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Am J Physiol Cell Physiol 290: C1209-C1220, 2006. First published November 23, 2005; doi:10.1152/ajpcell.00247.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2

Chung-Jiuan Jeng,1 Yu-Ting Chen,2 Yi-Wen Chen,2 and Chih-Yung Tang2

1School of Medicine, Fu Jen Catholic University, Hsin-Chuang, Taipei County; and 2Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan

Submitted 25 May 2005 ; accepted in final form 17 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming {alpha}1A-subunits of human P/Q-type Ca2+ channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human {alpha}1A-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of {alpha}1A-WT with each {alpha}1A-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba2+ currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of {alpha}1A-mutants increased, suggesting the presence of an {alpha}1A-mutant-specific suppression effect. When we coexpressed {alpha}1A-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of {alpha}1A-WT and mutant using a splice variant of {alpha}1A-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-{alpha}1A-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.

channelopathy; voltage clamp; Xenopus oocytes; cerebellum; splice variants

VOLTAGE-GATED P/Q-TYPE Ca2+ channels (Cav2.1 channels) comprise a principal pore-forming {alpha}1A-subunit in combination with auxiliary {alpha}2{delta}-, beta-, and perhaps {gamma}-subunits (5). Cav2.1 channels play an important role in neurotransmitter release, especially at cerebellar Purkinje cells and somatic motor nerve terminals of mammals (47, 51, 52, 60). Cav2.1 channels also participate in determining membrane excitability of the dendrosomatic region of postsynaptic neurons (49, 53). The expression of {alpha}1A-subunits is distributed widely throughout the brain with especially prominent density in the cerebellum (28, 57), highlighting the importance of Cav2.1 channels in the neurophysiological function of the cerebellum. For example, Cav2.1 channels have been shown to play a pivotal role in mediating glutamate release from the parallel fiber nerve terminals of granule cells and in generating complex Ca2+ spikes in the dendritic trees of Purkinje cells (30, 34, 41, 46), both of which serve as indispensable elements of the induction of long-term depression (16, 20), a process that is thought to underlie the synaptic mechanism of cerebellar motor learning (19, 39).

Episodic ataxia type 2 (EA2) is a rare autosomal dominant neurological disorder with distinct signs of cerebellar dysfunction, including interictal nystagmus and episodes of ataxia lasting for hours (21). Molecular genetic analyses have linked EA2 to mutations in a gene in chromosome 19p, CACNA1A, which encodes human {alpha}1A-subunits of Cav2.1 channels (36). To date, >20 mutations in human {alpha}1A-subunits have been identified in patients with familial or sporadic EA2, the majority of which are nonsense (truncation) mutations and the rest of which represent missense mutations and aberrant splicing (21, 24). Until now, only a few missense EA2 mutants have been shown to express functional human Cav2.1 channels. All of these missense mutations displayed significantly smaller ionic currents compared with human wild-type (WT)-Cav2.1 channels in the heterologous expression system (22, 45, 56), indicating that virtually all EA2 mutants result in loss-of-function phenotypes. Thus haploinsufficiency has long been suggested to be one of the major molecular mechanisms underlying EA2 (14, 36, 56).

Whether dominant-negative effects also underlie the molecular mechanism of EA2 mutations, however, remains controversial. One EA2 nonsense mutant (R1824x), when introduced into the rabbit {alpha}1A-subunit for heterologous expression, displayed pronounced dominant-negative effects (25). When introduced into the human {alpha}1A-subunit, the same EA2 nonsense mutant was shown not to possess significant dominant-negative effects, however (18). Similarly, one EA2 nonsense mutant that predicts a truncation mutation with a premature stop at the S1 segment of homologous domain 3 exhibited prominent dominant-negative effects when introduced into the rat {alpha}1A-subunit (37), whereas another EA2 nonsense mutant that also predicts a truncation mutation at the same homologous domain segment failed to demonstrate any dominant-negative effects when introduced into the human {alpha}1A-subunit (56). These discrepancies raise the possibility that dominant-negative effects may involve species differences and raise the question whether dominant-negative effects really occur in patients with EA2. Furthermore, whether missense EA2 mutants possess dominant-negative effects on their WT counterparts has never been determined.

The purpose of this study was to test the hypothesis that EA2 mutants might exert dominant-negative effects on human Cav2.1 channels. We systematically assessed the functional interaction between five EA2 mutants and the human WT {alpha}1A-subunit. Our results strongly suggest that for both missense and nonsense mutations, EA2 phenotypes may occur as a result of dominant-negative effects. The potency of the EA2 suppression effect varies significantly between two splice variants of human {alpha}1A-subunits, however.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC approval no. 20030120) of the National Taiwan University College of Medicine and College of Public Health.

