The Journal of Neuroscience, March 21, 2007, 27(12):3317-3327; doi:10.1523/JNEUROSCI.4566-06.2007
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Cellular/Molecular
Inhibition of Recombinant N-Type CaV Channels by the 
2 Subunit Involves Unfolded Protein Response (UPR)-Dependent and UPR-Independent Mechanisms
Alejandro Sandoval,1,3
Arturo Andrade,1
Aaron M. Beedle,4
Kevin P. Campbell,4 and
Ricardo Felix2
1Departments of Physiology, Biophysics, and Neuroscience, and 2Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City, 07300, Mexico, 3School of Medicine Faculty of Superior Studies Iztacala, National Autonomous University of Mexico, Tlalnepantla, 54090, Mexico, and 4Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101
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Abstract
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Auxiliary 
subunits are an important component of high-voltage-activated calcium (CaV) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the subunits on neuronal CaV channel activity and expression. We found that coexpression of 2 or 3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (CaV2.2/ß3/
2
) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by . Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, subunit expression altered the voltage dependence of channel activity. Second, subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal subunits significantly downregulate CaV2.2 channel activity, (2) uncover a role for the 2 subunit in CaV2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the subunits, supporting the notion that CaV channels constitute a class of difficult-to-fold proteins.
Key words: Ca2+ channels; ; subunit; genistein; HEK-293 cells; PERK; UPR
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Introduction
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Voltage-activated Ca2+ (CaV) channels are essential for basic neurophysiological processes including transmitter release, signal transduction, and gene expression (Berridge, 1998
; Hille, 2001). CaV channels may be broadly divided into low-voltage-activated (LVA) (T-type) and high-voltage-activated (HVA) channels (types L, N, P, Q, and R), with this classification indicative of their functional and pharmacological properties (Hille, 2001; Catterall et al., 2005). At the molecular level, it has been reported that as many as four subunits may form the CaV channel complex (Catterall et al., 2005; Felix, 2005). The CaV1 subunit is the ion-conducting element and contains the gating and voltage sensor machinery of the channel. The CaVß subunit is entirely cytoplasmic and participates in a wide range of regulatory actions. The CaV2 consist of an extracellular 2 peptide linked by disulfide bonds to a transmembrane domain, and is responsible for diverse regulatory actions on the membrane expression and functional activity of the pore-forming subunit. Lastly, the transmembrane CaV subunit has been found in skeletal muscle CaV channels, and related subunits are expressed in heart and brain. The effects of the neuronal CaV subunit on Ca2+ channels have been studied in heterologous systems, and although some of the results are discordant, the consensus seems to be that CaV downregulates channel activity (Black, 2003; Kang and Campbell, 2003).
The CaV family comprises eight tetraspan membrane glycoproteins with intracellular N and C termini (Black, 2003; Kang and Campbell, 2003). One of the most versatile members of this family of proteins is the neuronal CaV2 subunit. Recent studies have shown that, in addition to its role as a component of Ca2+ channels, CaV2 also serves as a chaperone for synaptic targeting of the AMPA receptor (Qiao and Meng, 2003; Letts, 2005) and can act as a cell adhesion molecule (Price et al., 2005). In addition, there has been some controversy about the function of the neuronal CaVs (including CaV2) as voltage-gated Ca2+ channel subunits (Moss et al., 2003), because their functional effects are relatively small in certain model systems. However, diverse studies agree that CaV2 inhibits Ca2+ current. Electrophysiological recordings have shown that CaV2 increases steady-state inactivation of neuronal CaV2.1 (P/Q-type) channels (Letts et al., 1998; Klugbauer et al., 2000; Rousset et al., 2001) and induces a significant decrease in the current amplitude through CaV2.2 (N-type) channels expressed in Xenopus oocytes (Kang et al., 2001).
In the current report, we show that the coexpression of neuronal CaV2 or CaV3 subunits substantially reduces whole-cell currents through recombinant CaV2.2 channels heterologously expressed in human embryonic kidney 293 (HEK-293) cells. Our data suggest that current inhibition may involve important alterations in the functional properties of the CaV2.2 channels as well as activate the unfolded protein response (UPR) to suppress channel translation.
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Materials and Methods
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Materials.
Chloroquine (catalog #C-6628) and genistein (catalog #G-6649) were obtained from Sigma (St. Louis, MO); benzyloxycarbonylleucyl-norleucinal (calpeptin; catalog #03-34-0051) and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132) (catalog #03-34-0051) were from Calbiochem (La Jolla, CA). N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-leucyl]-agmatine (E-64) (catalog #10874523001) was purchased from Roche (Basel, Switzerland), and DMSO (catalog #RES2166D) was from Research Organics (Cleveland, OH). All other chemicals were of reagent grade and obtained from different commercial sources.
cDNA clones.
