14-3-3τ Promotes Surface Expression of Cav2.2 (α1B) Ca2+ Channels*

Background: The mechanism and dynamics of Cav channels trafficking remains mysterious. Results: 14-3-3τ promotes Cav2.2 trafficking independent of Cav auxiliary subunits. Conclusion: 14-3-3τ enhances Cav2.2 trafficking by masking the ER retention signal at its proximal C-terminal region. Significance: Uncovering the regulation of Cav2.2 trafficking may contribute to understanding the Cav2.2 surface expression and functional control under physiological and pathophysiological conditions. Surface expression of voltage-gated Ca2+ (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the α1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, β and α2δ, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 α1B channel in transfected tsA-201 cells in the absence of any known Cav auxiliary subunit. Both the surface to total ratio of the expressed α1B protein and the current density of voltage step-evoked Ba2+ current were markedly suppressed by the coexpression of a 14-3-3 antagonist construct, pSCM138, but not its inactive control, pSCM174, as determined by immunofluorescence assay and whole cell voltage clamp recording, respectively. By contrast, coexpression with 14-3-3τ significantly enhanced the surface expression and current density of the Cav2.2 α1B channel. Importantly, we found that between the two previously identified 14-3-3 binding regions at the α1B C terminus, only the proximal region (amino acids 1706–1940), closer to the end of the last transmembrane domain, was retained by the endoplasmic reticulum and facilitated by 14-3-3 to traffic to plasma membrane. Additionally, we showed that the 14-3-3/Cav β subunit coregulated the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Altogether, our findings reveal a previously unidentified regulatory function of 14-3-3 proteins in promoting the surface expression of Cav2.2 α1B channels.


Surface expression of voltage-gated Ca 2؉ (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the ␣1
pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, ␤ and ␣2␦, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 ␣1B channel in transfected tsA-201 cells in the absence of any known Cav auxiliary subunit. Both the surface to total ratio of the expressed ␣1B protein and the current density of voltage stepevoked Ba 2؉ current were markedly suppressed by the coexpression of a 14-3-3 antagonist construct, pSCM138, but not its inactive control, pSCM174, as determined by immunofluorescence assay and whole cell voltage clamp recording, respectively. By contrast, coexpression with 14-3-3 significantly enhanced the surface expression and current density of the Cav2.2 ␣1B channel. Importantly, we found that between the two previously identified 14-3-3 binding regions at the ␣1B C terminus, only the proximal region (amino acids 1706 -1940), closer to the end of the last transmembrane domain, was retained by the endoplasmic reticulum and facilitated by 14-3-3 to traffic to plasma membrane. Additionally, we showed that the 14-3-3/Cav ␤ subunit coregulated the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Altogether, our findings reveal a previously unidentified regulatory function of 14-3-3 proteins in promoting the surface expression of Cav2.2 ␣1B channels.
Although coassembly with accessory proteins represents an important means of controlling the surface expression of Cav2 subfamily channels, these channels are regulated by interacting with other proteins that can influence their expression, trafficking, subcellular localization, stabilization, and biophysical properties. Previously, we have identified a protein-protein interaction between the Cav2.2 channel and 14-3-3 proteins, which are a family of homologous proteins that generally binds to targets containing specific phosphoserine motifs and participates in the regulation of a wide range of biological processes, including facilitating surface expression of several classes of ion channels and receptors (11)(12)(13)(14)(15)(16)(17)(18)(19). We have demonstrated that 14-3-3 binding to the C-terminal region of the ␣1B subunit leads to profound modulation of Cav2.2 channel inactivation kinetics (20,21). However, a potential role of 14-3-3 proteins in Cav2.2 channel trafficking has yet to be investigated.
