Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability and Working Memory

Abstract

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na⁺ influx through HCN channels activates Slack Na⁺-activated K⁺ (K_Na) channels to hyperpolarize the membrane. We have found that HCN and Slack K_Na channels coimmunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces K_Na current in pyramidal cells that express both HCN and Slack channels, but has no effect on K_Na currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K⁺ +current indirectly by lowering Na⁺ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca²⁺ reporter results in elevation of cytoplasmic Ca²⁺, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Keywords: HCN channel; Slack channel; neuronal excitability; working memory.

Figure 1. Slack channels co-immunoprecipitate and colocalize with HCN channels in mouse frontal cortex.

A. Coimmunoprecipitation of Slack and HCN channels from mouse frontal cortex. Frontal cortex lysates were subjected to immunoprecipitation using anti-Slack-B antibody or chicken IgY, followed by Western blotting with either Anti-HCN1 or Anti-Slack antibody. Input: sample before Slack or IgY immunoprecipitation; IP: Slack IP sample; IP Sup: supernatant after Slack IP; IgY: IgY IP Sample; IgY sup: supernatant after IgY IP. Left: The presence of HCN1 band in the Slack IP sample, but not in the IgY IP lane indicates interaction of HCN1 channel with Slack channel. The presence of HCN1 in the IgY sup confirms no binding of the negative IgY beads with HCN1. Right: The presence of Slack band in the IP lane but absence in the IgY lane, indicating specificity of Slack antibody immunoprecipitation. B. Immunolocalization of Slack and HCN1 in 2-month-old mouse mPFC. Triple immunolabeling experiments depicting colocalization between HCN1 (green) and Slack (red) at the somata (MAP2, purple) of cells in layer II-III of mouse mPFC. Scale bar = 50 μm for all the photographs except for the first one and the last three (scale bar = 25 μm). C. Immunolocalization of Slack and HCN1 in cultured frontal cortical neurons on DIV14. Triple immunolabeling experiments depicting colocalization between HCN1 (green) and Slack (red) at the soma and dendrites (MAP2, purple) of cultured cortical neuron. Scale bar, 50 μm.

Figure 2. Co-localization of HCN1 and Slack channels within dendritic spines in rat mPFC layer II/III.

A. Immunogold labeling (15 nm particles) reveals precise labeling of HCN1 within dendritic spines of pyramidal cells in rat prelimbic mPFC layer II/III. HCN1 channels are concentrated in the perisynaptic compartment adjacent to asymmetric, presumed glutamatergic-like synapses. B. Similar to HCN1 channel labeling, immunogold labeling (15 nm particle) for Slack channels is enriched along the plasma membranes bordering asymmetric axospinous synapses, receiving presumed glutamatergic input, within dendritic spines in rat mPFC layer II/III. C. Co-localization of HCN1 (15 nm particle) and Slack (5 nm particle) channels within dendritic spines in rat mPFC layer II/III. In dendritic spine heads, the immunoparticles for HCN1 and Slack overlap at perisynaptic locations. Synapses are between arrows. Color-coded arrowheads point to HCN1(red) and Slack (blue) immunoreactivity. Pro les are pseudocolored for clarity. Ax, axon; Sp, dendritic spine; Mit, mitochondria. Scale bars, 200 nm.

Figure 3. Slack-B channels reverse the cellular effects of HCN channel activation.

A. Diagram of components in HEK cells used for the assay. HEK-293 cells are transduced with bPAC (the actuator), HCN2 (ion channel that is cAMP coupled), and R-GECO1 (a Ca²⁺ sensor). Brief pulses of blue light (50 ms of 488nm light) activate bPAC to increase cAMP levels that results in opening of the HCN2 channel, producing a slow increase in Ca²⁺ uorescence signal (Thomas and Hughes 2020). B. Responses to light pulses in cells expressing HCN2 alone with those co-expressing Slack-B with HCN2 channels. Combined plots of normalized data comparing the responses of cells expressing HCN2 alone with those co-expressing Slack-B with HCN2 channels. Results are shown mean ± SEM for three independent experiments each of which measured responses of 52 independent cells for a total of 156 cells in each condition (p = 0.0149, Kolmogorov-Smirnov test).