Cell expression constructs were made by standard techniques, and their fidelity was verified by DNA sequencing. The 1B-pKCRH2 construct containing the cDNA clone encoding the rabbit brain N-type Ca2+ channel CaV2.2 pore-forming subunit (formerly 1B; GenBank accession number D14157) (Fujita et al., 1993) was kindly provided by B. Adams (Utah State University, Logan, UT). The cDNA coding the rat brain CaV2-1 (M86621
[GenBank]
) (Kim et al., 1992), rat brain CaVß3 (M88751
[GenBank]
) (Castellano et al., 1993), mouse brain CaV2 subunit (NM_007583
[GenBank]
) (Letts et al., 1998), and human skeletal muscle sarcospan (AF016028
[GenBank]
) (Crosbie et al., 1997) were subcloned into the pcDNA3 vector (Invitrogen, Carlsbad, CA). The mouse brain CaV3 (NM_019430
[GenBank]
) (Letts et al., 2005) cDNA was subcloned into the pIRES-hrGFP1a vector (Stratagene, La Jolla, CA).
The CaV2.2 cDNA was inserted into the pEGFP-C1 vector (Clontech, Mountain View, CA) downstream and in-frame with the enhanced fluorescent green protein (EGFP) to generate the EGFP-CaV2.2 construct. The CaV2 subunit was excised from the pcDNA3 vector using HindIII and BamHI and subcloned into the HindIII/BamHI sites of the pAd5CMVK-NpA shuttle vector as described previously (Arikkath et al., 2003). This construct was used as a template to generate the CaV2 N48Q mutant (see below).
The PKR-like endoplasmic reticulum (ER)-associated kinase (PERK) wild-type (PERK.WT.9E10.pCDNA.amp) and mutant constructs (PERK.K618A.9E10.pCDNAamp; PERK.dC.9E10.pCDNA.amp) cloned into the EcoRI/XhoI sites of the expression vector pcDNAI/Amp (Invitrogen) (Harding et al., 1999) were kindly provided by M. Oyadomari and D. Ron (Skirball Institute of Biomolecular Medicine, New York, NY).
Site-directed mutagenesis.
An asparagine-to-glutamine substitution at position 48 from the amino terminus of the CaV2 subunit was introduced to prevent N-linked glycosylation. This point mutation was introduced with 
40-mer synthetic oligonucleotides using the QuikChange XL mutagenesis kit (Stratagene). The cDNA of the mutant CaV2 channel subunit was sequenced on an automated Sequencer (ABI Prism 310; PerkinElmer Applied Biosystems, Norwalk, CT).
Cell culture and transfection.
HEK-293 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM-high glucose supplemented with 10% horse serum, 2 mM L-glutamine, 110 mg/L sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2/95% air humidified atmosphere. After splitting the cells on the previous day and seeding at 60% confluency, cells were transfected using the Lipofectamine Plus reagent (Invitrogen) with the cDNA clones mentioned previously, according to the manufacturer's instructions. All CaV channel subunits were transfected at the same time.
The human embryonic kidney cells (HEK-293 cells) stably expressing the CaV3.2 channel (GenBank accession number AF051946) (Cribbs et al., 1998) were grown as described previously (Avila et al., 2006). For electrophysiological recordings, cells were lifted off plates, reseeded on poly-L-lysine (0.05%)-precoated glass coverslips and used 2–6 h after plating.
Single dorsal root ganglion (DRG) neurons were isolated from 7- to 9-d-old BALB/c mice as described previously (Salceda et al., 2006) with slight modifications. In short, a dorsal laminectomy was performed and DRGs with the corresponding spinal roots were dissected out. After dissection, the ganglia were incubated with 1.25 mg/ml collagenase type IV and trypsin (Sigma), in DMEM culture medium at 37°C for 30 min. Thereafter, cells were dissociated by repeated pipetting. Dissociated DRG neurons were resuspended in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated on poly-L-lysine (0.05%)-precoated glass coverslips placed into 35 mm culture plates, and 24 h later were transfected with the CaV2 subunit cDNA subcloned into the pAd5CMVK-NpA shuttle vector using the Fugene transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Forty-eight hours after transfection, DRG neurons were subjected to electrophysiological recording.
Electrophysiology.
