA). Error bars indicate SEM.