The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2

Abstract

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

Figure 1. L-Type Ca²⁺ Channel Activity Is Decreased by Tonic Depolarization

(A) Ca²⁺ imaging of cultured cortical neurons subjected to a test pulse of 65 mM KCl following 15 min of pretreatment with 5 mM KCl (black) or 65 mM KCl (blue). Sample traces from eight cells (Ai and Aii) and average traces (Aiii) are shown for each condition (means ± SEM; n > 50 cells for each condition). (B) Ca²⁺ imaging of the [Ca²⁺]_i elevation in cultured cortical neurons subjected to a test pulse of 65 mM KCl following a 15 min depolarizing prepulse and recovery in 5 mM KCl for the indicated length of time (means ± SEM; n > 50 cells for each condition). (C) Time-course of recovery of [Ca²⁺]_i signals after a 15 min depolarizing prepulse. The y axis shows the ratio of the [Ca²⁺]_i rise triggered by the test stimulus to the [Ca²⁺]_i elevation triggered by the prepulse. The x axis shows the time between the end of the prepulse and the beginning of the test pulse (means ± SEM; n > 50 cells for each condition). (D) Whole-cell currents from hippocampal neurons elicited by 60 ms step depolarization to 0 mV from a holding potential of −80 mV, using 10 mM Ba²⁺ as the charge carrier. Na⁺ and K⁺ currents were blocked with extracellular tetrodotoxin and intracellular Cs⁺, respectively. Prior to recording, untransfected cells were left unstimulated (left), treated for 20 min in 65 mM KCl (middle), or treated for 20 min in 65 mM KCl, then allowed to recover for 20 min in 5 mM KCl (right). (E) Whole-cell current-voltage plot generated by a voltage ramp stimulus (1.6 mV/ms). Shown are traces from a control neuron (black) and a neuron depolarized for 20 min with 65 mM KCl (blue). (F) Whole-cell Ba²⁺ currents from hippocampal neurons expressing DN-dyn1 and left unstimulated prior to recording (left) or treated with 65 mM KCl for 30 min (right). (G) Normalized current densities recorded from cultured hippocampal neurons as in (D) and (F) (means ± SEM; *p < 0.0001; n = 3–8 neurons per condition).

Figure 2. Calcium Influx through L- and P/Q-Type Calcium Channels Is Reduced by Tonic Depolarization

(A) Ca²⁺ imaging of cultured cortical neurons stimulated with a 65 mM KCl test pulse in the presence (red) or absence (black) of 5 μM nimodipine following pretreatment with 5 mM (left) or 65 mM KCl (right) for 15 min (means ± SEM; n > 50 neurons for each condition). (B) Contribution of LTCs to the [Ca²⁺]_i elevation triggered by depolarization following pretreatment with 5 mM KCl (black) or 65 mM KCl (blue). (C) As in (A), but treating with 1 μM ω-conotoxin GVIA. (D) As in (B), but for P/Q-type VGCCs. (E) As in (A), but treating with 100 nM ω-Aga IVA (means ± SEM; n > 40 neurons for each condition). (F) As in (B), but for N-type VGCCs. (G) Ca²⁺ traces in (B), (D), and (F) were fit by linear regression to estimate the calcium influx mediated by different VGCCs. (H) Ca²⁺ influx mediated by L- (Ca_V1.2 and 1.3), N- (Ca_V2.2), and P/Q- (Ca_V2.1) type channels before (black bars) and after depolarization (blue bars) (*p < 0.05).