Ba2+ currents through recombinant N-type Ca2+ channels heterologously expressed in HEK-293 were recorded as described previously (Sandoval et al., 2004) using the whole-cell configuration of the patch-clamp technique (Marty and Neher, 1995). All experiments were performed at room temperature (RT) (22°C) and a holding potential (HP) of –80 mV. Currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Union City, CA) and filtered at 2 kHz (internal four-pole Bessel filter). Currents were digitized at 5.71 kHz, using a DigiData 1320A interface (Molecular Devices) and analyzed using the pCLAMP (Molecular Devices) and SigmaPlot (Systat Software, Richmond, CA) software. Linear capacitative currents were minimized via the capacitative transient cancellation feature of the amplifier. The remaining linear components were subtracted using a P/4 protocol. Membrane capacitance was determined as described previously (Avila et al., 2004) and used to normalize currents. The bath recording solution contained the following (in mM): 10 BaCl2, 125 TEA-Cl, 10 HEPES, and 15 glucose, pH 7.3. The internal solution consisted of the following (in mM): 110 CsCl, 5 MgCl2, 10 EGTA, 10 HEPES, 4 Na-ATP, and 0.1 Na-GTP, pH 7.3. Whole-cell patch-clamp endogenous K+ currents were obtained from an HP of –80 mV applying test pulses every 20 s as described previously (Avila et al., 2004). Current signals were filtered at 2 kHz and digitized at 5.71 kHz. Recording solutions mainly consisted of the following (in mM): 140 NaCl, 3 KCl, 5 CaCl2 (external solution) and 130 K-Asp, and 8 KCl (internal solution).
Flow cytometry.
Untransfected and CaV channel subunit-expressing HEK-293 cells were kept in culture for 48 h as described previously. Cells were dispersed by treatment with PBS/trypsin (0.05%) and harvested in PBS at RT. The dispersed cells were washed with PBS plus 2% EDTA (PBS/EDTA) and pelleted by low-speed centrifugation. Cells were resuspended in PBS/EDTA (400 µl aliquots). Cell-associated fluorescence was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) in two different assays. In the first series of experiments, cytometric analysis was performed directly in HEK-293 cells expressing the EGFP-CaV2.2 fusion protein using an excitation wavelength of 488 nm and an emission wavelength of 508 nm. In the second series of experiments, performed in HEK-293 cells expressing CaV2.2 channels (transfected in the pKCRH2 vector), an affinity-purified anti-CaV2 primary antibody (GP1) was used in a 1:1000 dilution. The rabbit anti-guinea pig secondary antibody (Zymed, San Francisco, CA) was conjugated with FITC. In this case, cells were first incubated with the antibodies raised against the extracellular CaV2 subunit for 30 min at RT, and then with the secondary antibody for additional 30 min at RT. Fluorescence was assayed using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Statistical analysis.
Curve fitting and statistical analyses were performed using the SigmaPlot 8.0 software package (SPSS, Chicago, IL). The significance of observed differences was evaluated by nonpaired Student's t test. A value of p < 0.05 was considered to be significant.
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Results
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Heterologous expression of CaV2 specifically inhibits IBa through recombinant CaV2.2 channels
To examine the effects of CaV2 on neuronal Ca2+ channels, this regulatory subunit was cotransfected with N-type channels (CaV2.2/ß3/2) in HEK-293 cells 48 h before current recordings. Representative traces of Ba2+ current (IBa) evoked by test pulses to +10 mV recorded from control (without CaV2) and cells expressing CaV2 are depicted in Figure 1A. Coexpression of CaV2 decreased IBa amplitude through N-type channels to nearly undetectable levels. Given the drastic effect of CaV2, it was necessary to rule out any potential interference of the pAd5CMVK-NpA construct with the transcriptional machinery of the cells; therefore, we generated a pIRES plasmid construct encoding the related CaV3 sequence (66% amino acid identity to mouse CaV2) (Letts et al., 2003) and examined its actions using the same approach. Expression of the CaV3 construct also resulted in a near complete loss of current amplitude (Fig. 1A,B).
Expression of SS (sarcospan), an unrelated tetraspan protein, with N-type channels had no effect on IBa, suggesting a specific functional interaction of CaVs with the channel complex (Fig. 1A,B). Likewise, CaV regulation was specific for high-voltage-activated Ca2+ channels as CaV expression had no effect on endogenous K+ currents (Fig. 1C), nor recombinant low-voltage-activated CaV3.2 (T-type) currents (Fig. 1D).
To confirm that current inhibition by the CaV subunits was not simply a nonspecific effect of overexpression, we next transfected HEK-293 cells using different concentrations of cDNAs. As we discuss later, this also allowed us to obtain currents large enough for accurate analysis. Hence, we fixed the cDNA concentration for CaV2.2, ß3, and 2 subunit transfection (1:1:1 molar ratio) and varied the CaV2 concentrations. Our results show that, on coexpression of the CaV2 cDNA in molar ratios ranging from 1:0.1 to 1:1.4 (with respect to the other CaV channel subunits), the amplitude of IBa through CaV2.2/ß3/2 channels decreased significantly as the relative molar ratio of CaV2 increased (Fig. 2A). These data suggested a CaV2-specific inhibitory effect. As shown in Figure 2B, current regulation was observed at virtually all voltages tested.