Molecular biology. Full-length cDNA for all constructs of the long isoform of human {alpha}1A-subunits (GenBank accession no. AF004884), as well as those for {alpha}2{delta}- and beta4-subunits, were kindly prepared and generously provided by Dr. Joanna Jen (Dept. of Neurology, University of California, Los Angeles, Los Angeles, CA) (22). In human cerebellum, approximately two-thirds of the {alpha}1A-subunit splice variant is the long isoform (44). As indicated in RESULTS, in one line of experimentation, we also used the short isoform of the human {alpha}1A-subunit (equivalent to GenBank accession no. AF004883, but with one splice deletion of tripeptide VEA) (56), which was kindly provided by Dr. Jörg Striessnig (Dept. of Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, Innsbruck, Austria). To avoid confusion, we adopted recently published nomenclature for the location of different mutations in {alpha}1A-subunits (21). To increase the level of protein expression in Xenopus oocytes, cDNA for the {alpha}1A-subunit (long isoform) and the {alpha}2{delta}-subunit were subcloned using BamHI and XbaI sites into the pGEMHE vector kindly provided by Dr. Emily Liman (University of Southern California, Los Angeles, Los Angeles, CA) (29).

For in vitro transcription, cDNA was linearized with XbaI (for {alpha}1A- and {alpha}2{delta}-subunits) or with HindIII (for beta4-subunits). Capped cRNA was transcribed in vitro from the linearized cDNA template using the mMessage mMachine T7 kit (Ambion, Austin, TX). Concentration of cRNA was determined using gel electrophoresis and verified using spectrophotometry (GeneQuant Pro RNA/DNA calculator; Amersham Biosciences, Piscataway, NJ).

cRNA injection in Xenopus oocytes. Adult female Xenopus laevis (African Xenopus Facility, Knysna, South Africa) were anesthetized by immersion in Tricaine (1.5 g/l). Ovarian follicles were removed from Xenopus frogs, cut into small pieces, and incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1.8 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.2). To remove the follicular membrane, Xenopus oocytes were incubated in Ca2+-free ND96 containing collagenase (2 mg/ml) on an orbital shaker (~200 rpm) for ~60–90 min at room temperature. After being washed several times with collagenase-free, Ca2+-free ND96, Xenopus oocytes were transferred into ND96 solution. Stage V-VI Xenopus oocytes were then selected for cRNA injection.

For all cRNA injection paradigms, the total volume of injection was always 41.4 nl/oocyte. To examine the phenotype of WT-Cav2.1 or mutant human Cav2.1 channels, Xenopus oocytes were injected with a 1:1:1 molar ratio combination of {alpha}1A, {alpha}2{delta}, and beta4 cRNA up to a maximal cRNA amount of 81 ng/oocyte. For coexpression experiments, it was imperative to find a submaximal cRNA concentration for WT-Cav2.1 channels that allowed us to add an extra amount of cRNA for mutant {alpha}1A-subunits. Therefore, we empirically set up a fixed concentration of cRNA for injection, known as the standard cRNA cocktail, in which {alpha}1A-, {alpha}2{delta}-, and beta4-cRNA were mixed in a molar ratio of 1:2:2, and the final injection amounts for {alpha}1A-, {alpha}2{delta}-, and beta4-cRNA were ~3.8 ng, ~3.8 ng, and ~1.9 ng/Xenopus oocyte, respectively. When necessary, a sixfold dilution of the standard cRNA cocktail, which we refer to as the low-cRNA cocktail, was instead used for Xenopus oocyte injection. Injected Xenopus oocytes were stored at 16°C in ND96 and functionally assayed 2–5 days after cRNA injection.

Electrophysiology and data analysis. For functional studies, Xenopus oocytes were transferred into a recording bath containing Cl–-free Ba2+ solution of the following composition (in mM): 40 Ba(OH)2, 50 NaOH, 2 CsOH, and 5 HEPES (pH 7.4 with methanesulfonic acid). To minimize the contribution of endogenous Ca2+-activated Cl– currents, niflumic acid (0.4 mM), which has been shown to display potent blocking effects on endogenous Ca2+-activated Cl– currents in Xenopus oocytes (58), was added to the bath solution. The removal of contaminating Cl– currents by niflumic acid was verified by the suppression of slow inward tail currents during repolarization after a depolarizing test pulse (6). The bath volume was ~200 µl. An agarose bridge was used to connect the bath solution to a ground chamber (containing 3 M KCl), into which two ground electrodes were inserted. Borosilicate electrodes (0.1–1 M{Omega}) used in voltage recording and current injection were filled with 3 M KCl. Ba2+ currents through Cav2.1 channels were acquired using the conventional two-electrode voltage-clamp technique with an OC-725C oocyte clamp (Warner Instrument, Hamden, CT). Data were filtered at 1 kHz (OC-725C oocyte clamp) and digitized at 100 µs per point using the Digidata 1332A/pCLAMP 8.0 data acquisition system (Axon Instruments, Foster City, CA). The holding potential was set at –90 mV, and 70-ms test pulses were typically applied in 10-mV increments from –80 to +70 mV. Passive membrane properties were compensated using the –P/4 leak subtraction method provided with pCLAMP 8.0 software. All recordings were performed at room temperature (20–22°C).

