Research Articles, Cellular/Molecular

Glucocorticoids Rapidly Modulate CaV1.2-Mediated Calcium Signals through Kv2.1 Channel Clusters in Hippocampal Neurons

Di Wan, Tongchuang Lu, Chenyang Li and Changlong Hu
Journal of Neuroscience 6 November 2024, 44 (45) e0179242024; https://doi.org/10.1523/JNEUROSCI.0179-24.2024

Abstract

The precise regulation of Ca2+ signals plays a crucial role in the physiological functions of neurons. Here, we investigated the rapid effect of glucocorticoids on Ca2+ signals in cultured hippocampal neurons from both female and male rats. In cultured hippocampal neurons, glucocorticoids inhibited the spontaneous somatic Ca2+ spikes generated by Kv2.1-organized Ca2+ microdomains. Furthermore, glucocorticoids rapidly reduced the cell surface expressions of Kv2.1 and CaV1.2 channels in hippocampal neurons. In HEK293 cells transfected with Kv2.1 alone, glucocorticoids significantly reduced the surface expression of Kv2.1 with little effect on K+ currents. In HEK293 cells transfected with CaV1.2 alone, glucocorticoids inhibited CaV1.2 currents but had no effect on the cell surface expression of CaV1.2. Notably, in the presence of wild-type Kv2.1, glucocorticoids caused a decrease in the surface expression of CaV1.2 channels in HEK293 cells. However, this effect was not observed in the presence of nonclustering Kv2.1S586A mutant channels. Live-cell imaging showed that glucocorticoids rapidly decreased Kv2.1 clusters on the plasma membrane. Correspondingly, Western blot results indicated a significant increase in the cytoplasmic level of Kv2.1, suggesting the endocytosis of Kv2.1 clusters. Glucocorticoids rapidly decreased the intracellular cAMP concentration and the phosphorylation level of PKA in hippocampal neurons. The PKA inhibitor H89 mimicked the effect of glucocorticoids on Kv2.1, while the PKA agonist forskolin abrogated the effect. In conclusion, glucocorticoids rapidly suppress CaV1.2-mediated Ca2+ signals in hippocampal neurons by promoting the endocytosis of Kv2.1 channel clusters through reducing PKA activity.

Significance Statement

Glucocorticoids are well known stress hormones, but their rapid nongenomic effects on the brain remain unclear. The hippocampus is one of the main effectors of glucocorticoids in the brain. Here, we demonstrate that glucocorticoids rapidly inhibit voltage-gated calcium (CaV) channel CaV1.2-mediated somatic calcium (Ca2+) spikes in hippocampal neurons. Glucocorticoids reduce the surface expression of Kv2.1 voltage-activated potassium (Kv) channel clusters but do not affect the surface expression of nonclustering Kv2.1. Moreover, glucocorticoids induce the endocytosis of CaV1.2 channels through wild-type Kv2.1. However, glucocorticoids cannot induce the endocytosis of CaV1.2 channels through nonclustering Kv2.1S586A mutant channels. This study elucidates the complex interaction between glucocorticoids, Kv2.1, and CaV1.2 channels, enhancing our understanding of glucocorticoid actions in the brain.

Introduction

Glucocorticoids, also known as stress hormones, are produced by the zona fasciculata cells of the adrenal cortex in a diurnal pattern. In the mammalian brain, glucocorticoids induce variable neuronal effects via both genomic and nongenomic signaling pathways. While the genomic pathway has been well studied, the rapid nongenomic effects of glucocorticoids, which usually occur within a few minutes, are still not fully understood. The hippocampus is one of the main effectors of glucocorticoids in the brain (Spiga et al., 2014). The level of glucocorticoids shows a distinct circadian and ultradian rhythm in the brain (Qian et al., 2012; Kalafatakis et al., 2019). Recent studies have shown that the ultradian rhythmicity of glucocorticoids is critical in regulating normal emotional and cognitive responses in humans (Kalafatakis et al., 2018). Glucocorticoids may modulate neural function through rapid nongenomic effects during these fluctuations.

The Ca2+ entry through L-type calcium channels, mainly CaV1.2 and CaV1.3 channels, is essential for the normal functioning of various mammalian brain neurons (Simms and Zamponi, 2014; Zamponi et al., 2015). Even though both mRNA expressions of CaV1.2 and CaV1.3 have been suggested in the hippocampus, L-type Ca2+ current in hippocampal neurons may be predominantly mediated by CaV1.2 channels (Hell et al., 1993; Sinnegger-Brauns et al., 2009; Schlick et al., 2010; Hofmann et al., 2014). Accordingly, the hippocampal pyramidal neurons in CaV1.2 knock-out mice displayed a nearly complete loss of L-type Ca2+ currents, and CaV1.2 but not CaV1.3 knock-out mice showed deficits in hippocampal long-term potentiation and memory formation (Clark et al., 2003; Moosmang et al., 2005; Temme et al., 2016).

The family of Kv2 channels, consisting of Kv2.1 and Kv2.2, plays a vital role in controlling neural excitability (Gutman et al., 2005; Johnston et al., 2010). The Kv2.1 channel is widely expressed throughout the mammalian brain and is the major conductor of rectifying potassium currents in hippocampal neurons (Murakoshi and Trimmer, 1999; Mohapatra et al., 2009). Kv2.1 channels in cell membrane exist in two forms: clustered and nonclustered (Fox et al., 2013) . Nonclustered Kv2.1 channels conduct K+ normally and are critical for maintaining repetitive firing of neurons (Guan et al., 2013; Liu and Bean, 2014). Clustered Kv2.1 channels are barely conductive and serve as a platform to organize neuronal endoplasmic reticulum/plasma membrane (ER/PM) junctions (Fox et al., 2015; Bishop et al., 2018; Johnson et al., 2018; Kirmiz et al., 2018a, b). ER/PM junctions are critical for lipid and Ca2+ homeostasis in eukaryotic cells (Gallo et al., 2016; Okeke et al., 2016) and are particularly abundant in the soma of mammalian brain neurons (Wu et al., 2017). Recent studies have shown that clustered Kv2.1 channels recruit CaV1.2 to somatic ER/PM junctions to form Ca2+ signaling microdomains. These microdomains control somatic Ca2+ signals and regulate excitation–transcription coupling in hippocampal neurons (Vierra et al., 2019, 2021).

In this study, we investigated the rapid effects of glucocorticoids on Ca2+ signals in hippocampal neurons. Our results show that glucocorticoids reduce cell surface expression of CaV1.2 by promoting endocytosis of clustered Kv2.1. This, in turn, inhibits somatic Ca2+ sparklets in hippocampal neurons through the PKA signaling pathway.