Regulation of whole-cell conductance and channel steady-state inactivation by the CaV2 subunit
Because the gating of CaV2.2 channels is influenced by the coexpression of auxiliary subunits, the reduction in IBa may reflect a CaV2-mediated effect on the voltage dependence of channel activity. To test this, we constructed activation and steady-state inactivation curves from analysis of macroscopic currents. Increasing CaV2 molar ratio clearly decreases the functional expression of recombinant CaV2.2-containing channels in a dose-dependent manner as shown by a significant decrease in macroscopic conductance (Fig. 3A). There was a significant shift in the half-maximal voltage (V1/2) of the G–V curves in the presence of increasing concentrations of CaV2, consistent with the idea that this auxiliary subunit affects the voltage sensitivity of CaV2.2 channels (Fig. 3B).
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Figure 3. The CaV2 subunit regulates whole-cell conductance. A, Voltage dependence of activation of recombinant N-type [CaV2.2(1B)/CaV2/CaVß3] channels in control cells (–2) or cells cotransfected with different concentrations of the cDNA encoding the CaV2 subunit. Conductance data were derived from Figure 2B using the expression: G = I/(Vm – Vrev), where I is current, G is conductance, Vm is the test potential, and Vrev is the extrapolated reversal potential. The mean data were fitted with Boltzmann functions of the form G = Gmax/(1 + exp[(Vm – V1/2)/k]–1), where Gmax is maximum conductance, Vm is the test potential, V1/2 is the potential for half-maximal activation of Gmax, and k is a slope factor. B, Comparison of the mean half-activation voltage (V1/2) for CaV2.2/CaV2/CaVß3 channels alone (–2) or coexpressed with the CaV2 subunit (n = 12–14). Error bars indicate SEM, and the asterisks denote significant differences (p < 0.05) compared with control.
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The voltage dependence of steady-state inactivation was also significantly affected by the presence of the of CaV2 subunit. As shown in Figure 4, there is a significant >10 mV hyperpolarizing shift in the CaV2.2 steady-state inactivation curve on coexpression with increasing molar ratios of the CaV2 subunit.
Regulation of CaV2.2 channel currents by CaV in the absence of CaV2 and CaVß subunits
Previous studies examining the influence of auxiliary CaV2 and CaVß subunits on the inhibitory actions of the neuronal CaV subunits have given contrasting results. Hence, although it has been reported that the CaV2-induced current inhibition is more pronounced with CaV2 expression (Kang et al., 2001), the CaV7 subunit inhibitory actions seem not to be dependent on the presence of the CaV2 subunit as part of the N-type channel complex (Moss et al., 2002). Therefore, we next tested the role of other Ca2+ channel regulatory subunits on CaV inhibition. To evaluate this, HEK-293 cells were cotransfected with CaV2.2/2 channels (in the absence of the CaVß subunit) and cDNA encoding CaV2 or CaV3 at a molar ratio of 1:1. As shown by representative current traces (Fig. 5A, top), the presence of CaV subunits results in a significant reduction of current amplitude. Consequently, the comparison of mean current density at +10 mV recorded in HEK-293 cells expressing CaV2.2/2 channels and cells coexpressing the CaV subunits resulted in current densities of 60 and 30%, respectively (Fig. 5A, bottom). We then performed this experiment in cells transfected with different concentrations of the CaV2 cDNA. As shown in Figure 5B, CaV2 coexpression led to suppression of CaV2.2 currents even at low CaV2 cDNA concentrations, although, as expected, the inhibitory effects were less pronounced as the relative molar ratio of CaV2 increased.
Similar suppression effects were observed when the HEK-293 cells expressing CaV2.2/ß channels were cotransfected with the cDNA coding for CaV2 or CaV3. Representative superimposed IBa traces show CaV-mediated reduction of N-type current (Fig. 6A, top). Notably, the inhibitory effect of the CaV subunits is evident even in the absence of the CaV2 subunit. Comparing the mean current densities at +10 mV, coexpression with CaV2 or CaV3 (molar ratios of 1:1) resulted in IBa densities of 15 and 25%, respectively (Fig. 6A, bottom). Lastly, when this experiment was performed in cells transfected with various concentrations of CaVß, coexpression of the CaV2 subunit decreased IBa density at all concentrations tested, and, as in the case of the CaV2 subunit, increased molar coexpression of CaVß did not counteract the inhibitory effects of CaV2 (Fig. 6B).