Immunofluorescence Assay-Cultured tsA-201 cells were transiently transfected using calcium phosphate transfection protocol according to the manufacturer's instructions. Surface and total immunofluorescence staining of HA-␣1B was performed as described (26) with minor modifications. Briefly, at least 2 days after transfection, cells were fixed at 37°C for 8 -10 min with 4% paraformaldehyde and 4% sucrose in PBS. The fixed cells were incubated in PBS containing either anti-HA or anti-Myc antibodies overnight at 4°C to label surface proteins and then probed using Alexa Fluor 546-conjugated secondary antibodies for 1 h at room temperature. After surface staining, cells were permeabilized with 0.5% Triton X-100 in blocking solution containing 5% fetal bovine serum in PBS for 30 min. Subsequently, intracellular HA-tagged ␣1B or Myc-tagged CD8␣ fusion protein was stained with the primary antibodies (either anti-HA or anti-Myc antibodies), followed by Alexa Fluor 633-conjugated secondary antibody for 1 h at room temperature to visualize intracellular HA-tagged ␣1B or Myctagged CD8␣ fusion protein. Fluorescence images were acquired using a Zeiss LSM-510 Meta confocal microscope (Zeiss, Germany) with a 40ϫ 1.3NA oil immersion lens in the inverted position, performed with identical gain, contrast, laser excitation, pinhole aperture, and laser scanning speed for each round of cultures. Image processing was performed using ImageJ software. Because the HA-or Myc-tagged protein is extracellular, signal intensity from nonpermeabilized cells was used as a measure of surface channel expression, and that from permeabilized cells was used as a measure of intracellular channel expression. The ratio of cell surface to total channel expression (surface expression to surface plus intracellular expression), representing either membrane:total HA-␣1B or membrane:total Myc-CD8␣, allowed comparisons between various conditions assayed from different batches of cells.
Antibodies, Coimmunoprecipitation, and Western Blot-The Western blotting and coimmunoprecipitation assays were performed as previously described with minor modifications (27). Plasmids encoding 14-3-3 and Flag-Myc-tagged CT fragments were transiently transfected into tsA-201 cells. At 48 h posttransfection, cells were lysed in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, 0.5% Nonidet P-40, 0.1 mM PMSF, and protease inhibitor mixture. The whole cell extracts were immunoprecipitated with anti-Flag beads (Sigma), and the coeluted pellets were resolved on SDS-PAGE and analyzed by Western blotting after transferring the resolved proteins to nitrocellulose membrane (Trans-Blot; Bio-Rad). Blots were probed with either anti-14-3-3 or anti-Flag antibodies, and the bands were detected by chemiluminescence using Lumigen PS-atto.
Size Fractionation-The fractionation of the cell extracts was carried out according to the manufacturer's instructions. Briefly, the tsA-201 cells expressing 14-3-3, Flag-Myc-CT1 were lysed by sonication in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40 and 1% dodecyl maltoside) supplemented with protease inhibitors. The lysates were then centrifuged at 4°C at 10,000 rpm for 10 min. The supernatants were transferred to a fresh tube, and protein concentrations were determined by using the BCA protein assay kit (Thermo Scientific). 4 -8 mg of protein was brought up to 0.5 ml of total volume using chromatography running buffer (PBS and 0.1% dodecyl maltoside, pH 7.4). The lysates were run on a Superose 6 10/300 GL size exclusion chromatography column (GE Healthcare) using the ÄKTA Purifier system (GE Healthcare), which was pre-equilibrated with PBS. After sample injection (using a 1-ml loop), the running buffer was set at a flow rate of 0.5 ml/min, and 1 ml per fraction was collected. Each fraction was separated by SDS-PAGE on 12% gels and fractionated proteins were detected with anti-Myc or 14-3-3 antibodies.
Primary Hippocampal Neuron Cultures-All animal procedures were carried out in accordance with the guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University School of Medicine and approved by the Institutional Animal Care and Use Committee. Cultures of embryonic hippocampal neurons were prepared using a standard procedure. Briefly, hippocampi were dissected from embryonic day 18 rats (Sprague-Dawley) with the aid of a stereo microscope. Isolated hippocampi were digested with papain for 15 min at 37°C. Isolated neurons were seeded on poly-D-lysine-precoated coverslips in appropriate density and cultured in neurobasal medium supplemented with B-27 and 0.5 mM glutamine at 37°C and 5% CO 2 . Cultures were used after 7-10 days in vitro.