Data analyses were performed using built-in analytical functions of pCLAMP 8.0 software. Peak Ba2+ current amplitudes measured at each test voltage level were plotted against voltage (current-voltage, or I-V, curve). Apparent reversal potentials (Vrev) were then determined by extrapolating the I-V curve to the voltage axis (typically ~65 mV), and macroscopic channel conductance (G) at each test potential was calculated using the equation: G = I/(V – Vrev), where I is the peak current amplitude and V is the test potential. The voltage dependence of activation was determined by fitting conductance-voltage (G-V) curves using a simple Boltzmann equation: G/Gmax = 1/{1 + exp[(V0.5 – V)/k]}, where Gmax is the maximum conductance, V0.5 is the half-maximal voltage for activation, and k is the slope factor of the G-V curve.

All data are means ± SE. The significance of differences between two means was analyzed using Student's t-test, whereas means from multiple groups were compared using one-way ANOVA. All statistical analyses were performed with Origin 7.0 software (Microcal, Northampton, MA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional expression of wild-type and mutant {alpha}1A-subunits in Xenopus oocytes. We focus on five EA2-related mutant {alpha}1A-subunits ({alpha}1A-mutants) that have been reported previously. Three of them involve nonsense mutations that introduce a premature stop codon at the site of mutation (R1281x, R1549x, or R1669x) (23, 24, 62), whereas the other two are missense mutations (F1406C and E1761K) (8, 22). As shown in Fig. 1A, all five mutations occurred in the homologous domains 3 and 4 region of the {alpha}1A-subunit. The functional properties of some of these mutants have been studied before using cell line expression systems such as human embryonic kidney (HEK) cells. However, an EA2 mutant that is nonfunctional when expressed in a HEK-derived cell line was found to exhibit detectable Ba2+ currents upon expression in Xenopus oocytes (56). We therefore reexamined the functional phenotype of the five aforementioned mutants using the Xenopus oocyte expression system. Figure 1B shows the typical Ba2+ currents recorded in Xenopus oocytes injected with either WT-{alpha}1A-subunit or missense {alpha}1A-mutants. Auxiliary {alpha}2{delta}- and beta4-subunits were coinjected with individual {alpha}1A-subunits into Xenopus oocytes at the molar ratio of 1:1:1 ({alpha}1A to {alpha}2{delta} to beta4). The maximal inward Ba2+ currents through human Cav2.1 channels were usually recorded at test potentials of +10 to +20 mV. Consistent with a previous functional analysis in which COS-7 cells were used as the expression system (22), the missense mutation F1406C produced dramatically reduced currents in Xenopus oocytes. The other missense mutation (E1761K), however, exhibited no significant Ba2+ currents in Xenopus oocytes. To the best of our knowledge, the present study is the first functional study to demonstrate that the E1761K mutation renders human Cav2.1 channels nonfunctional. Figure 1C shows the scatterplot of the amplitudes of Ba2+ currents at +20 mV recorded in oocytes expressing WT-Cav2.1 or mutant Cav2.1 channels. No detectable currents in Xenopus oocytes were observed in the three nonsense mutations (Fig. 1C), which is the typical phenotype expected of truncation mutations. Thus our functional expression studies show that all five {alpha}1A-mutants under investigation resulted in a significant loss of channel function in Xenopus oocytes.


Figure 1
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  		Fig. 1. Expression of wild-type (WT) and mutant pore-forming {alpha}1A-subunits of human P/Q-type Ca2+ channels (Cav2.1 channels) in Xenopus oocytes. A: schematic location of the 5 episodic ataxia type 2 (EA2) mutations under investigation within the {alpha}1A-subunit. Single-letter notation is used to denote the amino acid at the indicated position. Premature stop codon is represented by the letter X. I–IV, homologous domains 1–4. B: representative Ba2+ currents recorded from Xenopus oocytes expressing WT, F1406C, or E1761K Cav2.1 channels. The holding potential was –90 mV. The pulse protocol comprised 70-ms depolarizing test pulses ranging from –60 to +20 mV in 10-mV increments. C: distribution of peak current amplitudes at +20 mV measured in oocytes injected with water (H2O-injected) as well as those expressing various constructs of Cav2.1 channels. Numbers in parentheses refer to number of oocytes recorded for each construct. Mean peak current amplitude at +20 mV for WT, R1281x, F1406C, R1549x, R1669x, E1761K, and H2O-injected oocytes was (in µA, means ± SE) 1.04 ± 0.60, 0.04 ± 0.03, 0.12 ± 0.09, 0.02 ± 0.02, 0.04 ± 0.02, 0.03 ± 0.03, and 0.03 ± 0.03, respectively.