The actions of CaV2 and CaV3 are not prevented by inhibition of internalization
The preceding data indicated that heterologous expression of the CaV2 subunit substantially reduced IBa density through recombinant N-type Ca2+ channels in HEK-293 cells by altering the whole-cell conductance and the balance between channel availability and inactivation. However, the shift in the voltage dependence of channel inactivation (15 mV in the hyperpolarizing direction) only reduces the fraction of available channels by 10% at a holding potential of –80 mV, and thus cannot fully explain the drastic inhibitory effects observed in IBa density in the presence of the inhibitory subunit. This suggested the possibility that the channels could be removed from the cell surface after CaV2 expression. To analyze the possible participation of a CaV channel internalization-dependent mechanism, a series of inhibitors were used. First, we tested the lysosomal inhibitor chloroquine and the calpain antagonist calpeptin. Chloroquine is a weak base that accumulates in lysosomes, dissipating the acidic pH, whereas calpeptin is a general inhibitor of calpain-induced protein breakdown necessary for the activity of the ubiquitin–proteasome system. Both compounds have been used to inhibit internalization. The data in Figure 7, A and B, show that N-type IBa density was similarly reduced in cells transfected with CaV2 or CaV3 even in the presence of the internalization inhibitors, from a control value of –98.6 ± 18.4 to –5.5 ± 3.5 and –4.7 ± 1.6 pA/pF, in the case of chloroquine, and to –6.7 ± 3.2 and –2.5 ± 1.9 pA/pF in the case of calpeptin.
To examine in more detail whether the CaV-mediated current decrease involved internalization of the channels, we evaluated the effects of MG-132 and E-64. MG-132, a selective inhibitor of the 26S proteasome, failed to alter the inhibitory actions of CaV2 and CaV3 subunits as IBa reduction persisted after drug treatment (Fig. 7C). Similar results were obtained with E-64, a cysteine protease inhibitor that interferes with internalization. CaV2 decreased IBa density through CaV2.2/ß3/2 channels by 9.6-fold. This effect could not be prevented by the treatment with E-64 (Fig. 7D). Together, these data indicate that the CaV-mediated reduction of N-type current does not involve a mechanism of enhancing channel internalization.
N-linked protein glycosylation is not a major determinant for CaV2 regulation of recombinant CaV2.2 channels
Previous studies have shown that at least one consensus site for N-linked glycosylation is present in all eight CaV subunits (Arikkath and Campbell, 2003; Kang and Campbell, 2003), suggesting that such posttranslational modification may have important functions in the CaV channel complex. Interestingly, mutation of this site alters the role of CaV2 in cell aggregation presumably by preventing the protein from localizing to the plasma membrane (Price et al., 2005). To define the role of this consensus N-glycosylation site in plasma membrane targeting and downregulation of CaV channels, we generated a mutant subunit by substituting glutamine for asparagine at amino acid position 48 of CaV2 (N48Q) (Fig. 8A, top). In the absence of CaV2, control current levels ranged up to 1.3 nA, with an average peak current density of –110 ± 23 pA/pF. Unexpectedly, coexpression of the CaV2N48Q variant led to near complete inhibition of IBa, similar to that of the wild-type CaV2 subunit (–1.5 ± 0.4 and –1.4 ± 0.5 pA/pF, respectively) (Fig. 8B). These data suggest that N-type current downregulation may not require efficient CaV2 trafficking to the cell membrane.
CaV2-mediated decrease in IBa density may involve altered CaV channel synthesis and activation of the UPR
CaV2 effects on N-type channel properties (see above) demonstrate that CaV2 influences channels at the cell surface. However, our data indicated that IBa reduction after CaV2 transfection may also involve alterations in surface expression or interference with the synthesis of new channels. Because we ruled out the possibility that CaV2 could increase channel internalization, we next investigated a potential inhibition of channel synthesis. To examine this, an EGFP-CaV2.2 fusion construct was heterologously expressed in the HEK-293 cells together with the CaVß and CaV2 subunits, and the fluorescence emissions in presence and absence of CaV2 were quantified using flow cytometry. Coexpression of CaV2 with the tagged N-type channel reduced the percentage of GFP-expressing cells to 39 ± 2 from 48 ± 3% obtained in the absence of CaV2 (data not shown). Interestingly, coexpression of CaV2 also reduced the average fluorescence intensity per fluorescence-positive HEK-293 cells to 285 ± 9 from 354 ± 8 arbitrary units in the control cells.