Electrophysiology-Calcium channel currents were recorded in tsA-201 cells as described previously (20) using the whole cell configuration of the patch-clamp technique. Briefly, a standard calcium phosphate protocol was used to transfect the cells with cDNAs encoding Cav2.2 ␣1B-EGFP, either alone or in combination with 14-3-3-pEBFP-N1, pSCM138, or pSCM174 as indicated. At least 2 days after transfection, Ca 2ϩ channel currents were recorded from transfected cells with an EPC10 amplifier (HEKA). ␣1Band 14-3-3-cotransfected cells bearing both green and blue fluorescence were identified with the FITC and blue fluorescent protein (BFP) filter sets on the Olympus IX71 inverted fluorescence microscope. To ensure that the BFP tag did not affect 14-3-3 function, similar experiments were performed by transfecting cells with ␣1B-pRc/CMV and 14-3-3-pIRES2-EGFP. Under this condition, recordings were done from green cells. Recording microelectrodes with resistances of 3-5 megohms were pulled from thin walled borosilicate glass with inner filament (Sutter Instrument) and filled with the intracellular solution containing 135 mM CsCl, 10 mM EGTA, 4 mM Mg-ATP, and 10 mM HEPES (pH 7.5 with CsOH). The extracellular solution contained 125 mM NaCl, 20 mM BaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES (pH 7.3 with NaOH). Whole cell currents, which were filtered at 4 kHz and sampled at 50 kHz, were elicited by 400-ms depolarizing steps from Ϫ80 to ϩ60 mV in 5-mV increments every 5 s from a holding potential of Ϫ80 mV (to allow full recovery from inactivation). Data analysis was performed using Clampfit 10.0 (Axon Instruments), Prism 5.0 (GraphPad), and Origin 8.0 (OriginLab) software. Current densities (pA/pF) were obtained for each cell by normalization of the whole cell current to cell capacitance to account for differences in cell membrane surface area. Activation curve were fitted using the Boltzmann equation: where G is conductance, G max is the maximum conductance, V m is membrane voltage, V1 ⁄ 2 is the half-activation potential, and k is the slope factor.
Statistical Analysis-Statistical analyses of surface and intracellular staining of HA-␣1B and Myc-CD8␣ were performed using ImageJ software. The data are expressed as means Ϯ S.E. with statistical significance assessed by Student's t test for two group comparison or one-way analysis of variance (ANOVA) tests for multiple comparisons. The value of p Ͻ 0.05 was considered to have statistically significant difference.

14-3-3 Enhances Membrane Expression of Cav2.2 Independent of Cav Auxiliary Subunits-Functional
Cav channel expression is known to be promoted by the auxiliary subunits (1, 2, 4 -7, 9, 28). To specifically assess the effect of 14-3-3 proteins on surface expression of the Cav2.2 channel, we expressed Cav2.2 ␣1B subunits in tsA-201 cells in which neither endogenous ␤ or ␣2␦ subunit was expressed ( Fig. 1A and Ref. 25). In addition, we did not detect any voltage-gated Cav channel current in the absence of exogenously expressed ␣1B subunit (Fig.  1B), suggesting a lack of endogenous Cav channel activity in the tsA-201 cells.