Figure 4. Blocking HCN channels with ZD7288 reduces outward K⁺ currents in mPFC pyramidal neurons.

A. Representative current traces from whole cell voltage clamp recordings in cultured frontal cortical neurons on DIV14 induced by voltage steps from −90 to +50 mV in 10 mV increments before and after application of HCN channel blocker ZD7288 (10 μM, 10 min). B. Summary data show the outward K⁺ currents for each voltage step in neurons before and after application of ZD7288. Data are shown as mean ± SEM (n = 12–14, two-way ANOVA). C. Maximal K⁺ current at +50 mV for each condition. Data are shown as mean ± SEM (n = 12–14, Student’s t test). D and E. Representative current traces and summary data from whole cell voltage clamp recordings in mPFC pyramidal neurons of 2-month-old mice induced by voltage steps from −120 to +200 mV in 20 mV increments before and after application of ZD7288 (10 μM, 10 min). Data are shown as mean ± SEM (n = 6, two-way ANOVA). F. Maximal K⁺ current at +200 mV for each condition. Data are shown as mean ± SEM (n = 6, Student’s t test). G and H. Representative current traces and summary data from whole cell voltage clamp recordings in cultured frontal cortical neurons on DIV14 induced by voltage steps from −90 to +50 mV in 10 mV increments before and after application of Slack channel blocker SLK-01 (10 μM, 10 min) or SLK-01 with ZD7288 (10 μM, 10 min). Data are shown as mean ± SEM (n =7–12, two-way ANOVA). I and J. Representative current traces and summary data from whole cell voltage clamp recordings in HEK cells stably expressing Slack induced by voltage steps from −120 to +80 mV in 20 mV increments before and after application of ZD7288 (10 μM, 10 min). Data are shown as mean ± SEM (n = 4, two-way ANOVA). K. Maximal K⁺ current at +80 mV for each condition. Data are shown as mean ± SEM (n = 4, Student’s t test). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 5. SLK-01 inhibits Slack currents.

A. Structure of SLK-01. B. Representative traces of currents elicited by voltage steps between −100 and +60 mV from a holding potential of −80 mV, in control medium and afer application of 40 mM SLK-01. C. Concentration-response relationship for SLK-01 at +40 mV. Compound was sequentially applied to cells (n = 7) and the peak current measured. The inhibition increased during depolarizing commands producing near complete blockade after 50 ms at the highest concentration (40 mM). D. Current-voltage relationships for cells treated with 40 mM SLK-01. E. Use-dependence of block of Slack currents by SLK-01. Currents were evoked by step depolarizations (220 ms) from a holding potential of −80 mV to +40 mV at rates of 1, 2 or 5 Hz. Plots show the progressive change in current amplitude recorded at the end of each pulse in the absence (n = 4) or presence of 40 mM SLK-01 (n = 3). F. SLK-01 has no effect on BK currents. Representative traces of currents in CHO cells stably expressing rat BK channels before and after application of 40 mM SLK-01. Currents were evoked by voltage steps between −100 and +60 mV from a holding potential of −80 mV. G. Group data quanti ying the mean amplitude of currents recorded at +60 mV before and after SLK-01 (40 mM n = 6).

Figure 6. Blockade of Slack channels with infusion of SLK-01 into rat mPFC improved delayed alternation performance in a T maze.

A. Diagram of the delayed alternation task in a T maze for testing spatial working memory performance in the rat. On the first trial, the rat can choose either arm and be rewarded, on subsequent trials the rat must alternate its response. The rat spends the delay period in the start box, and thus must remember its previous spatial response over this delay. B. Infusion of SLK-01 (20–50 μM) produced a dose-related improvement in working memory performance, where a best dose of either 30 or 50 μM significantly improved performance compared to vehicle control (**p < 0.01, Student t-test).