 
Because EA2 is an autosomal dominant disease, most patients with EA2 are expected to carry the heterozygous CACNA1A genotype: one copy each of the WT and mutant allele. Regarding the molecular mechanism of EA2, a loss-of-function phenotype of the mutant CACNA1A gene certainly supports haploinsufficiency as a reasonable mechanism causing the neurological disorder. It is likely, however, that {alpha}1A-mutants may influence the proper function of {alpha}1A-WT negatively by, for example, disrupting channel assembly by competing for the binding of auxiliary subunits, which are known to upregulate the functional expression of voltage-gated Ca2+ channels in the heterologous expression system (2, 50). We therefore wanted to explore the possibility that dominant-negative effects may serve as a major molecular mechanism underlying EA2.

Coexpression of equimolar ratios of {alpha}1A-WT and {alpha}1A-mutants in Xenopus oocytes. By definition, pure haploinsufficiency effects are expected of nonfunctional {alpha}1A-mutants that display neither functional nor structural interactions with {alpha}1A-WT. Accordingly, upon coexpressing {alpha}1A-WT and {alpha}1A-mutant in a 1:1 molar ratio in Xenopus oocytes, a significant reduction of the Ca2+ channel function (e.g., amplitude of Ba2+ currents) relative to that of expressing {alpha}1A-WT alone would support the idea that the {alpha}1A-mutant possesses dominant-negative effects. On the other hand, if the current amplitude of {alpha}1A-WT turned out not to be affected significantly by the presence of the {alpha}1A-mutant, it would be counted as evidence against dominant-negative effects. We thus set out to perform experiments to assess the coexpression of {alpha}1A-WT and {alpha}1A-mutant.

We first empirically established a standard cRNA concentration for Xenopus oocyte injection, which we called the standard cRNA cocktail (see MATERIALS AND METHODS), to ensure that the amplitude of Ba2+ currents through human WT-Cav2.1 channels would be large enough to conduct detailed analyses. In the standard cRNA cocktail, a fixed concentration of cRNA for {alpha}1A-WT, {alpha}2{delta}-, and beta4-subunits was mixed in a 1:2:2 molar ratio before being injected into oocytes. In other words, the standard cRNA cocktail can be regarded as a control for haploinsufficiency effects, because it is equivalent to coexpressing {alpha}1A-WT with a hypothetical nonfunctional {alpha}1A-mutant that does not exert dominant-negative effects. To evaluate the dominant-negative effects of the five EA2 mutants, Xenopus oocytes were coinjected with {alpha}1A-WT and {alpha}1A-mutant cRNA mixed at a molar ratio of 1:1 (i.e., one copy of cRNA for {alpha}1A-mutant was added to standard cRNA cocktail). As stated above, for all cRNA injection paradigms, the total volume of injection per oocyte was identical (41.4 nl).

The representative data shown in Fig. 2A indicate that the amplitude of Ba2+ currents recorded from Xenopus oocytes coexpressing {alpha}1A-WT with any of the five EA2 mutants was significantly smaller than that for oocytes expressing {alpha}1A-WT alone. Because it is not uncommon to observe a wide variation in the amplitude of Ba2+ currents through WT-Cav2.1 channels among different oocytes (see Fig. 1C), we adopted a normalization procedure to perform an objective comparison of the expression levels of Cav2.1 channels. With the same batch of oocytes on a given day of experimentation, the average value of the peak Ba2+ current amplitudes at +20 mV was calculated in 10 or more oocytes expressing WT-Cav2.1 channels. The average value was then used to normalize the peak +20-mV current amplitudes measured in Xenopus oocytes subjected to different coinjection paradigms. Normalized data from different batches of Xenopus oocytes on different dates were later pooled for comprehensive analysis. In addition, to avoid biased data due to unhealthy Xenopus oocytes, we analyzed only data collected during experiments in which there was no apparent difference between different injection paradigms regarding the viability of Xenopus oocytes. As shown in Fig. 2B, in the presence of nonsense and missense EA2 mutants, the current amplitude of WT-Cav2.1 channels decreased by ~60–80%, suggesting that the functional expression of WT-Cav2.1 channels was significantly lower in the presence of {alpha}1A-mutants.