Materials and Methods

Cell culture and transfection

Human embryonic kidney (HEK293) cells were obtained from the cell bank of the Chinese Academy of Science. These cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution. Plasmids for rat GFP-tagged Kv2.1 channels and mouse CaV1.2 with β3 and α2δ1 subunits were transiently transfected into HEK293 cells using jetPRIME (Polyplus). For patch-clamp recordings, the cells were used after 24 h transfection. Hippocampal neurons from Sprague Dawley rats of either sex on Postnatal Day 0–1 were used for primary culture. Briefly, hippocampi were dissected and dissociated into single cells with trypsin, and then the cells were plated onto poly-L-lysine–coated glass coverslips inside a six-well plate. All experiments were conducted on neurons between 7 and 14 d in vitro (DIV) and were performed on neurons from a minimum of three separate cultures.

All protocols used were approved by the Committee on the Ethics of Animal Experiments of Fudan University and were in strict accordance with the NIH Guidelines for the Care and Use of Animals.

Molecular biology

Plasmids for rat GFP-tagged Kv2.1 (Zhang et al., 2021) channels and mouse CaV1.2 (Yang et al., 2020) were as previously reported. The Kv2.1S586A point mutant was generated using the GFP-Kv2.1 wild-type (WT) plasmid as a template. Site-directed S586A mutagenesis was performed on the Kv2.1 channel using the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene). Kv2.1S586A mutation was confirmed by sequencing. The shRNA plasmids targeting glucocorticoid receptors (GR; #sc-35505-SH; Gazorpak et al., 2023) and the control shRNA Plasmid-A (#sc-108060) were purchased from Santa Cruz Biotechnology. For the knockdown of GR, HEK293 cells were transfected with the shRNA plasmids or control plasmids for 24 h.

Live-cell imaging

Hippocampal neurons and HEK293 cells (transfected with GFP-tagged Kv2.1) were plated onto poly-L-lysine–coated glass bottom dishes. Imaging was performed in HBSS (14025092, Invitrogen) solution with 20 mM HEPES (H3375, Sigma-Aldrich). Hippocampal neurons were first incubated in regular culture medium with 2 µM fluorescent calcium indicator Cal-590 AM (20510, AAT Bioquest) for 60 min at 37°C. Dye-containing medium was then aspirated, followed by two washes in HBSS which had been warmed to 37°C. Cells were then incubated in HBSS for an additional 30 min at 37°C prior to imaging. Images were acquired on a Nikon Eclipse Ti total internal reflection fluorescence (TIRF)/widefield microscope equipped with an Andor iXon EMCCD camera and a Nikon LUA4 laser launch with 405, 488, 561, and 647 nm lasers, using a 100×/1.49 NA PlanApo TIRF objective and NIS Elements software. Images were analyzed by Fiji (https://fiji.sc). The Fiji plugin xySpark (Steele and Steele, 2014) was used for automated spark detection and analysis.

Western blot

The cytosolic and membrane proteins from HEK293 and hippocampal cells were isolated using the Pierce Cell Surface Protein Biotinylation and Isolation Kit (A44390, Thermo Fisher Scientific). The cell lysate was passed through a NeutrAvidin agarose column to collect membrane proteins, while the eluate contained the cytosolic proteins. The protein samples were resolved using 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes. The membranes were immunoblotted with anti-Kv2.1 antibody (1:1,000, ab192761, Abcam), anti-CaV1.2 antibody (1:1,000, ab84814, Abcam), anti-CaV1.2 antibody (1:200, ACC-003, Alomone Labs), anti-GR antibody (1:1,000, 3660, Cell Signaling Technology), anti-Na+-K+ ATPase (1:1,000, ab76020, Abcam), anti-beta-tubulin antibody (1:2,000, ab179511, Abcam), anti-PKA antibody (1:1,000, 4782, Cell Signaling Technology), and anti-phospho-PKA antibody (1:1,000, 5661, Cell Signaling Technology). The blots were developed using enhanced chemiluminescence reagents and imaged using either the ChemiDoc XRS+ imaging system from Bio-Rad Laboratories or the e-BLOT Touch Imager (e-BLOT Life Sciences) with manufacturer’s software. Molecular weight marker and chemiluminescent signal images were automatically overlaid by the manufacturer’s software. Quantitative analysis of detected bands was performed using Fiji (https://fiji.sc/).

Immunofluorescence

Hippocampal neurons on glass coverslips were fixed in 4% paraformaldehyde for 15 min at room temperature, washed with cold PBS, and incubated in 0.5% Tween 20 in PBS (PBST) for 10 min. Next, the neurons were blocked with 10% horse serum and 0.3% Triton X-100 in PBS for 2 h at room temperature. The primary antibodies used were mouse anti-Kv2.1 (1:500, ab192761, Abcam) and rabbit CaV1.2 (1:500, ab234438, Abcam), and they were incubated with the cells for 1 d at 4°C in a solution containing 1% horse serum and 0.3% PBST. Cells were washed three times with PBS and incubated overnight at 4°C in a secondary antibody solution containing Cy3-labeled goat anti-mouse/rabbit IgG(H + L) or Alexa Fluor 488-labeled goat anti-mouse IgG(H + L; 1:500, Beyotime Biotech), 1% horse serum, and 0.3% PBST. Following another wash with PBS, the cells were imaged using the Nikon A1+ Confocal Microscope System. The images were subjected to rolling ball background subtraction and subsequently converted into a binary mask by thresholding. The sizes of Kv2.1 clusters were measured using the “analyze particles” feature of Fiji.

cAMP assay

Cultured hippocampal neurons were treated with 1 μM corticosterone (Cort) for 1, 5, and 10 min, respectively. The cells were lysed in 0.1 M HCl. cAMP levels in hippocampal neurons were measured using a cAMP ELISA Kit (NewEast Biosciences) following the manufacturer's instructions.

Electrophysiology

Whole-cell currents in cultured hippocampal neurons and HEK293 cells were recorded using an Axopatch 200B amplifier (Axon Instruments) and data were collected with PCLAMP 10.7 software (Axon Instruments). The bath solution for potassium current recording contained the following (in mM): 140 NaCl, 2.5 KCl, 0.001 TTX and 5 4-AP, 10 glucose, 10 HEPES, and 1 MgCl2, pH adjusted to 7.4 (using NaOH). The internal solution contained the following (in mM): 135 potassium gluconate, 10 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 2 Mg-ATP, and 10 EGTA, pH adjusted to 7.3 (using KOH). For calcium current recording, the bath solution contained the following (in mM): 140 TEACl, 10 BaCl2 and 5 4-AP, 10 glucose, 10 HEPES, and 2 MgCl2, pH adjusted to 7.4 (using TEAOH). The internal solution contained the following (in mM): 125 Cs-methanesulfonate, 1 MgCl2, 10 HEPES, 5 Mg-ATP, 0.3 Tris-GTP, and 10 EGTA, pH adjusted to 7.2 (using CsOH). The pipettes were made from capillary tubing (BRAND) and had resistances of 4 to 6 MΩ under these solution conditions. Currents were sampled at 10 kHz and filtered at 2 kHz and corrected online for leak and residual capacitance transients using a P/4 protocol. All recordings were performed at room temperature.