14-3-3 proteins bind to the C terminus of Cav2.2 ␣1B subunit at two putative regions (20). To test whether the interaction with 14-3-3 affects Cav2.2 channel trafficking, we used a previously characterized high affinity 14-3-3 antagonist expression construct, pSCM138, which encodes EYFP-tagged R18 peptide dimer (29). The construct coding for the inactive mutant of the R18 peptide pSCM174 was used as a negative control (20,29). The surface expression of Cav2.2 was examined by transfection into tsA-201 cells of a HA-tagged ␣1B construct (HA-␣1B), in which the HA epitope was inserted into the second extracellular loop (between transmembrane segments 5 and 6) of the domain II transmembrane repeat of the ␣1B coding sequence. This allowed detection of surface expressed and total Cav2.2 ␣1B protein levels by immunofluorescence labeling under nonpermeablized and permeabilized conditions, respectively. As shown in Fig. 1C, the surface expressed HA-␣1B, as labeled by the anti-HA antibody and the Alexa Fluor 546-conjugated secondary antibody under nonpermeabilized conditions, was decreased by the cotransfection of pSCM138, but not pSCM174. Quantification of fluorescence intensity ratio (nonpermeabilized intensity to nonpermeabilized plus permeabilized intensity) revealed that inhibiting 14-3-3 by pSCM138 reduced the plasma membrane expression of Cav2.2 ␣1 subunit by ϳ40% (Fig. 1D). By contrast, coexpression of exogenous 14-3-3 significantly enhanced the surface expression of HA-␣1B, which was completely abolished by cotransfected pSCM138, but not pSCM174 (Fig. 1, C and D). The 14-3-3-dependent changes in the surface expression were also reflected by alterations in the densities of Cav2.2 whole cell currents over a wide range of test potentials in the transfected tsA-201 cells. The current density of Cav2.2 was decreased by ϳ70% with cotransfected pSCM138, whereas it more than doubled with 14-3-3 coexpression (Fig. 1E). Again, the enhancing effect of 14-3-3 on Cav2.2 current density and the ratio of surface expressed Cav2.2 ␣1 subunit was completely prevented by the cotransfection with pSCM138, suggesting a requirement for 14-3-3 binding. All the 14-3-3 isoforms were tested in this study, 14-3-3 exhibited quantitatively strongest ratio of sur- Expression by 14-3-3 face expression and increased current density of Cav2.2, and this particular isoform was thus utilized in most of the subsequent experiments. Moreover, there were no differences in the half-maximal activation (V1 ⁄ 2 : ␣1B alone, 7.79 Ϯ 1.00 mV, n ϭ 9; ␣1B plus 14-3-3,7.01 Ϯ 0.83 mV, n ϭ 11) and slope factors (k: ␣1B alone, 5.63 Ϯ 0.25 mV, n ϭ 9; ␣1B plus 14-3-3, 5.45 Ϯ 0.27 mV, n ϭ 11) of steady-state activation between tsA-201 cells that expressed ␣1B alone and 14-3-3 plus ␣1B. This indicates that the enhancement in current density did not result from 14-3-3-induced changes in gating properties of Cav2.2 channels. Collectively, these results demonstrate that the 14-3-3 proteins increase cell surface expression of the Cav2.2 pore forming ␣1B subunit.

Regulation of Cav2.2 Surface
14-3-3 Regulates Endoplasmic Reticulum Retention of Cav2.2 ␣1B-14-3-3 proteins have been shown to modulate surface expression of several membrane proteins by interfering the interaction between the coat protein I (COPI) complex and cargo. To determine whether such mechanism applies to 14-3-3-mediated surface expression of Cav2.2 ␣1B channel, we examined the influence of ␣1B C-terminal fragments on retention of the rat CD8␣ subunit, which is a transmembrane glycoprotein predominantly expressed on the cell surface. Two regions (CT1 and CT2, encompassing amino acids 1706 -1940 and 2102-2220, respectively; Fig. 2A) of the ␣1B C terminus previously shown to bind to 14-3-3 were fused individually to the C terminus of rat CD8␣ that contained a Myc tag at its N terminus. Another ␣1B C-terminal fragment (Myc-CD8␣-CT3; Fig. 2A) was tested in parallel as a negative control. Consistent with our previous finding (20), CT1 and CT2, but not CT3, exhibited robust binding to 14-3-3 as shown by coimmunoprecipitation between 14-3-3 and these Flag-Myc-tagged CT fragments in cotransfected tsA-201 cells (Fig. 2B). To further strengthen this observation, we analyzed 14-3-3 binding to Flag-Myc-tagged CT1 using size exclusion chromatography, which is another widely established method for assessing protein complexes. As shown in Fig. 2C, either the 14-3-3 or Flag-Myc-CT1 protein eluted in fractions that correspond to monomeric and/or dimeric forms of the respective proteins (Fig. 2C,  fractions 17 and 18), whereas the mixture of Flag-Myc-CT1 and 14-3-3 proteins coeluted in fractions with higher molecular weights (Fig. 2C, fractions 16 -18). These observations demonstrate that 14-3-3 and CT1 coexist in the same protein complex under this condition. Subsequently, we assessed the cell surface expression of the three Myc-CD8␣ fusion proteins by immunofluorescence labeling of the transfected tsA-201 cells under nonpermeabilized and permeabilized conditions. The ratio of membrane to total Myc-CD8␣-CT1 was significantly lower than that of Myc-CD8␣-CT2 and Myc-CD8␣-CT3 (Fig.  2, D and E), suggesting that CT1 was able to obstruct CD8␣ from trafficking to the plasma membrane. The effect of CT1 on CD8␣ surface expression is in agreement with previous studies showing that the Cav2.2 C terminus contains one ER retention motif at its proximal region (7,9). It thus raises a possibility that 14-3-3 may facilitate Cav2.2 channel trafficking by promoting its ER export.