Figure 2
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  		Fig. 2. Equimolar coexpression with {alpha}1A-mutants significantly decreased current amplitudes of WT-Cav2.1 channels in Xenopus oocytes. A: representative Ba2+ currents recorded from Xenopus oocytes subjected to different cRNA coinjection paradigms. WT-Cav2.1 channels correspond to oocytes injected with standard cRNA cocktail in which {alpha}1A-WT, {alpha}2{delta}-, and beta4-cRNA were mixed at a molar ratio of 1:2:2 (see text and MATERIALS AND METHODs for more details). For coexpression experiments, one copy of cRNA for {alpha}1A-mutant was added to the standard cRNA cocktail for oocyte injections, resulting in a 1:1 molar ratio of {alpha}1A-WT and {alpha}1A-mutant. Holding potential was –90 mV. Pulse protocol comprised 70-ms depolarizing test pulses ranging from –60 to +50 mV in 10-mV increments. B: normalized mean Ba2+ current amplitudes of WT-Cav2.1 channels in absence or presence of {alpha}1A-mutants. Peak +20-mV current amplitude measured in each Xenopus oocyte coexpressing {alpha}1A-WT and {alpha}1A-mutant was normalized to corresponding mean +20-mV Ba2+ current amplitude of WT-Cav2.1 channels measured in same batch of oocytes on same day of experimentation; i.e., mean +20-mV Ba2+ current amplitude of WT-Cav2.1 channels on any given day of experimentation was always normalized as unity. Normalized data from different batches of Xenopus oocytes or from different days of experimentation were later pooled for comprehensive analysis. In the presence of nonsense or missense EA2 mutants, the current amplitude of WT-Cav2.1 channels became significantly smaller. *P < 0.05; t-test. Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 
Because the gating of Cav2.1 channels is determined by the membrane potential (Vm) experienced by the voltage sensors located in {alpha}1A-subunits, the reduction in Ba2+ currents might have arisen from the alteration of the voltage dependence of activation of {alpha}1A-WT by the EA2 mutants. To examine this possibility, we compared the steady-state activation property (conductance-voltage curve, or G-V curve) of Cav2.1 channels. As shown in Fig. 3 and Table 1, no significant shift in the G-V curves was noted in the absence or presence of {alpha}1A-mutants, consistent with the idea that the EA2 mutants failed to affect the voltage sensitivity of WT-Cav2.1 channels. Likewise, other biophysical properties of human Cav2.1 channels, such as activation and inactivation kinetics, were not significantly affected by the presence of the EA2 mutants (data not shown).


Figure 3
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  		Fig. 3. Voltage activation curves of WT-Cav2.1 channels were not significantly affected by the presence of {alpha}1A-mutants. Macroscopic channel conductance (G) at each test potential (V) was calculated using the equation G = I/(V – Vrev), where I is the peak Ba2+ current amplitude measured at each test voltage and Vrev is the apparent reversal potential determined by extrapolating current-voltage (I-V) curve to the voltage axis. For each oocyte, the channel conductance at each test potential was normalized to the corresponding peak conductance. Vm, membrane potential.

 

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  		Table 1. Steady-state voltage-dependence property of wild-type Cav2.1 channels in the absence or presence of equimolar coexpressions of {alpha}1A mutants

 
Therefore, coexpression of {alpha}1A-WT with any of the five EA2 mutants under investigation resulted in significant suppression of the current amplitudes of WT-Cav2.1 channels that cannot be explained by a haploinsufficiency effect, which is consistent with the idea that the EA2 mutants may exert dominant-negative effects.

Increasing the relative molar ratios of {alpha}1A-mutants reveals significant dominant-negative effects of EA2 mutations. The decreased amplitude of Cav2.1 currents in the presence of {alpha}1A-mutants presumably reflects an {alpha}1A-mutant-specific suppression of the functional expression of WT-Cav2.1 channels. If there is indeed an {alpha}1A-mutant-specific suppression effect, an increase in the relative molar ratio {alpha}1A-mutant should enhance this suppression effect. We therefore addressed this issue by testing the effect of coexpressing {alpha}1A-WT and {alpha}1A-mutants in the molar ratios of 1:1, 1:3, 1:5, and 1:10 by increasing the amount of {alpha}1A-mutant cRNA added to the standard cRNA cocktail. For all cRNA injection paradigms, the total volume of injection per oocyte was identical (41.4 nl).