Figure 3. Ca_V1.2 Surface Levels Are Regulated by Electrical Activity

(A) Cultured cortical neurons containing YH-Ca_V1.2 and stained using an anti-HA antibody with (left column) or without (right column) membrane permeabilization. (B) Profiles of the intensity of YFP and anti-HA signals along the lines drawn through the neurons in (A). The anti-HA signal in unpermeabilized cells shows sharp peaks at the edges of the cell, whereas the other traces remain elevated throughout the cell. (C) Relationship between the total HA and YFP intensities in permeabilized cortical neurons and fit by a linear equation (n = 35 cells). (D) Cultured cortical neurons containing YH-Ca_V1.2 and treated with 5 mM KCl or 65 mM KCl for 30 min and stained using an anti-HA antibody without permeabilization. Line scans of the fluorescence intensity through the cell bodies are shown in the insets. (E) Cultured cortical neurons containing GFP/HA-K_V1.2 and treated with 5 mM KCl or 65 mM KCl for 30 min and stained using an anti-HA antibody without membrane permeabilization. (F) Histogram of the Ca_V1.2 SF in cortical neurons treated with 5 mM KCl (white bars) or 65 mM KCl (red bars) for 30 min (n > 50 cells for each condition; population medians differ with p < 0.005 by Mann-Whitney test). (G) K_V1.2 SF in cortical neurons treated for 30 min with 5 mM KCl or 65 mM KCl (means ± SEM; n > 50 for each condition). (H) Ca_V1.2 SF in cortical neurons treated for 30 min with 5 mM KCl (bar 1) or 65 mM KCl (bars 2–5) in the presence of 2 mM extracellular Ca²⁺, DN-dyn1, 0 mM extracellular Ca²⁺, or 100 μM diltiazem. Ca_V1.2 SF in cells treated with 1 μM ionomycin (bar 6) or 50 μM glutamate (bar 7; means ± SEM; n > 30 for each condition; *p < 0.05 by Mann-Whitney test).

Figure 4. Ca_V1.2 Is Endocytosed in Response to Electrical Activity

(A) Schematic of manipulations involved in the biotin endocytosis assay (top) and surface biotinylation assay (bottom). (B) Western blot analysis using an anti-Ca_V1.2 antibody of fractions taken from biotin endocytosis assays (lanes 1–5) or surface biotinylation assays (lanes 6–8). The presence of a band in lane 2 suggests that Ca_V1.2 channels were endocytosed by stimulation with glutamate. Bands in lanes 4 and 5 show that short stimulation with glutamate has no effect on total Ca_V1.2 levels. The bands in lanes 7 and 8 show Ca_V1.2 surface levels in untreated and glutamate stimulated neurons, respectively. Band intensities are quantified below each lane in the gel. These gels are representative of the data from three separate experiments. (C) Depolarization triggers Ca_V1.2 endocytosis as measured in single cells. Schematic for the fluorescent endocytosis assay (top bar). YFP or anti-HA antibody fluorescence of neurons expressing YH-Ca_V1.2 and treated with 5 mM KCl or 65 mM KCl for 30 min. HA fluorescence indicates endocytosed channels. Inset shows a magnified view of internalized Ca_v1.2 channels in vesicles near the plasma membrane. (D) Quantification of anti-HA staining in neurons from the fluorescence endocytosis assay shown in part (C) (bars 1 and 2) or control experiments in which the unlabeled blocking antibody or the permeabilization step was omitted (means ± SEM; n > 70 for each condition; *p < 0.05 by Student’s t test).

Figure 5. Ca_V1.2-Containing Vesicles Move Rapidly Close to the Membrane and Contain Endocytosed Channels

(A) Time-lapse TIRF microscopy images of Neuro2a cells containing YFP-Ca_V1.2 (rows 1 and 2) or myrisotylated YFP (row 3) and treated with 5 mM KCl (row 1) or 65 mM KCl (rows 2 and 3) for 10 min. Depolarized cells show a pronounced decrease in YFP-Ca_V1.2 levels at the membrane. (B) Average TIRF signal in Neuro2a cells containing YFP-Ca_V1.2 treated with either 5 mM KCl (red) or 65 mM KCl (black) or containing myristoylated YFP (blue; n = 10 cells for each condition). (C) Images of a cortical neuron containing mCherry-Ca_V1.2 taken with epifluorescence illumination (left) and TIRF illumination (right). (D) Images of a Neuro2a cell containing YH-Ca_V1.2 taken with epifluorescence illumination (left) and TIRF illumination (right). (E) Images of a cortical neuron (left) and Neuro2a cell (right) containing YH-Ca_V1.2 and CFP-TfR (pseudocolored red). Arrows mark examples of vesicles containing both fluorescent proteins, and arrowheads mark examples of vesicles containing only YH-Ca_V1.2. (F) Images of Neuro2a cells containing either YFP-Ca_V1.2 or YB-Ca_V1.2 and incubated with Alexa 594-conjugated bungarotoxin for 1 hr under resting conditions. An arrow indicates an example of a vesicle containing both YB-Ca_V1.2 and bungarotoxin showing channel internalization.