Cortisol-BSA conjugate (CSB-MC00371b0105) was purchased from CUSABIO. Cortisol (B1951), H89 (B2190), and Forskolin (B1421) were purchased from APExBIO. Cort (HY-B1618), CORT125281 (HY-117880), RU486 (HY-13683), and PKI (HY-P0222) were purchased from MedChemExpress. All drugs were dissolved in DMSO with a final concentration not exceeding 0.1%.

Statistical analysis

Data analysis was performed using Clampfit 10.7 (Axon Instruments) and GraphPad Prism (v9.4, GraphPad Software). Normality of the data was checked by the Shapiro–Wilk test. The two-tailed paired or unpaired t test was used to compare two samples, and one-way or two-way ANOVA with Bonferroni’s post hoc test was employed for comparing multiple samples. Data are presented as means ± SEM, where “n” indicates the number of tested cells or independent tests. p < 0.05 was considered statistically significant.

Results

Glucocorticoids reduce somatic spontaneous Ca2+ signals and Kv2.1 clusters in cultured hippocampal neurons

First, we investigated the effect of glucocorticoids on somatic spontaneous Ca2+ signals in cultured hippocampal neurons under TIRF microscopy. The fluorescent calcium indicator Cal-590 AM was used to detect the spontaneous somatic Ca2+ signals in cultured hippocampal neurons. Continuous exposure to excitation light can lead to photobleaching and phototoxicity in fluorescence live-cell imaging. To mitigate these effects, we designed our recording protocol as follows: We first recorded for 1 min to capture baseline automatic calcium spike events. This was followed by a 5 min interval where cells were either exposed to 1 µM Cort (Lovell et al., 2004) or equal amount of DMSO. After this interval, we recorded for another 1 min to capture the drug effect. Bath application of 1 µM Cort for 5 min significantly reduced the frequency and amplitude of spontaneous somatic Ca2+ sparks in hippocampal neurons, with no effect on the spark width (Fig. 1A–C; Movies 1, 2).

Figure 1.
Figure 1.

Effect of glucocorticoids on spontaneous Ca2+ sparks in cultured rat hippocampal neurons. A, Representative TIRF images showing somatic Ca2+ sparks in hippocampal neurons (loaded with Cal-590 AM) under the vehicle control (0.01% DMSO) condition and in the presence of 1 μM Cort for 5 min in the same hippocampal neuron. Arrows indicate regions of interest (ROIs) where spontaneous Ca2+ signals were detected (scale bar, 10 μm). Fluorescence live-cell imaging was recorded for 1 min to capture baseline automatic calcium spike events. This was followed by a 5 min interval where cells were either exposed to 1 μM Cort or equal amount of DMSO. After this interval, we recorded for another 1 min to capture the drug effect. (Movies 1, 2). B, Fluorescence intensity traces (top panels) and kymographs (bottom panels) corresponding to the two ROIs indicated in panel A. C, Statistics for the frequency, full-width at half-maximum (FWHM) and amplitude of all spatially distinct localized Ca2+ sparks recorded from hippocampal neurons before and after 1 μM Cort or equal amount of DMSO (0.01%) treatment for 5 min using a two-way ANOVA with Bonferroni’s post hoc test (DMSO, n = 22; Cort, n = 20). Frequency, F(1, 40) = 8.175; **p = 0.0037; amplitude, F(1, 40) = 4.350; **p = 0.0049. n.s., not significant (p > 0.05). Each point corresponds to a single neuron. D, Representative examples of immunofluorescence images showing the Kv2.1 channel clusters in rat hippocampal neurons with treatment of 1 µM Cort or an equal volume of DMSO for 5 min (scale bars, 20 µm). E, Statistics for the percentage area of Kv2.1 clusters and the number of Kv2.1 clusters in the somatic region of cultured hippocampal neurons from D using a two-tailed unpaired t test (n = 18). ****p < 0.0001; t(34) = 4.792; **p = 0.0018; t(34) = 3.380.

Spontaneous somatic Ca2+ signals in hippocampal neurons are usually generated at Kv2.1 cluster-associated ER/PM junction sites, where Kv2.1 recruits CaV1.2 to form local Ca2+-release microdomains (Vierra et al., 2019). Therefore, we investigated whether Cort regulates Ca2+ signaling via Kv2.1 clusters. Kv2.1 clusters with a diameter >0.5 μm were calculated (Antonucci et al., 2001). Treatment with 1 µM Cort for 5 min reduced Kv2.1 clusters in the soma of hippocampal neurons (Fig. 1D,E). This suggests that Cort caused Kv2.1 declustering or endocytosis. To further investigate the mechanism by which glucocorticoids reduce somatic spontaneous Ca2+ signals in cultured hippocampal neurons, we tested the effect of Cort on Ca2+ and K+ channel currents in cultured hippocampal neurons.

Glucocorticoids rapidly inhibit calcium and potassium channel currents in hippocampal neurons

Since the predominant calcium channel currents in hippocampal neurons are conducted by L-type CaV1.2 channels (Moosmang et al., 2005), Ba2+ was used as the charge carrier to minimize Ca2+-induced inactivation. Whole-cell Ba2+ currents in cultured hippocampal neurons were elicited by a 100 ms depolarizing pulse to 0 mV from a holding potential of −90 mV at 10 s intervals. Bath application of 1 µM Cort significantly inhibited the Ba2+ currents in hippocampal neurons (Fig. 2A). The inhibitory effect of Cort on IBa was rapid, reaching its maximum within ∼5 min (Fig. 2B) and was irreversible upon washing. Kv2.1 channels are the major contributor of delayed rectifier potassium currents (Ik) in hippocampal neurons (Murakoshi and Trimmer, 1999). Ik in hippocampal neurons was elicited by a 200 ms depolarizing pulse to +40 mV from a holding potential of −80 mV at 10 s intervals. One micrometer Cort had a small inhibitory effect on Ik in hippocampal neurons (inhibition percentage, 15.4 ± 2.7%; Fig. 2C). Previous studies have reported that Kv2.1 declustering is usually accompanied by a large hyperpolarizing shift in the steady-state activation of IK in hippocampal neurons (Misonou et al., 2005b; Mohapatra and Trimmer, 2006). We found that extracellular application of Cort did not alter the steady-state activation of Ik in hippocampal neurons (Fig. 2D). This suggests that Cort may cause Kv2.1 cluster endocytosis instead of declustering. Therefore, we tested the effect of Cort on surface expression of Kv2.1 channels.

Figure 2.
Figure 2.