Figure 4A shows the representative Ba2+ currents recorded from Xenopus oocytes coinjected with different molar ratios of cRNA for WT and R1281x {alpha}1A-subunits, clearly demonstrating that R1281x mutant decreased the expression of WT-Cav2.1 channels in a dose-dependent manner. Compared with the mean current amplitude at +20 mV for the expression of {alpha}1A-WT alone, coexpression with the {alpha}1A-mutant in the molar ratios of 1:1, 1:3, 1:5, and 1:10 resulted in normalized amplitudes of ~36%, ~21%, ~16%, and ~14%, respectively (Fig. 4B), indicating that the amplitude of Ba2+ currents decreased as the relative molar ratio of R1281x increased. The presence of an increased amount of R1281x did not result in any significant shift in the current-voltage relationship (i.e., I-V curves) of Ba2+ currents (Fig. 4C), suggesting that the foregoing reduction of Ba2+ current was not due to a change in the voltage dependence of WT-Cav2.1 channels. In addition, there was no apparent difference in the viability of oocytes between different coinjection paradigms (data not shown), suggesting that the observed decline in Cav2.1 channel expression did not arise from a deterioration of the viability of Xenopus oocytes as a result of an increased amount of injected cRNA.


Figure 4
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  		Fig. 4. Effects of enhanced coexpression of R1281x mutant on current amplitudes of WT-Cav2.1 channels in Xenopus oocytes. A: representative Ba2+ currents recorded from Xenopus oocytes coexpressing different molar ratios of R1281x mutant. To achieve indicated molar ratios of {alpha}1A-WT and {alpha}1A-mutant, an appropriate amount of cRNA for R1281x was added to standard cRNA cocktail for oocyte injections. Holding potential was –90 mV. Pulse protocol comprised 70-ms depolarizing test pulses ranging from –60 to +50 mV in 10-mV increments. B: normalized mean Ba2+ current amplitudes of WT-Cav2.1 channels in the presence of different molar ratios of R1281x. Data were normalized to mean +20-mV Ba2+ current amplitude of WT-Cav2.1 channels in the absence of R1281x (1:0) using the procedure described in Fig. 2B. Mean current amplitudes of coexpressing {alpha}1A-WT and R1281x in the molar ratios 1:1, 1:3, 1:5, and 1:10 were significantly different. P < 0.05; 1-way ANOVA. Furthermore, when individually compared with coexpression ratio 1:1, mean current amplitude for molar ratio 1:3, 1:5, or 1:10 was significantly smaller (*P < 0.05; t-test), indicating that the amplitude of Ba2+ currents decreased as relative molar ratio of R1281x increased. Number of oocytes measured for each coexpression paradigm is shown in parentheses. C: normalized I-V curves of WT-Cav2.1 channels in the presence of different molar ratios of mutation R1281x. Peak Ba2+ current amplitude measured at each test voltage was normalized to mean current amplitude at +20 mV of WT-Cav2.1 channels. Increased molar coexpression of R1281x failed to shift position of I-V curves of Cav2.1 channels significantly along the voltage axis.

 
Similar suppression effects on {alpha}1A-WT were also observed in the other nonsense as well as missense mutants (Fig. 5). For instance, with the F1406C mutant, the normalized mean current amplitude at +20 mV decreased from ~38% at a 1:1 coexpression ratio to only 3% at a 1:10 coexpression ratio. Likewise, with the R1669x mutant, the mean current amplitude at 1:1 and 1:10 molar ratios was ~22% and ~4%, respectively. Increased molar coexpression of the EA2 mutants did not significantly shift the position of the I-V curves of Cav2.1 channels along the voltage axis (data not shown).