Figure 6. Electrical Activity Regulates the Dynamics of Ca_V1.2 Vesicles Near the Membrane

(A) Images of a cortical neuron under TIRF illumination at the indicated times during a time-lapse experiment. The final image is a pseudocolor probability map that indicates how frequently a channel was present in each location in the cell. The red circle marks a single vesicle that disappears and reappears during the experiment. (B) Images of a Neuro2a cell under TIRF illumination at the indicated points during a time-lapse experiment. The final image is a probability map that indicates how frequently a channel was present at each location in the cell. The red and blue circles mark single vesicles that disappear and reappear during the experiment. (C) Images of a single vesicle over the course of a time-lapse experiment within the red region of interest in the cell shown in (B). (D) Plot of fluorescence intensity versus time for the vesicle shown in (C) (black) and for an adjacent background region (red). The 3σ threshold (red line) was set to exclude 99.7% of the signal from the background region. (E) A binary representation of the fluorescence intensity trace in (D). Time points classified as ‘‘present’’ exceeded the threshold, whereas time points classified as absent fell below it. (F) Histograms of the membrane residence time of Ca_V1.2 vesicles in Neuro2a cells under control (top) and depolarizing (bottom) conditions (n > 50 vesicles for each condition). For each histogram, the distribution was well fit by a single exponential equation. Medians under control and depolarized conditions were found to differ with p < 0.005 by Mann-Whitney test. (G) Histograms of the membrane absence time of Ca_V1.2 vesicles in Neuro2a cells under control (top) and depolarizing (bottom) conditions (n > 50 vesicles for each condition). For each histogram, the distribution was well fit by a single exponential. (H) Cumulative surface probability of Ca_V1.2 vesicles under control (bar 1) or depolarizing (bar 2) conditions (means ± sem; *p < 0.01 by Student’s t test).

Figure 7. The Ca_V1.2 II–III Intracellular Loop Binds to eIF3e in a Calcium-Dependent Manner

(A) Ca_V1.2 SF in cortical neurons containing YH-Ca_V1.2 and a vector (bars 1 and 2) or the Ca_V1.2 II–III intracellular loop (bar 3). Neurons were treated with 5 mM KCl (bar 1) or 65 mM KCl (bars 2–3) (means ± SEM; *p < 0.05 by Mann-Whitney test). (B) Epifluorescence images of HEK293T cells containing CFP-eIF3e (pseudocolored red) and either YFP or LTC-YFP. An arrow indicates an example region of colocalization at the membrane between CFP-eIF3e and LTC-YFP. (C) Ca_V1.2 immunoprecipitates eIF3e-myc. Left panels show Ca_V1.2 and eIF3e-myc levels in the lysates from HEK293T cells. Right panels show the levels of Ca_V1.2 and eIF3e following immunoprecipitation with an anti-Ca_V1.2 antibody. (D) eIF3e-myc mmunoprecipitates Ca_V1.2. Left panels show Ca_V1.2 and eIF3e-myc levels in the lysates from HEK293T cells. Right panels show the levels of Ca_V1.2 and eIF3e following immunoprecipitation with an anti-myc antibody. (E) A purified Ca_V1.2 II–III loop immunoprecipitates eIF3e. Autoradiograms showing in vitro-translated eIF3e (left) and immunoprecipitation of this eIF3e with GST or GST fused to the II–III loop (right). The bottom panel shows Coomassie staining of the purified GST and GST-II–III that was incubated with radiolabeled eIF3e. (F) Endogenous Ca_V1.2 immunoprecipitates eIF3e in the brain. Immunoblotting for Ca_V1.2 and eIF3e was performed on membrane fractions from P5 rat brain before immunoprecipitation (lane 1) and after immunoprecipitation with a control antibody or an antibody against Ca_V1.2. (G) Ca_V1.2 immunoprecipitation of eIF3e is inducible. Immunoprecipitation of Ca_V1.2 from Neuro2a cells containing Flag/myc-Ca_V1.2 and eIF3e-myc following treatment with 5 mM KCl or 65 mM KCl with or without extracellular Ca²⁺.

