Protein kinase modulation of dendritic K+ channels in hippocampus involves a mitogen-activated protein kinase pathway

Abstract

We investigated mitogen-activated protein kinase (MAPK) modulation of dendritic, A-type K+ channels in CA1 pyramidal neurons in the hippocampus. Activation of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) leads to an increase in the amplitude of backpropagating action potentials in distal dendrites through downregulation of transient K+ channels in CA1 pyramidal neurons in the hippocampus. We show here that both of these signaling pathways converge on extracellular-regulated kinases (ERK)-specific MAPK in mediating this reduction in dendritic K+ current, which is confirmed, in parallel, by biochemical assays using phosphospecific antibodies against the ppERK and pKv4.2. Furthermore, immunostaining indicates dendritic localization of ppERK and pKv4.2. Taken together, these results demonstrate that dendritic, A-type K+ channels are dually regulated by PKA and PKC through a common downstream pathway involving MAPK, and the modulation of these K+ channels may be accounted for by the phosphorylation of Kv4.2 subunits.

Fig. 1.

Examples of CA1 pyramidal neurons and whole-cell and cell-attached recordings from soma and dendrites. A,A pyramidal neuron in hippocampal CA1 region filled with biocytin through a recording electrode patched in the dendrites. Locations for patch electrodes are indicated. B, Representative traces for action potentials (AP) and outward transient K⁺ currents (I_K(A)) from the soma and a dendrite 340 μm from the soma (current traces from Hoffman et al., 1997). In the soma, the AP amplitude is ∼120 mV, whereas I_A is ∼11 pA. In the dendrites the amplitude of the AP has declined to ∼26 mV, whereasI_A is almost sixfold bigger than in the soma. C, Summary data for AP amplitude as a function of recording distance from the soma (recording data are binned to 50 μm segments). The number of cells for each group is inparentheses.

Fig. 2.

Isoproterenol application produced a time- and kinase-dependent increase in action potential amplitude in distal dendrites. A, At 32°C, iso application (1 μm) resulted in a 105% increase in amplitude of an AP recorded 250 μm from the soma. Prolonged application sometimes resulted in a broad, presumably Ca²⁺-dependent AP (arrow) after the Na⁺ AP.B, In a more proximal recording location (160 μm from the soma), iso had no effect, even at 32°C. C,Preincubation of the slices in U0126 (20 μm) for 30 min blocked the effect of iso on AP amplitude in a dendrite 250 μm from the soma. D, Summary data. Percentage of changes in action potential amplitude and initial rate of rise were measured when responses got stable, usually between 15 and 25 min. The number of cells for each group is in parentheses. Recordings were made from distal dendrites (250–350 μm). E, Wash-in of 20 μm U0126 for 30 min caused a reduction of AP amplitude at a 200 μm dendritic location. F, Summary data. Percentage of changes in AP amplitude and initial rate of rise are indicated from recordings made from 150–225 μm on dendrites (n = 5).

Fig. 3.

Effects of direct activation of PKA or PKC were blocked by MEK inhibitors. A, Antidromically initiated APs (recorded 270 μm from the soma) at various time points before and after the application of 50 μm forskolin (FSK). FSK increased the AP amplitude in distal dendrites by 77.9 ± 10.5% (n = 7).B, Previous incubation with MEK inhibitor U0126 (20 μm) blocked the effect of FSK (n = 4) (different cell, 250 μm from the soma). C, APs recorded 275 μm from the soma before and during the presence of PKC activator PDA (10 μm). Cells were held hyperpolarized to −80 mV to remove Na⁺ channel inactivation before antidromic stimulation. D, Another recording 250 μm from the soma in the slice preincubated in U0126 for 30 min, AP amplitudes are shown at various time points of PDA application. Cell was held −80 mV before antidromic stimulation. E,Summary data. Percentage of changes in action potential amplitude and maximum rate of rise were measured when responses got stable, usually between 15 and 25 min, and the number of cells for each group is inparentheses. Recordings were made from distal dendrites (250–350 μm). Significant difference revealed by a two-samplet test is designated by anasterisk.

Fig. 4.

Effects of bath application of PDA.A, Responses recorded from a 250 μm dendritic location in the absence (top traces) and in the presence of PDA (20 μm) (bottom traces) were superimposed. PDA application increased frequency of spontaneous EPSPs recorded after the antidromic AP (truncated). B, In PDA, in response to single antidromic stimulation, sometimes more than one AP (top trace) and possible Ca²⁺-dependent AP (bottom trace, arrow) can be observed. C,A train of antidromic APs was evoked at a rate of 50 Hz. PDA greatly decreased the attenuation of dendritic APs during a train.D, Summary data for C. Percentage of changes of normalized 10th/first AP in a train of antidromic APs are compared between control, PDA (n = 5), and PDA + U0126 (n = 4).