Effect of glucocorticoids on Ca2+ and K+ channel currents and membrane protein expressions in cultured hippocampal neurons. A, Left, Representative IBa traces induced by a depolarization pulse from −90 to 0 mV under the vehicle control (0.01% DMSO) condition and subsequently in the presence of 1 μM Cort and after Cort washout in the same hippocampal neuron. Right, Statistical analysis of the effect of Cort on IBa using a two-tailed paired t test (n = 6; t(5) = 7.303; ***p = 7.5 × 10−4). B, The time course of the IBa inhibition by Cort. C, Top, Representative current traces show the inhibitory effect of 1 μM Cort on IK. Bottom, Statistics for the amplitude of IK from the left using a two-tailed paired t test (n = 11; t(10) = 5.687; ***p = 2.0 × 10−4). D, Left, Representative IK traces induced by a depolarization pulse from −80 to +80 mV under the vehicle control condition and the application of 1 μM Cort in the same hippocampal neuron. Middle, IK activation curves for the DMSO (0.01%) group (n = 6 for each data point; blue) and the Cort-treated group (n = 6 for each data point; pink). Right, Statistics of the half-activation potential (V1/2) of Ik using a two-tailed paired t test (n = 6; t(5) = 1.417; p = 0.2156). n.s., not significant. E, Left, Representative Western blot of the Kv2.1 channel surface expression after 10 min Cort treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in cultured hippocampal neurons. Right, Statistics from 15 independent experiments using a two-tailed unpaired t test (t(28) = 5.256; ****p = 1.38 × 10−5). Na+, K+-ATPase as a membrane protein loading control. F, Left, Representative Western blot of the CaV1.2 channel surface expression after 10 min Cort treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in cultured hippocampal neurons. Right, Statistics from 11 independent experiments using a two-tailed unpaired t test (t(20) = 6.903; ****p = 1.05 × 10−6). Uncropped Western blot images of panels E and F are provided in Extended Data Figure 2-1.

Figure 2-1

Uncropped western blot images of Figure 2E and F. Top (Figure 2.E), representative western blot of the Kv2.1 channel surface expression after 10-min corticosterone treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in cultured hippocampal neurons. Na+, K + -ATPase as a membrane protein loading control. Bottom (Figure 2.F), representative western blot of the CaV1.2 channel surface expression after 10-min corticosterone treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in cultured hippocampal neurons. Download Figure 2-1, TIF file.

Glucocorticoids reduce membrane protein expressions of Kv2.1 and CaV1.2 in cultured hippocampal neurons

Treatment of cultured hippocampal neurons with 1 µM Cort for a duration of 10 min resulted in a significant reduction in the cell surface expression of Kv2.1 channels (Fig. 2E). Additionally, the application of 1 µM Cort also induced a substantial decrease in the cell surface expression of CaV1.2 channels in cultured hippocampal neurons (Fig. 2F). To further explore the mechanism of glucocorticoids regulating Kv2.1 and CaV1.2 channels, we tested the inhibitory effect of glucocorticoids in heterologous HEK293 cells, which lack endogenous Kv2.1 and CaV1.2 channels (Berjukow et al., 1996; Zhang et al., 2021).

Glucocorticoids have little effect on Kv2.1 currents but significantly reduce the cell surface expression of Kv2.1 channels in HEK293 cells

The primary glucocorticoids in mice and rats are Cort, while in humans, the primary glucocorticoids are cortisol. Therefore, we used cortisol on HEK293 cells transfected with Kv2.1 channels. Extracellular application of cortisol (0.1–10 μM; Zaki and Barrett-Jolley, 2002) had little effect on Kv2.1 currents in HEK293 cells (Fig. 3A,B). Similar to the effect of Cort on Kv2.1 channels in hippocampal neurons, 1 µM cortisol significantly reduced the cell surface expression of Kv2.1 in HEK293 cells (Fig. 3C). Consequently, the level of Kv2.1 channel proteins in the cell cytoplasm increased (Fig. 3D). Additionally, the effect of cortisol on Kv2.1 channels in HEK293 cells was dose-dependent (Fig. 3E). These results suggest that cortisol caused Kv2.1 endocytosis rather than declustering. It is well known that clustered Kv2.1 channels are nonconducting. Since the effect of cortisol on the surface expression of the Kv2.1 channel is much greater than the effect on the current amplitude, it is reasonable to assume that the endocytosed Kv2.1 channels induced by cortisol are from non-K+ –conducting Kv2.1 clusters. Therefore, we tested the effect of cortisol on Kv2.1 clusters. We used TIRF microscopy to image the change of Kv2.1 (N-terminal GFP fusion of Kv2.1) clusters in HEK 293 cells. Consistent with previous studies (Fox et al., 2013), large GFP-Kv2.1 clusters were clearly detected by TIRF (Fig. 3F). Extracellular application of 1 µM cortisol quickly reduced Kv2.1 clusters in HEK293 cells (Fig. 3F). Furthermore, we tested the effect of cortisol on mutant Kv2.1S586A channels, which lost the ability to form clusters (Lim et al., 2000). Extracellular application of 1 µM cortisol for 10 min did not alter the surface expression of Kv2.1S586A channels (Fig. 3G). Furthermore, extracellular application of 1 µM cortisol did not alter Kv2.1S586A channel currents (Fig. 3H). The data suggest that glucocorticoids mainly promote the endocytose of Kv2.1 clusters.

Figure 3.
Figure 3.

Effect of glucocorticoids on Kv2.1 channels in HEK293 cells. A, Left, Representative Kv2.1 current traces induced by a depolarization pulse from −80 to +40 mV under the vehicle control (0.01% DMSO) condition and subsequently in the presence of 1 μM cortisol (Cort) in the same HEK293 cell. Right, Statistics for the amplitude of Kv2.1 current from Left using a two-tailed paired t test (n = 9; t(8) = 1.322; p = 0.2226). n.s., not significant. B, Statistics for the effect of different concentrations of cortisol on the Kv2.1 current using a one-way ANOVA with Bonferroni’s post hoc test (n = 8 for each concentration; F(3, 28) = 0.3760; p = 0.7710). n.s., not significant. C, Left, Representative Western blot of the Kv2.1 channel surface expression after a 10 min treatment with cortisol (1 μM) in the extracellular solution or vehicle control (0.01% DMSO) in HEK293 cells. Right, Statistics from 14 independent experiments using a two-tailed unpaired t test (t(26) = 6.623; ****p = 5.03 × 10−7). Na+, K+-ATPase as a membrane protein loading control. D, Left, Representative Western blot of the cytosol protein level of Kv2.1 channels after 10 min cortisol treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in HEK293 cells. Right, Statistics from eight independent experiments using a two-tailed unpaired t test (t(14) = 3.536; **p = 0.0033). β-Tubulin as an internal control. E, Top, Representative Western blot illustrating Kv2.1 surface expression in HEK 293 cells treated with varying concentrations of cortisol. Bottom, Statistics from seven independent experiments using one-way ANOVA with Bonferroni’s post hoc test (F(3, 24) = 10.80; p = 0.0001). **p = 0.0046; ***p = 0.0003; n.s., not significant. F, Representative TIRF images showing the effect of 1 μM cortisol on Kv2.1 clusters in HEK293 cells. G, Left, Representative Western blot of the Kv2.1S586A mutant channel surface expression after 10 min cortisol treatment or vehicle control (0.01% DMSO) treatment in the extracellular solution in HEK293 cells. Right, Statistics from six independent experiments using a two-tailed unpaired t test (t(10) = 0.6263; p = 0.5451). H, Left, Representative Kv2.1S586A current traces induced by a depolarization pulse from −80 to +40 mV under the control condition (0.01% DMSO) and in the presence of 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of the Kv2.1S586A current using a two-tailed paired t test (n = 7; t(6) = 0.5454; p = 0.6051). n.s., not significant. Uncropped Western blot images of panels C, D, E, and G are provided in Extended Data Figure 3-1.