To test this hypothesis, we first utilized the 14-3-3 antagonist to assess the participation of endogenous 14-3-3 proteins in forward transport of the Myc-CD8␣ and Cav2.2-CT fusion. Cotransfection of pSCM138, but not pSCM174, significantly reduced the surface expression of Myc-CD8␣-CT1 (Fig. 3, A  and B). Moreover, the level of surface Myc-CD8␣-CT1 was increased by coexpression of 14-3-3, an effect that was also abolished by cotransfection of pSCM138 (Fig. 3, A and B). By contrast, neither exogenous 14-3-3, pSCM138 nor pSCM174 affected the surface expression of Myc-CD8␣-CT2 (Fig. 3, C and D), which also binds to 14-3-3 but does not contain an ER retention motif (Fig. 2B). Together, these data suggest that 14-3-3 may promote forward transport of the Cav2.2 channel by masking the ER retention signal at its proximal C-terminal region (CT1).
In addition, we have previously showed that phosphorylation of a serine residue (Ser-2126) situated within CT2 of ␣1B was involved in the interaction with 14-3-3 proteins (20). Consistent with the idea that only CT1, but not CT2, of Cav2.2 ␣1B is involved in the ER retention regulated by 14-3-3, the current density of the ␣1B S2126A mutant was enhanced by 14-3-3 to a similar degree as that of the wild type channel (Fig. 4A), despite the obvious increase in the rate of current inactivation as previously described (20). Furthermore, coexpression of S2126␣1B with the 14-3-3 antagonist construct pSCM138, but not its nonfunctional control pSCM174, dramatically reduced the current density regardless of whether 14-3-3 was coexpressed or not (Fig. 4B). These observations provide additional evidence to support that 14-3-3 binding to the CT1 region, but not the CT2 region, of the Cav2.2 ␣1B subunit is responsible for 14-3-3-mediated regulation of Cav2.2 surface expression.
14-3-3 and Cav ␤ Subunit Coregulate Surface Expression of Cav2.2 Channels-As reported previously (20), 14-3-3 and the Cav ␤ subunit can simultaneously bind to the ␣1B subunit. Considering that 14-3-3 and the ␤ subunit may mask ER retention signals localized to different regions of the ␣1B subunit (7,9), we anticipated the possibility that they could act in concert to regulate the surface expression of Cav ␣1 subunits. Thus, we compared the Cav current density in tsA-201 cells that were cotransfected Cav ␣1B and ␤1 with either pSCM138 or pSCM174. Coexpression of pSCM138, but not pSCM174, significantly decreased the current density of Cav2.2 channel (Fig.  5A), suggesting that endogenous 14-3-3 and Cav ␤1 subunit may coregulate the surface expression of Cav channels in tsA-201 cells. Next, we tested whether endogenous 14-3-3 and Cav ␤ subunits comodulate Cav2.2 channels in neurons, where both Cav2.2 channels and 14-3-3 proteins are abundantly expressed (20). Cultured neurons were transfected with either pSCM138 or pSCM174. Two days following transfection, endogenous Ca 2ϩ channel currents were recorded from these neurons with bath solution contained tetrodotoxin (1 M) to block Na ϩ FIGURE 2. An ER retention signal is present in the 14-3-3 binding region at the proximal C terminus of Cav2.2 ␣1B. A, schematic diagram showing the relative positions of the three C-terminal fragments [CT1 (red), CT2 (yellow), and CT3 (blue). Note: the green color represents the overlapping area between CT2 and CT3] of Cav2.2 ␣1B fused to the C terminus of Myc-CD8␣ or dual Flag-Myc epitope tags. B, association of ␣1B C-terminal fragments with 14-3-3. Lysates of tsA-201 cells cotransfected with 14-3-3 and Flag-Myc-tagged CT1, CT2, and CT3 were subjected to immunoprecipitation (IP) with the anti-Flag beads at 48 h post-transfection. The precipitants were analyzed by Western blotting using anti-14-3-3 or anti-Flag antibody as indicated. C, Western blots show the patterns of the 14-3-3, Flag-Myc-tagged-CT1, and the mixture of 14-3-3 and CT1 proteins eluted from Superose 6 10/300 GL size column. Whole cell extract (4 mg) was prepared from tsA-201 cells transiently expressing 14-3-3, and Flag-Myc-CT1 or their mixture and was loaded onto a Superose 6 10/300 GL gel filtration column. D, representative images of tsA-201 cells transfected