Fig. 5.

Stimulation of β-adrenergic receptors, the PKA cascade, or PKC leads to increased activation of ERK2 and increased ERK phosphorylation of Kv4.2 in hippocampal area CA1. A,β-adrenergic receptor activation by isoproterenol (ISO) application leads to Kv4.2 phosphorylation by ERK. Slices were exposed to vehicle [control (C)] or iso, with or without the MEK inhibitor U0126. Shown are representative blots of a membrane fraction prepared from area CA1 tissue, probed with JPA170 (top, left) or α-ppERK (top, right). Two bands revealed by α-ppERK represent ERK1/2 (p42 and p44). Below the blots are summary densitometric analyses, showing increased immunoreactivity following stimulation with iso, as detected with antibody JPA170 (215.3 ± 41.0% of vehicle-treated control; n= 11; p < 0.01) or α-ppERK (177.2 ± 20.2%; n = 20; p < 0.001). Conjunctive application of U0126 blocked the isoproterenol-induced increase in ERK activation and phospho-Kv4.2 immunoreactivity (JPA170: 88.5 ± 11.6%, n = 11, p< 0.01; α–ppERK: 19.8 ± 4.7%, n = 20,p < 0.001). B, Activation of the PKA cascade by forskolin (FSK) leads to Kv4.2 phosphorylation by ERK. Slices were exposed to vehicle (C) or FSK, with or without the MEK inhibitor U0126. Shown are representative blots of a membrane fraction prepared from area CA1 tissue, probed with JPA170 (middle, left) or α–ppERK (middle, right). Below the blots are summary densitometric analyses, showing increased immunoreactivity after stimulation with FSK, as detected with antibody JPA170 (152.1 ± 14.0% of vehicle-treated control; n= 8; p < 0.01) or α-ppERK (298.2 ± 44.0%;n = 13; p < 0.001). Conjunctive application of U0126 blocked the FSK-induced increase (JPA170: 55.1 ± 8.6%, n = 8,p < 0.001; α-ppERK: 24.5 ± 16.0,n = 13, p < 0.001).C, PKC activation by phorbol ester application leads to Kv4.2 phosphorylation by ERK. Slices were exposed to vehicle (C) or phorbol 12,13-diacetate (PDA), with or without the MEK inhibitor U0126. Shown are representative blots of a membrane fraction prepared from area CA1 tissue, probed with JPA170 (bottom, left) or α-ppERK (bottom, right). Below the blots is summary densitometric analysis, showing increased immunoreactivity following stimulation with PDA, as detected with antibody JPA170 (153.0 ± 18.3% of vehicle-treated control; n = 9; p < 0.01) or α-ppERK (316.5 ± 28.9; n = 13;p < 0.001). Conjunctive application of U0126 blocked the PDA-induced increase relative to U0126-treated control (JPA170: 100.9 ± 14.9%,n = 9, p < 0.05; α-ppERK: 19.4 ± 8.2%, n = 13, p< 0.001). For all experiments, the immunoreactivity of the dual-phospho-ERK antibody, α-ppERK, was increased in both the membrane and cytosolic fractions (data not shown) in response to activator application. There was no JPA immunoactivity detected in control experiments for cytosolic fraction. Activator + U0126 immunoreactivity was normalized to U0126-alone control.

Fig. 6.

Distribution of ppERK and pKv4.2 in adult rat hippocampal slices. A, ppERK staining at proximal (top, left) and distal dendrites (top, right) in stratum pyramidal (s.p.), stratum radiatum (s.r.), but not in stratum oriens (s.o..) and stratum lacunosum-moleculare (s.l.-m.) of CA1 region of hippocampus. Scale bar, 50 μm. B, pKv4.2 staining in the same region of hippocampus. Scale bar, 50 μm.

Fig. 7.

Kv4.2 subunit is the target of MAPK signaling pathway. A, Schematic diagram of Kv4.2 subunit, showing six transmembrane domains and the intracellular N and C termini domains. Approximate locations of known ERK (*) and PKA (⋄) phosphorylation sites are highlighted. B, Our work suggests the following signal transduction pathways leading to Kv4.2 phosphorylation. PKA and PKC activation by neurotransmitters converge onto MAPK, which phosphorylates Kv4.2, resulting in changes in channel biophysical properties. PKA and PKC can also phosphorylate Kv4.2 directly without going through MAPK pathway, possibly serving as channel targeting mechanisms.