It should be noted that, under these conditions, flow cytometry is not well suited to distinguish fluorescence originating from the plasma membrane versus the cytoplasm. To investigate whether the number of the channels expressed at the cell surface was also altered, we performed flow cytometry experiments using an affinity-purified primary antibody against an extracellular epitope in the CaV2 subunit. As summarized in Figure 9, both the percentage of CaV2-immunopositive cells and the mean fluorescence intensity significantly decreased when CaV2 was coexpressed. The proportion of immunoreactive cells was reduced to 23 ± 9% in the CaV2-expressing cells from 47 ± 13% observed in the controls. Likewise, coexpression of the CaV2 subunit decreased the average fluorescence intensity 70% compared with control cells (+CaV2, 77 ± 31; –CaV2, 247 ± 13 arbitrary units), which may help to explain the reduction in N-type current density on CaV2 expression.
Together, these results strongly suggested that the decrease in IBa density caused by CaV2 expression involved an inhibition of CaV channel synthesis without compensatory alterations in channel internalization. In this regard, it is worth mentioning that the expression of truncated versions of CaV2.2 causes channel synthesis arrest at an early stage (Raghib et al., 2001). Likewise, it has been reported that the expression of a short variant of CaV2.1 markedly suppresses currents presumably through the activation of an endoplasmic reticulum-resident RNA-dependent kinase (PERK), which activates components of the UPR to suppress CaV2.1 translation (Page et al., 2004).
Notably, our results with the CaV2 subunit bear a striking resemblance to the dominant-negative synthesis suppression of the CaV2 subunits induced by the coexpression of truncated constructs. Therefore, we tested whether CaV2.2 current suppression by CaV2 involved activation of the UPR. Initially, we investigated the effects of genistein, a tyrosine kinase inhibitor that prevents full expression of the UPR through inactivation of the transcription factor NF-Y (nuclear factor Y) (Zhou and Lee, 1998; Nyfeler et al., 2003). After 6 h treatment with the drug (140 µM), there was no significant change in IBa density of CaV2-expressing cells (Fig. 10). In contrast, longer exposure (24 h) to genistein resulted in a significant reduction of the suppressive effect of CaV2. These findings suggested that activation of the UPR might play a role in the suppression of CaV2.2 currents by the CaV2 auxiliary subunit.
To confirm that CaV2 expression promotes N-type channel regulation via the unfolded protein response mechanism, we used a strategy analogous to that reported by Dolphin and colleagues (Page et al., 2004). This approach consisted in the expression of two dominant-negative PERK constructs. The first construct encodes a PERK lacking the C-terminal kinase domain (PERK 
C), whereas the second construct encodes a PERK with a point mutation in the catalytic site (PERK K618A). Both constructs have been shown to prevent the activation of endogenous PERK (Harding et al., 1999) and therefore inhibit the UPR. When dominant-negative PERK constructs were expressed with the CaV2 subunit and N-type (CaV2.2/ß3/2) channels, there was a significant reversal of CaV2-mediated current suppression (Fig. 11). With the PERK C mutation, CaV2-mediated IBa inhibition was significantly diminished from 94 to 74%, whereas PERK K618A also reduced the suppressive effect of CaV2 to 66% (Fig. 11B). These results indicate that activation of PERK may play an important role in the suppression of CaV2.2 currents by the CaV2 regulatory subunit.
CaV2 inhibits Ca2+ channel activity in DRG neurons
We lastly sought to determine whether the inhibitory actions of CaV2 were also present on native neuronal CaV channels. To this end, we analyzed the IBa density in neonatal mouse DRG neurons transiently transfected with GFP or with the pAd5CMVK-NpA construct (containing the CaV2 subunit plus the GFP). IBa through native Ca2+ channels were analyzed by the whole-cell mode of the patch-clamp technique (Fig. 12). Only cells with a robust GFP fluorescence signal were used for electrophysiological recording. DRG neurons transfected with GFP construct produced current amplitudes of 1 nA. Interestingly, the exogenous expression of the CaV2 produced significantly smaller current amplitudes (Fig. 12B). Likewise, the expression of the auxiliary subunit in DRG neurons did not significantly change the capacitance of the cells (data not shown). As a result, in comparison with the control condition, the expression of CaV2 causes a substantial decrease in current density through CaV channels in mouse neonatal DRG neurons.
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Discussion
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In the current report, we show that the neuronal CaV2 and CaV3 subunits exert an important inhibitory effect on currents through recombinant N-type Ca2+ channels heterologously expressed in HEK-293 cells. This inhibition is (1) specific to CaV channels given that CaV subunit coexpression did not affect endogenous K+ current, (2) restricted to HVA channels because currents through recombinant LVA channels were not affected, (3) not mimicked by an unrelated tetraspan protein, and (4) observed in the absence of the other CaV regulatory subunits. These findings are consistent with previous reports that CaV subunit coexpression generally reduces current density through recombinant channels (Kang et al., 2001; Moss et al., 2002; Arikkath et al., 2003; Hansen et al., 2004).