Figure 3-1

Uncropped western blot images of Figure 3C, D, E and G. (Figure3.C) Left, representative western blot of the Kv2.1 channel surface expression after a 10-min treatment with cortisol (1 μM) in the extracellular solution or vehicle control (0.01% DMSO) in HEK293 cells. Na+, K + -ATPase as a membrane protein loading control (right). (Figure3.D) Left, representative western blot of the cytosol protein level of Kv2.1 channels after 10-min cortisol treatment (1 μM) or vehicle control (0.01% DMSO) treatment in the extracellular solution in HEK293 cells. β-tubulin as an internal control (right). (Figure3.E) Representative western blot illustrating Kv2.1 surface expression in HEK 293 cells treated with varying concentrations of cortisol. (Figure3.G) Left, representative western blot of the Kv2.1S586A mutant channel surface expression after 10-minute cortisol treatment or vehicle control (0.01% DMSO) treatment in the extracellular solution in HEK293 cells. Na+, K + -ATPase as a membrane protein loading control (right). Download Figure 3-1, TIF file.

Glucocorticoids significantly inhibit CaV1.2 currents while having little effect on the surface expression of CaV1.2 channels in HEK293 cells

When compared with its effect on calcium channel currents in hippocampal neurons, glucocorticoids also rapidly reduced the CaV1.2 currents but with smaller amplitude in HEK293 cells (Fig. 4A). Cortisol inhibited CaV1.2 currents in a dose-dependent manner (Fig. 4A). Moreover, cortisol inhibited CaV1.2 currents at all testing potentials that were more positive than −10 mV and did not alter the steady-state activation of CaV1.2 currents (Fig. 4C). Unlike the effect on CaV1.2 in hippocampal neurons, glucocorticoids (1 µM cortisol) did not alter the cell surface expression of CaV1.2 in HEK293 cells (Fig. 4D).

Figure 4.
Figure 4.

Effect of glucocorticoids on CaV1.2 channels in HEK293 cells. A, Left, Representative IBa traces induced by a depolarization pulse from −90 to 0 mV under the control (0.01% DMSO) condition and subsequently in the presence of 1 μM cortisol in the same HEK293 cell. Right, Statistics for the effect of different concentrations of cortisol on the CaV1.2 current using a one-way ANOVA with Bonferroni’s post hoc test (n = 5–7 for each concentration). F(4, 26) = 43.26; ****p < 0.0001; n.s., not significant. B, I–V curve of CaV1.2 currents in the absence or presence of 1 μM cortisol in HEK293 cell. C, Left, Plot of CaV1.2 activation curves in the control (0.01% DMSO) group (n = 7 for each data point) and cortisol-treated group (n = 7 for each data point). Right, Statistical analysis of the half-activation potential (V1/2) of CaV1.2 using a two-tailed paired t test (t(6) = 1.681; p = 0.1438). n.s., not significant. D, Left, Representative Western blot of the CaV1.2 channel surface expression after a 10 min treatment with either 1 μM cortisol or 0.01% DMSO vehicle control in the extracellular solution in HEK293 cells. Right, Statistics from four independent experiments using a two-tailed unpaired t test (t(8) = 2.079; p = 0.0713). n.s., not significant. Uncropped Western blot images of panel D are provided in Extended Data Figure 4-1.

Figure 4-1

Uncropped western blot images of Figure 4D. Left, representative western blot of the CaV1.2 channel surface expression after a 10-min treatment with either 1μM cortisol or 0.01% DMSO vehicle control in the extracellular solution in HEK293 cells. Na+, K + -ATPase as a membrane protein loading control (right). Download Figure 4-1, TIF file.

Glucocorticoids reduce the cell surface expression of CaV1.2 channels when Kv2.1 and CaV1.2 are coexpressed in HEK293 cells

Previous studies have demonstrated a spatial correlation between Kv2.1 clusters and CaV1.2 channels in cultured hippocampal neurons (Vierra et al., 2019, 2021). Therefore, we hypothesized that glucocorticoids affect CaV1.2 surface expression through Kv2.1 channel clusters. To test our hypothesis, we cotransfected CaV1.2 with either WT Kv2.1 or mutant Kv2.1S586A expression vectors in HEK293 cells. Our results showed that 1 µM cortisol significantly reduced the cell surface express of CaV1.2 channels in the presence of WT Kv2.1 channels in the HEK293 cells (Fig. 5A), as seen in cultured hippocampal neurons. Additionally, 1 µM cortisol had a greater inhibitory effect on CaV1.2 currents in the cotransfected cells than the cells transfected with CaV1.2 alone (Fig. 5B). Furthermore, we found that 1 µM cortisol did not alter the cell surface express of CaV1.2 channels in the presence of mutant Kv2.1S586A channels in HEK293 cells (Fig.5C). This suggests that glucocorticoids induce CaV1.2 channel endocytosis through Kv2.1 clusters.

Figure 5.
Figure 5.

Effect of glucocorticoids on CaV1.2 channels in HEK293 cells expressing Kv2.1. A, Left, Representative Western blot of the CaV1.2 channel surface expression after a 10 min cortisol treatment with either 1 μM cortisol or 0.01% DMSO vehicle control in the extracellular solution in HEK293 cells expressing both CaV1.2 and Kv2.1 channels. Right, Statistics from five independent experiments using a two-tailed unpaired t test. Kv2.1, t(8) = 4.486; **p = 0.0020; CaV1.2, t(8) = 4.819; **p = 0.0013. B, Left, Representative IBa traces induced by a depolarization pulse from −90 to 0 mV under the control (0.01% DMSO) condition and subsequently in the presence of 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of Kv2.1 current from the left using a two-tailed paired t test (n = 4; t(3) = 4.372; *p = 0.0221). C, Representative Western blots (left) and statistical analysis (right) show the effect of cortisol on the surface expression of the CaV1.2 channel when CaV1.2 and Kv2.1S586A channels are coexpressed in HEK293 cells. Two-tailed unpaired t test; n = 10. Kv2.1S586A, t(18) = 1.255; p = 0.2255; CaV1.2, t(18) = 0.06056; p = 0.9524. Uncropped Western blot images of panels A and C are provided in Extended Data Figure 5-1.

Figure 5-1

Uncropped western blot images of Figure 5A and C. (Figure5.A) Representative western blots of the CaV1.2 and Kv2.1 channel surface expression after a 10-min cortisol treatment with either 1μM cortisol or 0.01% DMSO vehicle control in the extracellular solution in HEK293 cells expressing both CaV1.2 and Kv2.1 channels. (Figure5.C) Representative western blots show the effect of cortisol on the surface expression of the CaV1.2 channel when CaV1.2 and Kv2.1S586A channels are co-expressed in HEK293 cells. Download Figure 5-1, TIF file.