Figure 8. The II–III Loop of Ca_V1.2 Contains an eIF3e-Binding Domain

(A) Schematic representation of Ca_V1.2 (top). Schematic of Ca_V1.2 II–III loop deletion constructs (left) and their performance as bait vectors in yeast interaction studies with eIF3e (right). All constructs support growth on nonselective media (right), whereas only fragments capable of interacting with eIF3e support growth on selective media (left). (B) Alignment of the region surrounding the eIF3e-binding domain of Ca_V1.2 in six species with the conserved binding site highlighted in blue. (C) Yeast two-hybrid analysis of eIF3e binding for the II–III intracellular loops of rat Ca_V1.2, Ca_V2.2, and Ca_V1.3. All channel loops support growth on nonselective media (right), but only Ca_V1.2 and Ca_V1.3 support growth on selective media (left). (D) Phylogenetic tree of Ca_V channels organized by their sequence in the eIF3e binding domain. (E) eIF3e immunoprecipitates Ca_V1.2, but not Ca_V1.2(CLS). Western blot analysis of HEK293T cells containing eIF3e-myc together with Ca_V1.2 (lane 1) or Ca_V1.2(CLS) (lane 2). Membranes were immunoblotted before (bottom two panels) and after (top two panels) immunoprecipitation with a Ca_V1.2 antibody.

Figure 9. Binding of eIF3e Is Required for the Activity-Dependent Internalization of Ca_V1.2

(A) Cultured cortical neurons stained without permeabilization using an anti-HA antibody. Neurons contained YH-Ca_V1.2(CLS), a control shRNA (third row), or sheIF3e (bottom row) and were treated as indicated for 30 min. (B) Ca_V1.2 SF in cortical neurons containing YH-Ca_V1.2(CLS) and treated with 5 mM KCl (bar 1) or 65 mM KCl (bar 2) (means ± SEM; n > 35 for each condition). (C) Ca_V1.2 SF in cortical neurons treated with 65 mM KCl and containing a control shRNA (red), sheIF3e (blue), a control vector (black), or p56 (orange) (means ± SEM; n > 35 for each condition; *p < 0.05 by Mann-Whitney test). (D) Average TIRF signal in Neuro2a cells containing YFP-Ca_V1.2 (black) or YFP-Ca_V1.2(CLS) (red) during 10 min of treatment with 65 mM KCl. Data for Ca_V1.2 are shown in 2B and reprinted here for comparison. (n = 10 for each condition). (E) Histograms of the membrane residence time of Ca_V1.2(CLS) vesicles in Neuro2a cells under control (top) and depolarizing (bottom) conditions (n > 50 vesicles for each condition). For each histogram, the distribution was well fit by a single exponential equation. (F) Cumulative surface probability of Ca_V1.2(CLS) vesicles under control (bar 1) or depolarizing (bar 2) conditions (means ± SEM). (G) Calcium imaging of cortical neurons containing a control vector (black), a plasmid encoding eIF3e, or a plasmid encoding sheIF3e (blue) and treated with 65 mM KCl for 1000 s (means ± SEM; n > 20 cells for each condition). (H) Currents recorded from cultured hippocampal neurons expressing sheIF3e without stimulation (black) or treated with 65 mM KCl for 30 min (red). (I) Normalized current densities recorded from cultured hippocampal neurons producing sheIF3e without stimulation or treated with 65 mM KCl for 20 min (means ± SEM; n = 3–8 neurons per condition).