An interesting issue to be clarified relates to cell pathways by which this CaV-mediated inhibition occurs. As we discuss in the following sections, distinct mechanisms could underlie the decrease in IBa density after CaV expression.
Alteration in the functioning of the channels
In addition to decreasing peak current density, patch-clamp recordings showed that CaV subunits alter the voltage dependency of activation and inactivation of recombinant CaV2.2/ß3/2 channels. Interestingly, a specific effect of CaV2 on current inactivation has previously been shown for CaV2.1 (Letts et al., 1998; Klugbauer et al., 2000) and CaV1.2 (Klugbauer et al., 2000), suggesting that this property may not be restricted to a particular CaV1 ion-conducting subunit.
Increased channel internalization
Given that regulation of preexisting channels was not sufficient to fully explain CaV-mediated suppression of N-type currents, a key mechanistic question was whether the channels were removed from the cell surface after coexpression of the auxiliary subunit. According to this, accelerated channel degradation/internalization would lead to a decrease in current density. However, our data seem to firmly rule out this possibility given that the inhibitory actions of the CaV2/3 subunits remained unaltered after treatment of the cells with a series of inhibitors of degradation/internalization (chloroquine, calpeptin, MG-132, and E-64).
Inhibition of channel targeting to the cell membrane
Because CaV-mediated suppression of N-type currents seemed not to involve increased protein internalization, we investigated whether CaV2 might interfere with the trafficking of CaV2.2 subunits to the cell surface. It is well known that N-linked glycosylation is crucial for the correct folding, subcellular targeting, and stability of numerous proteins (Kleene and Schachner, 2004), including CaV channels (Gurnett et al., 1996; Sandoval et al., 2004). Recently, it has been shown that a mutation in a consensus site for N-glycosylation in the CaV2 sequence disrupts the ability of the protein to act as a claudin mediating cell–cell adhesion (Price et al., 2005). Consequently, we used the same construct (N48Q) to investigate whether this putative N-glycosylation site mutation could abolish the capability of CaV2 to act as a regulatory subunit of CaV channels. We reasoned that, if CaV2 produced a reduction in IBa by reducing channel targeting to the plasma membrane, then transfection of the N48Q mutant should prevent suppression of N-type channel activity. However, our results showed that the suppressive effects of the CaV2 remained unaltered, suggesting that the mutation may alter some of the functional properties of CaV2, but may not be important to determine inhibition of N-type channel functional expression. This is consistent with previous studies showing that treatment with the antibiotic tunicamycin inhibits N-linked glycosylation of the skeletal muscle CaV1 subunit but cannot inhibit the association of the auxiliary subunit to the CaV1.1 subunit (Arikkath et al., 2003).
Thus, CaV2 may alter Ca2+ channel function via two distinct pathways. The first pathway, discussed previously, involves modulation of preexisting channels and is suitable for dynamically regulating Ca2+ channel activity on a rapid timescale. The second pathway, discussed below, may involve interference with CaV2.2 subunit proper folding and synthesis, and would require protein turnover before functional effects are apparent, a mechanism well suited for setting the steady-state level of channels.
Suppression of channel protein synthesis and prevention of the correct folding of CaV2.2
Seminal work by Dolphin and colleagues indicated that coexpression of the CaV7 subunit specifically abolished IBa through heterologously expressed CaV2.2 channels and significantly reduced Ba2+ conductance in non-N type (CaV2.1 and CaV1.2) channels, although the mechanism by which the auxiliary subunit was acting was not determined (Moss et al., 2002). Previous work by these authors also had shown that truncated constructs of the CaV2.2 subunit suppressed CaV2.2 currents and reduced full-length CaV2.2 protein levels by a mechanism that involved decreased synthesis (Raghib et al., 2001). More recently, they found that current suppression required an interaction between the truncated construct and the full-length channel which activates PERK, a component of the UPR, to suppress translation (Page et al., 2004). Remarkably, CaV2-mediated suppression of N-type currents expressed in HEK-293 cells shows resemblance to the inhibition of the CaV2.2 currents induced by the coexpression of truncated CaV2.2 constructs.
When mammalian cells are subjected to ER stress, a specific signaling pathway termed the UPR is triggered. When UPR is initiated, an immediate consequence is the activation of PERK to inhibit protein biosynthesis through phosphorylation of the eukaryotic translation initiation factor eIF2 (Harding et al., 2002; Marciniak and Ron, 2006). In our study, genistein partially prevented the CaV2-mediated suppression of CaV2.2 currents, suggesting the participation of the UPR. Likewise, we found that the use of protease inhibitors as well as blockers of the proteasome activity did not increase the amount of CaV2.2 currents observed in the presence of CaV2, suggesting that the mechanism does not involve enhanced proteolysis. From these results, the most likely explanation for current suppression by CaV2 is that synthesis of the CaV2.2 subunit was arrested. In support of this, flow cytometry analysis indicated that the inhibition of CaV2.2 currents by CaV2 may be attributable to a reduction in the number of channels, because there was a significantly reduction in the percentage of fluorescent cells and mean fluorescence intensity of cells coexpressing EGFP-CaV2.2 and the auxiliary subunit.