Glucocorticoids regulate Kv2.1 channels through membrane-associated receptors

Finally, we investigated how glucocorticoids regulate the membrane expression of Kv2.1 channels. The rapid effect of glucocorticoids is normally mediated by membrane-associated receptors (Panettieri et al., 2019). We tested the effect of cortisol-BSA conjugate (cortisol-BSA), which cannot pass through cell membranes, on Kv2.1 channels. We found that 1 µM cortisol-BSA had similar effects on the cell surface expression of Kv2.1 as 1 µM cortisol in cultured hippocampal neurons (Fig. 6A), indicating that glucocorticoids regulate cell surface expression of Kv2.1 channels through membrane-associated receptors. Interestingly, the GR inhibitors CORT125281 and RU486 did not alter the effect of cortisol on Kv2.1 channels (Fig. 6A,B), suggesting that the cortisol-induced effect is not via GRs. To further investigate, we used RNA interference to knock down GR expression. The knockdown did not alter the cortisol-induced internalization of Kv2.1 channels (Fig. 6C,D), indicating that this effect is independent of traditional GRs. Additionally, we assessed the duration of the cortisol effect and found that Kv2.1 channels returned to the cell surface 1 h after cortisol washout (Fig. 6E).

Figure 6.
Figure 6.

Glucocorticoids modulate the cell surface expression of Kv2.1 channels through membrane-associated receptors. A, Top, Representative Western blot images show the effects of cortisol-BSA and cortisol with/without the GR inhibitor CORT125281 on Kv2.1 channel surface expression in cultured hippocampal neurons. Bottom, Statistical analysis of the effects of the various treatments on the cell surface expression of Kv2.1 using a one-way ANOVA with Bonferroni’s post hoc test (n = 5; F(3, 16) = 10.14; p = 0.0006). **p < 0.01. n.s., not significant. B, Top, Representative western blot images show the effects of 1 μM cortisol with/without the GR inhibitor CORT125281(10 μM) and RU486 (10 μM) on Kv2.1 channel surface expression in HEK293 cells. Bottom, Statistical analysis of the effects of various treatments on the cell surface expression of Kv2.1 using a one-way ANOVA with Bonferroni’s post hoc test (n = 6; F(3, 20) = 11.02; p = 0.0002). DMSO versus cortisol, **p = 0.0043; DMSO versus CORT125281 + Cort, **p = 0.0013; DMSO versus RU486 + Cort, ***p = 0.0002. n.s., not significant. C, Top, Representative Western blot illustrating GR expression in HEK 293 cells transfected with control plasmids (Ctrl) or shRNA plasmids targeting GR (shGR). β-Tubulin as protein loading control. Bottom, Statistics from four independent experiments using a two-tailed unpaired t test (t(6) = 10.37; ****p = 4.7 × 10−5). D, Top, Representative Western blot showing the effect of knockdown GR on cortisol-induced internalization of Kv2.1 channels in HEK 293 cells. Na+, K+-ATPase serves as the membrane protein loading control. Bottom, Statistics from six independent experiments using a two-way ANOVA with Bonferroni’s post hoc test (F(1, 20) = 40.03; p = 0.000004). ***p = 0.0004; **p = 0.004; n.s., not significant. E, Left, Representative Western blot images show Kv2.1 channel surface expression in HEK293 cells treated with 1 μM cortisol for 10 min, and after cortisol washout for 0.5 h, 1 h, and 2 h. Right, Statistical analysis of the cell surface expression of Kv2.1 under various conditions from the left using a one-way ANOVA with Bonferroni’s post hoc test (n = 6; F(4, 25) = 5.741; p = 0.00203). DMSO versus Cort, **p = 0.0090; DMSO versus 0.5 h, **p = 0.0049. n.s., not significant. Uncropped Western blot images of panels A, B, C, D, and E are provided in Extended Data Figures 6-1 and 6-2.

Figure 6-1

Uncropped western blot images of Figure 6A, B, C and D. (Figure6.A) Representative western blot images show the effects of cortisol-BSA and cortisol with/without the glucocorticoid receptor inhibitor CORT125281 on Kv2.1 channel surface expression in cultured hippocampal neurons. Na+, K + -ATPase as a membrane protein loading control. (Figure6.B) Representative western blot images show the effects of 1 μM cortisol with/without the glucocorticoid receptor inhibitor CORT125281(10 μM) and RU486 (10 μM) on Kv2.1 channel surface expression in HEK293 cells. (Figure6.C) Representative western blot illustrating glucocorticoid receptor (GR) expression in HEK 293 cells transfected with control plasmids or shRNA plasmids targeting GR (siGR). β-tubulin as protein loading control. (Figure6.D) Representative western blot showing the effect of knockdown GR on cortisol-induced internalization of Kv2.1 channels in HEK 293 cells. Download Figure 6-1, TIF file.

Figure 6-2

Uncropped western blot images of Figure 6E. (Figure6.E) Representative western blot images show Kv2.1 channel surface expression in HEK293 cells treated with 1 μM cortisol for 10 minutes, and after cortisol washout for 0.5 hour, 1 hour, and 2 hours. Na+, K + -ATPase as a membrane protein loading control. Download Figure 6-2, TIF file.

Glucocorticoids regulate Kv2.1 and CaV1.2 channels through PKA signaling pathway

Several signaling pathways have been reported for the rapid effects of glucocorticoids through membrane-associated receptors, including PKC, ERK, and especially the cAMP-PKA signaling pathway (Panettieri et al., 2019; Nunez et al., 2020). In cultured hippocampal neurons, we observed that 1 μM Cort rapidly reduced cAMP levels (Fig. 7A) and inhibited PKA activity, as confirmed by a decrease in phosphorylated PKA levels (Fig. 7B). Furthermore, the effect of Cort on cell surface expression of Kv2.1 channels in hippocampal neurons was mimicked by PKA inhibitor H89 (1 μM) and abrogated by PKA stimulator forskolin (20 μM; Fig. 7C).

Figure 7.
Figure 7.