with Myc-CD8␣-tagged CT1, CT2, and CT3 by immunofluorescence assay under nonpermeabilized conditions as indicated. Scale bar, 10 m. E, quantification of membrane:total ratios of Myc-CD8␣. ***, p Ͻ 0.001 compared with Myc-CD8␣-CT3, one-way ANOVA followed by Tukey's post hoc test. The data are means Ϯ S.E. from four to six experiments; the total numbers of cells analyzed (n) ranged from 24 to 61 cells per condition. channels and nifedepine (10 M) and -agatoxin IVA (200 nM) to block Cav1 and Cav2.1 channels, respectively. Under these conditions, the inward currents are mediated primarily by Cav2.2 channels (20). Transfection of pSCM138, but not pSCM174, significantly reduced the density of endogenous Cav2.2 channel currents (Fig. 5B). This is in line with our obser-  vation in tsA-201 cells and indicates the involvement of endogenous 14-3-3 in promoting surface expression of Cav2.2 channels. Thus, these data support that the surface expression of Cav2.2 channels could be coordinately regulated by both 14-3-3 and Cav ␤ subunits.

DISCUSSION
Cav2.2-mediated signaling is determined by the channel abundance at the cell surface. Thus, appropriate cellular trafficking and localization are crucial for the physiological function of Cav2.2 channels. The foregoing results provide evidence and elucidate the possible mechanisms underlying the effect of 14-3-3 on Cav2.2 surface expression in transfected tsA-201 cells in the absence of known Cav auxiliary subunits. Coexpression of 14-3-3 led to enhanced surface expression of Cav2.2 ␣1B channels via binding of its proximal C-terminal region (CT1, amino acids 1706 -1940) to the 14-3-3 protein, which may modulate the export of ␣1B from the ER. Consistent with the immunofluorescence assay, biochemical analysis and electrophysiological recordings in transfected tsA-201 cells demonstrated that the CT1 region of the Cav2.2 ␣1B C terminus accounted for the observed effect of 14-3-3 on Cav2.2 ␣1B surface expression. In addition, the 14-3-3/Cav ␤ subunit coregulates the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Our data thus show a critical role for the CT1 region of Cav2.2 ␣1B C terminus in regulating channel trafficking to the plasma membrane, and this regula-tion appears to be mediated by 14-3-3 proteins via direct protein-protein interaction.
The Cav channels are thought to be heteromultimers composed of the pore-forming ␣1 subunit and auxiliary ␤ and ␣2␦ subunits. The ␤ subunit has been proposed to both enhance the functional expression and influence the biophysical properties of the Cav1 and Cav2 channels. Whereas some studies have described that ␤ subunit hyperpolarizes the voltage dependence of activation, augments the maximal open probability, and consequently results in increased current through individual channel and macroscopic current density (3,10,30,31), others observed that the ␤ subunit either promotes the insertion of Cav channels into the plasma membrane, as determined by gating charge measurements, imaging, and biochemical methods (7,(32)(33)(34)(35)(36)(37) or has no effect on membrane insertion at all (38). The mechanism of the Cav ␤ subunit effect on surface expression has generally been attributed to masking an ER retention signal in the ␣1 subunits (7,8). However, it was reported recently that the Cav ␤ subunit enhanced the channel translocation from ER to plasma membrane via preventing its degradation by the proteasomal pathway and thereby leading to increased cell surface expression of ␣1 subunits rather than masking an ER retention signal (9, 10). Our current results suggest that 14-3-3 enhances surface expression of Cav2.2 ␣1B by binding to the CT1 region at the ␣1B C terminus via possible masking its ER retention effect. This would fit well with the previous reports showing that the Cav ␤ subunits mask an unidentified ER retention signal on the ␣1 subunit (7,9). In this context, our findings support that 14-3-3 binding to CT1 of the Cav2.2 ␣1B C terminus is critical for mediating the surface expression of Cav2.2 channels via masking the ER retention signal at this site and consequently enabling the channel to escape the ER.