To examine whether the mechanism of suppression of CaV2.2 expression by CaV2 indeed involved the UPR, we used two mutant PERK constructs, PERK C and PERK K618A, both of which lack kinase activity. Their dominant-negative behavior arises from their capability to form nonfunctional dimers with endogenous PERK (Harding et al., 1999). Notably, the mutant PERK constructs prevented the inhibition of CaV2.2 expression by the CaV2 subunit, implicating activation of the endogenous kinase, and therefore the UPR, in the process of current suppression.
Likewise, it is interesting to consider what physiological role the CaV2/3 subunit-induced suppression of N-type CaV channels may play. It is well known that this type of channels is expressed predominantly in presynaptic nerve terminals and play a key role in neurotransmitter release (Fisher and Bourque, 2001; Hille, 2001). Given that the CaV2/3 subunits significantly decrease N-type channel activity and expression, they might provide a negative feedback on current amplitude and neurotransmission. In addition, it has been reported that N-type channel expression is an important cue in the genesis of synaptic transmission (Jones et al., 1997; Vance et al., 1998). Therefore, it is also possible that CaV2/3 might play a role as a negative regulator for N-type channel expression during brain ontogeny. Likewise, the expression levels and cell distribution of CaV channel auxiliary subunits can change in diseases such as diabetes (Iwashima et al., 2001), temporal lobe epilepsy (Lie et al., 1999), and neuropathic pain (Luo et al., 2001). Analogously, the CaV subunits could also play important roles in the pathophysiology of certain channelopathies. In line with this, it has been shown hat the removal of the CaV2 protein from the brain causes numerous disorders, including spontaneous absence seizures and ataxia in the stargazer mouse mutant (Letts, 2005).
Lastly, considering the role of CaV2 as a chaperone protein for proper folding and surface expression of AMPA receptors (Qiao and Meng, 2003), our results suggest some clues concerning a possible multifaceted role for this protein in an individual cell. For instance, in a physiological context in which CaV2 expression was low, Ca2+ influx through CaV2.2 channels would provide a favorable local environment for the stabilization of AMPA receptors at the postsynapsis. However, when CaV2 expression was high, CaV2.2 channel synthesis would be negatively modulated, which may result in decreased Ca2+ influx and a favorable local environment for the lateral movement of AMPA receptors. It is worth mentioning that it has been suggested that indeed changes in local intracellular Ca2+ may regulate the lateral movement of AMPA receptors in hippocampal neurons (Borgdorff and Choquet, 2002). In this way, the expression of the CaV2 subunit could play a role in the molecular mechanism underlying synaptic plasticity.
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Footnotes
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Received Oct. 20, 2006;
revised Feb. 15, 2007;
accepted Feb. 16, 2007.
This work was supported by grants from the National Council for Science and Technology (Conacyt) and the Miguel Aleman Foundation (R.F.). Doctoral fellowships from Conacyt to A.S. and A.A. are gratefully acknowledged. K.P.C. is an Investigator of the Howard Hughes Medical Institute. We gratefully appreciate the technical expertise of B. E. Reyes, L. Escobedo, M. Oliva, M. Urban, and G. Aguilar. We also thank M. Oyadomari and D. Ron (Skirball Institute of Biomolecular Medicine, New York, NY), who generously provided the PERK cDNA constructs, as well as L. M. Alvarez-Salas (Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City, Mexico) for the pEGFP-C1 vector, J. C. Gomora (Institute of Cellular Physiology–National Autonomous University of Mexico, Mexico City, Mexico) for the CaV3.2 channel stably expressing HEK-293 cell line, and A. Almanza for helpful advice on the DRG neuron culture.
Correspondence should be addressed to Dr. Ricardo Felix, Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Avenida Instituto Politécnico Nacional #2508, Colonia Zacatenco, México D.F., CP 07300, México. Email: rfelix{at}fisio.cinvestav.mx
Copyright © 2007 Society for Neuroscience 0270-6474/07/273317-11$15.00/0
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Abstract
Introduction
Q mutant (N48Q). Currents were generated by applying 140 ms activating pulses at +10 mV from a holding potential of –80 mV. B, Mean IBa density in HEK-293 cells as in A. Bars denote mean ± SE; the number of recorded cells is indicated in parentheses. *p < 0.05 compared with control.
Gene