Glucocorticoids reduce the cell surface expression of Kv2.1 and CaV1.2 channels through PKA signaling pathway. A, Extracellular application of 1 μM Cort rapidly reduced cAMP levels in cultured hippocampal neurons. (One-way ANOVA with Bonferroni’s post hoc test, n = 3; F(3, 8) = 4.946; p = 0.0314). *p < 0.05. B, Left, Representative Western blot of the PKA phosphorylation level after 10 min Cort treatment or vehicle control (0.01% DMSO) treatment in cultured hippocampal neurons. Right, Statistics from seven independent experiments using a two-tailed unpaired t test (t(12) = 2.486; *p = 0.0286). C, Representative Western blots (left) and statistical analysis (right) showing the effect of PKA inhibitor H89 and agonist forskolin (Fsk) on the Kv2.1 channel surface expression in cultured hippocampal neurons. n = 8; F(3, 28) = 12.93; ****p < 0.0001; **p = 0.0031; n.s., not significant; one-way ANOVA with Bonferroni’s post hoc test. D, Left, Representative CaV1.2 current traces induced by a depolarization pulse from −90 to 0 mV under the control (0.01% DMSO) condition, in the presence of 1 μM H89, and in the presence of an additional 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of the CaV1.2 current using a RM one-way ANOVA with Bonferroni’s post hoc test (n = 6; F(2, 10) = 15.87; p = 0.0008). DMSO versus H89, **p = 0.0027; DMSO versus Cort, **p = 0.0014; H89 versus Cort, p > 0.9999. E, Left, Representative CaV1.2 current traces induced by a depolarization pulse from −90 to 0 mV under the control (0.01% DMSO) condition, in the presence of PKI (10 μM), and in the presence of an additional 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of the CaV1.2 current using a RM one-way ANOVA with Bonferroni’s post hoc test (n = 5; F(2, 8) = 20.48; p = 0.0007). DMSO versus PKI, **p = 0.0031; DMSO versus Cort, **p = 0.0010; PKI versus Cort, p > 0.9999; n.s., not significant. F, Left, Representative CaV1.2 current traces induced by a depolarization pulse from −90 to 0 mV in the presence of 10 μM RU486 and subsequently in the presence of an additional 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of CaV1.2 current from the left using a paired t test (n = 6; t(5) = 5.633; **p = 0.0024). G, Left, Representative CaV1.2 current traces induced by a depolarization pulse from −90 to 0 mV in the presence of 10 μM CORT125281 and subsequently in the presence of an additional 1 μM cortisol in the same HEK293 cell. Right, Statistics for the amplitude of CaV1.2 current from the left using a paired t test (n = 7; t(6) = 6.449; ***p = 0.0007). Uncropped Western blot images of panels B and C are provided in Extended Data Figure 7-1.

Figure 7-1

Uncropped western blot images of Figure 7B and C. (Figure7.B) Representative Western blot of the PKA phosphorylation level after 10-min corticosterone treatment or vehicle control (0.01% DMSO) treatment in cultured hippocampal neurons. (Figure7.C) Representative western blots showing the effect of PKA inhibitor H89 and agonist Forskolin (Fsk) on the Kv2.1 channel surface expression in cultured hippocampal neurons. Download Figure 7-1, TIF file.

Cortisol inhibited CaV1.2 currents but had little effect on the cell surface expression of CaV1.2 in HEK293 cells (Fig. 4), suggesting glucocorticoids regulate CaV1.2 activity besides promoting the channel endocytosis. Previous studies indicate that PKA can directly phosphorylate CaV1.2, and this phosphorylation increases CaV1.2 activity (Sculptoreanu et al., 1993). We investigated whether the inhibitory effect of glucocorticoids on CaV1.2 currents was via the PKA signaling pathway. Extracellular application of PKA-specific inhibitor H89 (1 μM) or PKI (10 μM) mimicked the effect of cortisol on CaV1.2 currents in HEK293 cells. Subsequent application of 1 μM cortisol did not produce any additional effect (Fig. 7D,E), indicating that glucocorticoids inhibit CaV1.2 currents by reducing PKA phosphorylation of the channel. Furthermore, we investigated whether this inhibition involves GRs. Preincubating HEK293 cells with RU486 or CORT125281 for 10 min did not modify the cortisol-induced inhibition of CaV1.2 currents (Fig. 7F,G).

TTX prevents the glucocorticoid-induced endocytosis of Kv2.1 in HEK293 cells

The actin cytoskeleton plays a crucial role in the formation and maintenance of Kv2.1 clusters (Tamkun et al., 2007; Weigel et al., 2010). Previous studies have shown that TTX enhances Kv2.1 clustering (Cerda and Trimmer, 2011; Romer et al., 2019) and stabilizes the binding between α-actin and CaV1.2 (Hall et al., 2013). Our findings indicate that preincubation with TTX prevents the glucocorticoid-induced endocytosis of Kv2.1 in HEK293 cells (Fig. 8), suggesting that glucocorticoids may exert their effects by altering the actin cytoskeleton in the cell membrane.

Figure 8.
Figure 8.

TTX blocked the effect of cortisol on the cell surface expression of Kv2.1 channels in HEK293 cells. A, Representative western blots (left) and statistical analysis (right) show the effects of cortisol (1 μM) and TTX (1 μM) on the Kv2.1 channel surface expression in HEK293 cells. One-way ANOVA with Bonferroni’s post hoc test (n = 7; F(3, 24) = 12.83; p < 0.0001). DMSO versus Cort, **p = 0.0078; DMSO versus TTX, p > 0.9999; DMSO versus TTX + Cort, p > 0.9999. n.s., not significant. Uncropped Western blot images of panel A are provided in Extended Data Figure 8-1.

Figure 8-1

Uncropped western blot images of Figure 8A. Representative western blots show the effects of cortisol (1 μM) and TTX (1 μM) on the Kv2.1 channel surface expression in HEK293 cells. Download Figure 8-1, TIF file.

Movie 1.

Representative video showing the effect of vehicle control (0.01% DMSO for 5 min) on spontaneous somatic Ca2+ sparks in hippocampal neurons. [View online]

Movie 2.

Representative video showing the effect of Cort (1 μM for 5 min) on spontaneous somatic Ca2+ sparks in hippocampal neurons. [View online]

Discussion

This study demonstrates that glucocorticoids regulate Ca2+ signals in mammalian hippocampal neurons through the PKA signaling pathway in two ways: by inhibiting PKA to reduce CaV1.2 channel activities and by promoting Kv2.1 clusters endocytosis to increase CaV1.2 channel internalization.

Mammalian brain Kv2.1 channels are typically found in clusters on the somas and proximal dendrites of neurons (Trimmer, 1991; Kirizs et al., 2014). Increasing evidence suggests that these clustered Kv2.1 channels play important nonconducting roles. Previous studies have shown that Kv2.1 clusters play a structural role in inducing the formation of ER/PM junctions in both cultured hippocampal neurons and transfected HEK293 cells through binding with vesicle-associated membrane protein-associated protein A and B (Fox et al., 2015; Kirmiz et al., 2018a). Clustered Kv2.1 channels at ER/PM junctions recruit CaV1.2 to form Ca2+ microdomains with ryanodine receptor ER Ca2+ release channels, which generate local spontaneous Ca2+ release at the soma of hippocampal neurons (Vierra et al., 2019). In this study, we found that glucocorticoids rapidly inhibited spontaneous Ca2+ sparks. Glucocorticoids not only inhibit CaV1.2 channel currents but also disrupt the Ca2+ microdomains by targeting clustered Kv2.1 at the soma of cultured hippocampal neurons. The major target of glucocorticoids is clustered Kv2.1 rather than dispersed Kv2.1, as supported by two pieces of evidence. First, glucocorticoids reduced the cell surface expression of Kv2.1 by 30.2% but had little effect of Kv2.1 currents in HEK293. This can be explained by the fact that clustered Kv2.1 conducts little potassium (O'Connell et al., 2010). Second, in the presence of WT Kv2.1, glucocorticoids caused a decrease in the surface expression of CaV1.2 channels in HEK293 cells. However, this effect was not observed in the presence of nonclustering Kv2.1S586A mutant channels.