Our previous study revealed two putative 14-3-3 interaction sites in the C terminus of the ␣1B subunit, including a phosphoserine-containing motif that directly binds to 14-3-3 and an upstream region near the EF hand and IQ domain (20). Using immunofluorescence assay and electrophysiological recording, we confirmed that 14-3-3 binding to ␣1B C terminus is important for Cav2.2 channel surface expression, as reflected by decreases in the ratio of surface to total Cav2.2 ␣1B protein expression and the current density of the Cav2.2 channel caused by coexpression with the 14-3-3 antagonist, pSCM138 (Fig. 1, C-E). The mutant S2126A ␣1B subunit did not affect the effect of 14-3-3 on Cav2.2 current density (Fig. 4), suggesting that the Ser-2126 phosphoserine is not involved in the 14-3-3-mediated regulation on Cav2.2 ␣1B surface trafficking. This is consistent with the finding using Myc-CD8␣ fusion proteins that although both interact with 14-3-3 proteins, only CT1, but not CT2 (which contains Ser-2126), fragment of the ␣1B C terminus is involved in ER retention, the effect that can be masked by 14-3-3 proteins (Figs. 2 and 3). Taken together, our results indicate that only one of the putative 14-3-3-binding sites at ␣1B C terminus, the one closer to the last transmembrane domain (amino acids 1706 -1940), contains the ER retention signal that is subjected to regulation by 14-3-3 proteins, which enable the channel to escape from the ER. The other more downstream 14-3-3-binding site that contains the phosphoserine may be more dedicated to regulation of biophysical properties of Cav2.2 channels (20).
Based on our previous study, 14-3-3 and Cav ␤ subunits are known to simultaneously bind to the pore-forming ␣1B subunit of Cav2.2 (20). We showed that the current density of Cav2.2 channels can be regulated by both 14-3-3 and Cav ␤ subunit in either tsA-201 cells or neurons (Fig. 5), suggesting that the surface expression of Cav2.2 could be coordinately regulated by Cav ␤ subunit and 14-3-3. Certainly, these results may not be sufficient to establish a direct correlation between 14-3-3 and Cav ␤ subunits. Therefore, it will be interesting to determine whether 14-3-3 and Cav ␤ subunit might cooperatively or competitively regulate the surface expression of Cav2.2 channels and elucidate the detailed mechanism on 14-3-3-dependent modulation of the Cav2.2 channel trafficking in the future.
14-3-3 protein interaction with ion channels has been shown to not only regulate the functional properties of the ion channels (20, 39 -41) but also to modulate their trafficking without affecting their biophysical properties (14 -18). Interestingly, our previous and current results suggest that 14-3-3 modifies both function (20) and trafficking of Cav2.2 ␣1B channels. In light of the findings reported here, we propose a model for 14-3-3-mediated Cav2.2 ␣1B surface expression through a possible pathway that masks an ER retention signal and consequently enables the channel to escape the ER in the absence of known Cav auxiliary subunits. The proximal C-terminal region of ␣1B that binds to 14-3-3 proteins contains the ER retention motif important for the functional surface expression of the Cav ␣1B subunit. Together, 14-3-3 binding to the C-terminal ER retention signal of Cav2.2 ␣1B plays a critical role in the functionality of the Cav channel because of its regulation on channel protein trafficking to the cell surface. Uncovering the regulation of forward trafficking of Cav2.2 channels is pivotal for understanding the molecular mechanisms underlying their surface expression and functional control under physiological and pathophysiological conditions. This knowledge is fundamental for the development of therapeutic approaches to treat human diseases caused by dysfunctions in channel surface expression.