Figure 5
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  		Fig. 5. Effects of enhanced coexpression of missense and nonsense EA2 mutants on current amplitudes of WT-Cav2.1 channels in Xenopus oocytes. Normalized mean Ba2+ current amplitudes at +20 mV of WT-Cav2.1 channels in the presence of different molar ratios of mutants F1406C, R1549x, R1669x, and E1761K. Mean current amplitudes of coexpressing {alpha}1A-WT and {alpha}1A-mutants in molar ratios 1:1, 1:3, 1:5, and 1:10 were significantly different (P < 0.05; 1-way ANOVA). For all four EA2 mutants, mean current amplitude for coexpression ratio 1:3, 1:5, or 1:10 was significantly smaller than that for molar ratio 1:1 (*P < 0.05; t-test), demonstrating that amplitude of Ba2+ currents decreased as relative molar ratio of EA2 mutants increased. Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 
An alternative explanation for the suppression effects of the EA2 mutants discussed above is the exhaustion of the cellular machinery for protein synthesis as a result of excessive amounts of cRNA injected into Xenopus oocytes. To address this issue, we repeated the experiments by coexpressing WT-Cav2.1 channels with two different types of proteins: densin-180 and rat olfactory channels (ROLF). Densin-180 is a 180-kDa transmembrane protein that is tightly associated with postsynaptic density in neurons and is postulated to function as a synaptic adhesion molecule (1, 55). As demonstrated in Fig. 6A, the average size of Ba2+ currents through WT-Cav2.1 channels did not decrease significantly in the presence of an excessive amount of densin-180. Because all previously reported heterologous expression of densin-180 involved only mammalian expression systems, one might argue that the lack of suppression effect reported herein could be interpreted as a failure of the functional expression of densin-180 in Xenopus oocytes. In light of this possibility, we performed the coexpression experiment using ROLF, which is a cyclic nucleotide-gated ion channel that can be expressed functionally in oocytes (48). As shown in Fig. 6B, the mean current amplitude of WT-Cav2.1 channels did not decrease significantly in the presence of an excessive amount of ROLF. Altogether, these results are consistent with the idea that coexpression of {alpha}1A-WT and {alpha}1A-mutants fail to deplete the cellular machinery for protein synthesis in Xenopus oocytes.


Figure 6
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  		Fig. 6. Densin-180 and rat olfactory channel (ROLF) failed to display dominant-negative effects on WT-Cav2.1 channels in Xenopus oocytes. Normalized mean Ba2+ current amplitudes at +20 mV of Cav2.1 channels in presence of different molar ratios of densin-180 (A) and ROLF (B). Mean amplitudes of Ba2+ currents through Cav2.1 channels in the absence (1:0) or presence (1:1, 1:3, 1:5, 1:10) of densin-180/ROLF were not significantly different (P > 0.05; 1-way ANOVA). Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 
Another possibility to explain the suppression effects of the EA2 mutants is that in Xenopus oocytes an {alpha}1A-subunit-specific suppression effect exists in either the cellular machinery for protein synthesis or the posttranslational processing of Cav2.1 channels per se; that is, both {alpha}1A-WT and {alpha}1A-mutant may confer this suppression effect. To test this hypothesis, perhaps we should have evaluated the effect of adding up to a 10-fold extra amount of {alpha}1A-WT cRNA for oocyte injection. However, because the amount of {alpha}1A-WT cRNA was rather high in the standard cRNA cocktail, the viability of Xenopus oocytes significantly deteriorated by merely doubling the amount of {alpha}1A-WT cRNA (equivalent to 1:1 coinjection ratio), presumably as a consequence of Ca2+-mediated cytotoxic effects resulting from overexpression of WT-Cav2.1 channels in Xenopus oocytes. We therefore performed this experiment using the low-cRNA cocktail for Xenopus oocyte injection, in which the amount of cRNA for {alpha}1A-, {alpha}2{delta}-, and beta4-subunits was only one-sixth that used for the standard cRNA cocktail. As shown in Fig. 7A, at low-cRNA concentration, increasing the amount of {alpha}1A-WT cRNA injected into Xenopus oocytes did not lead to suppression of Cav2.1 currents. Instead, the expression of Cav2.1 channels increased. For example, when we enhanced the amount of cRNA for {alpha}1A-WT sixfold (coinjection ratio of 1:5), which is equivalent to the amount of {alpha}1A-WT in the standard cRNA cocktail, the mean amplitude of Ba2+ currents at +20 mV increased by ~83%. When we enhanced the amount of {alpha}1A-WT cRNA further by 11-fold (coinjection ratio of 1:10), the expression of Cav2.1 channels did not increase accordingly but was still 57% higher than the control condition. This apparent reversal of the trend of increased expression of Cav2.1 channels at a 1:10 coinjection ratio, which is equivalent to doubling the amount of {alpha}1A-WT cRNA in the standard cRNA cocktail, was concurrent with the presence of the deterioration of the viability of Xenopus oocytes as a result the presumable Ca2+-mediated cytotoxic effects mentioned above and thus should not be attributed to a suppression of the expression of Cav2.1 channels. In contrast, when we coexpressed WT and mutant {alpha}1A-subunits in a 1:1 molar ratio using the low-cRNA cocktail, the suppression effect of EA2 mutations was still significant, ranging from 50 to 88% (Fig. 7B). In fact, except for E1761K, the suppression effects of EA2 mutations using the low-cRNA cocktail were actually more prominent than those using the standard cRNA cocktail (compare Fig. 2B with 7B). Altogether, our results indicate that the decreased expression of human WT-Cav2.1 channels in the presence of the EA2 mutants is indeed conferred by the specific dominant-negative effects of the {alpha}1A-mutants.