Glucocorticoids have been shown to rapidly regulate neuronal membrane protein trafficking, including that of α1 adrenergic receptors, AMPA receptors, and NMDA receptors. Cort, through rapid nongenomic effects, promotes the norepinephrine-induced internalization of α1 adrenergic receptors in corticotropin-releasing hormone neurons (Jiang et al., 2022) . Additionally, Cort rapidly stimulates the surface trafficking of GluR2-containing AMPA receptors in hippocampal neurons via membrane-associated receptors (Groc et al., 2008) and enhances both AMPA and NMDA receptor trafficking in prefrontal cortex pyramidal neurons (Yuen et al., 2011). Neuronal Kv2.1 clusters can be declustered by stimuli, including kainite, glutamate, hypoxia, and ischemia (Misonou et al., 2004; Misonou et al., 2005a, b), while none of them affect the total surface expression of Kv2.1. We found that glucocorticoids affect the surface expression of Kv2.1. Previous studies showed that the activation properties of Kv2.1 were altered by channel dephosphorylation, but not by actin depolymerization (O'Connell et al., 2010). The actin cytoskeleton is important for forming and maintaining Kv2.1 clusters (Tamkun et al., 2007; Weigel et al., 2010). Our data suggest that glucocorticoids may act by changing the cell membrane actin skeleton.

The rapid effects of glucocorticoid are commonly mediated by membrane-associated GRs (Panettieri et al., 2019). Our study found that cortisol-BSA mimicked cortisol's effect on Kv2.1 channels, while the GR antagonist CORT125281 did not alter this effect. Additionally, knockdown of GRs did not affect cortisol-induced internalization of Kv2.1 channels. These findings suggest that glucocorticoids inhibit Kv2.1 through unidentified membrane receptors. Glucocorticoids exhibit cell-specific effects on PKA activity: they suppress PKA in mouse pituitary tumor AtT20 cells (Iwasaki et al., 1997) but stimulate PKA in hypothalamic paraventricular nucleus neurosecretory neurons (Malcher-Lopes et al., 2006). PKA phosphorylation significantly influences CaV1.2 activity. Previous studies suggest that AKAP-anchored PKA is essential for maintaining neuronal CaV1.2 channel activity and NFAT transcriptional signaling (Murphy et al., 2014). Additionally, Qian et al. demonstrated that β-adrenergic receptor agonist isoproterenol increases CaV1.2 activity by stimulating PKA in mouse hippocampal neurons (Qian et al., 2017). Similarly, our findings indicate that glucocorticoids decrease cAMP levels and PKA activity, thereby inhibiting CaV1.2 currents in rat hippocampal neurons and HEK293 cells. In contrast, ffrench-Mullin (ffrench-Mullen, 1995) reported that cortisol inhibits L-type calcium channels in guinea pig hippocampal neurons via PKC activation, suggesting a different regulatory mechanism. This discrepancy might stem from species differences in hippocampal neuron signaling pathways, as cortisol is the principal glucocorticoid in guinea pigs, while Cort predominates in rats and mice. A recent study showed that glucocorticoids reduce cAMP levels by activating the adhesion G-protein–coupled receptor GPR97, with cortisol (but not Cort) activating GPR97 (Ping et al., 2021). Nevertheless, we found that both cortisol and Cort reduce Kv2.1 surface expression. Further research is necessary to identify the membrane-associated receptors responsible for the glucocorticoid-induced effects on Kv2.1 channels.

Glucocorticoids are produced in a diurnal pattern controlled by the hypothalamic–pituitary–adrenal (HPA) axis. The precise functions of rapid glucocorticoid actions in the brain remain partially understood. However, a key role is the swift feedback regulation of the HPA axis. Glucocorticoids modulate stress-induced HPA activation through rapid effects on the hippocampus (Tasker and Herman, 2011) and prefrontal cortex (McKlveen et al., 2013). It is now well known that the glucocorticoid circadian variation is actually composed of an underlying ultradian rhythm. This rhythm occurs <60 min for rodents (Spiga et al., 2011) and every 60–120 min in human (Gavrila et al., 2003; Russell et al., 2010). Furthermore, the ultradian rhythm of glucocorticoids is not affected by the disruption of circadian inputs to the HPA activity (Waite et al., 2012). Recent studies show that the ultradian rhythmicity of glucocorticoids is critical in regulating normal emotional and cognitive responses in humans (Kalafatakis et al., 2018). Wu et al. have revealed that Kv2.1-organized ER/PM junction sites represent ∼12% of the somatic surface in mammalian brain neurons (Wu et al., 2017). At ER/PM junctions, Kv2.1 control somatic Ca2+ signals by recruiting CaV1.2, thereby regulating neuronal excitation–transcription coupling (Yap and Greenberg, 2018; Vierra et al., 2021). Since both Kv2.1 and CaV1.2 are widely expressed in mammalian brain, the rapid effect of glucocorticoids on the Kv2.1-CaV1.2 unit may play an import functional role in the ultradian rhythm of glucocorticoids.

In conclusion, glucocorticoids rapidly regulate Ca2+ signals in hippocampal neurons in two ways. First, they inhibit CaV1.2 channel activity (regardless of whether it is coupled with Kv2.1 or not) by reducing PKA activity, which results in a reduction of the overall Ca2+ signal. Second, they reduce the surface expression of CaV1.2 channels by promoting the endocytosis of Kv2.1 channels. This disruption of clustered Kv2.1-mediated Ca2+ reduces the local spontaneous somatic Ca2+ signals that would otherwise regulate excitation–transcription coupling. This study provides further insight into the rapid effect of glucocorticoids in the central nervous system.

Footnotes

  • This work was supported by the National Key Research & Development Program of China (2022YFC3602702), the Science and Technology Innovation 2030 - Brain Science and Brain- Inspired Intelligence Project (2021ZD0201301), the Natural Science Foundation of Shanghai (23ZR1425900), and the National Natural Science Foundation of China (31771282).

  • ↵*D.W. and T.L. contributed equally to this work.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Changlong Hu at clhu{at}fudan.edu.cn.

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Glucocorticoids Rapidly Modulate CaV1.2-Mediated Calcium Signals through Kv2.1 Channel Clusters in Hippocampal Neurons
Di Wan, Tongchuang Lu, Chenyang Li, Changlong Hu
Journal of Neuroscience 6 November 2024, 44 (45) e0179242024; DOI: 10.1523/JNEUROSCI.0179-24.2024
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