Figure 7
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  		Fig. 7. Increased amount of {alpha}1A-WT cRNA injection did not reduce mean current amplitudes of Cav2.1 channels. A: normalized mean Ba2+ current amplitudes recorded from Xenopus oocytes expressing different concentrations of {alpha}1A-WT cRNA. Low-cRNA cocktail, in which amount of cRNA for {alpha}1A-, {alpha}2{delta}-, and beta4-subunits was only one-sixth that for standard cRNA cocktail, was used as control group (1:0) for oocyte injections. Appropriate amount of additional {alpha}1A-WT cRNA was then added to low-cRNA cocktail to mimic miscellaneous coexpression paradigms of {alpha}1A-WT and {alpha}1A-mutants described in Figs. 4 and 5. The mean amplitudes of Ba2+ currents recorded from Xenopus oocytes expressing different concentrations of {alpha}1A-WT cRNA (1:0, 1:1, 1:3, 1:5, and 1:10) were significantly different (P < 0.05; 1-way ANOVA). In particular, enhancing the amount of {alpha}1A-WT cRNA 6-fold (1:5) or 11-fold (1:10) resulted in mean Cav2.1 current amplitudes significantly larger (*P < 0.05; t-test) than those in the control group (1:0). Data were normalized to mean +20-mV Ba2+ current amplitude of WT-Cav2.1 channels (1:0) using procedure described in Fig. 2B. Number of oocytes measured for each coexpression paradigm is shown in parentheses. B: equimolar coexpression of EA2 mutants with WT-Cav2.1 channels using low-cRNA cocktail. Mean Ba2+ current amplitudes in the presence of EA2 mutants were significantly smaller (*P < 0.05; t-test) than those expressing WT-Cav2.1 channels alone. Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 
Enhancing the amount of auxiliary subunits partially reverses the dominant-negative effects of nonsense EA2 mutants. It is known that one of the major roles of Ca2+ channel auxiliary subunits is to facilitate membrane insertion of {alpha}1A-subunits, thereby ensuring proper functional expression of Cav2.1 channels (2, 50). Consequently, EA2 mutants may suppress the functional expression of their WT counterpart by competing for the availability of auxiliary {alpha}2{delta}- and beta4-subunits. To test this hypothesis, we asked whether the suppression effect bestowed by the five EA2 mutants could be reversed by the presence of an excessive amount of auxiliary {alpha}2{delta}- and beta4-subunits. As shown in Fig. 8A, with the three nonsense mutants, up to a fourfold increase of the auxiliary subunits resulted in a small (~20%) reversal of the dominant-negative effects. This partial reversal of suppression effects is not likely due to direct upregulation of the expression of {alpha}1A-WT by the auxiliary {alpha}2{delta}- and beta4-subunits, because the expression level of WT-Cav2.1 channels did not significantly alter with an up to eightfold increase of auxiliary subunits (data not shown). To the contrary, in the presence of additional auxiliary subunits, the two missense mutants showed no significant change in the suppression effects (Fig. 8B). Thus our results demonstrate that competition for the availability of auxiliary {alpha}2{delta}- and beta4-subunits can partially account for the dominant-negative effects of nonsense but not missense mutants.


Figure 8
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  		Fig. 8. Effects of enhancing molar ratio of auxiliary subunits on dominant-negative effects of EA2 mutants. A: increasing amount of cRNA for auxiliary subunits partially reversed suppression effects of nonsense EA2 mutants. Data were normalized to mean +20-mV Ba2+ current amplitude of WT-Cav2.1 channels using standard cRNA cocktail (1:0:2:2), in which cRNA for {alpha}1A-WT, {alpha}2{delta}-, and beta4-subunits were mixed at a molar ratio of 1:2:2 before being injected into oocytes. Mean current amplitudes of equimolar coexpression of nonsense EA2 mutants using standard (1:1:2:2), double (1:1:4:4), and quadruple (1:1:8:8) auxiliary concentrations were significantly different (P < 0.05; 1-way ANOVA). *P < 0.05 (t-test), significant increase in current amplitude relative to that for standard auxiliary concentration (1:1:2:2). B: increasing amount of cRNA for auxiliary subunits did not affect suppression effects of missense EA2 mutants. Mean current amplitudes for the standard (1:1:2:2), double (1:1:4:4), and quadruple (1:1:8:8) auxiliary concentrations were not significantly different (P > 0.05; 1-way ANOVA). Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 
Dominant-negative effect of R1281x is dramatically reduced for the short isoform of human {alpha}1A-subunits. R1281x mutant predicts a truncation mutation with a premature stop at the S1 segment of homologous domain 3 (Fig. 1A). Our finding that R1281x displays potent dominant-negative effects (Fig. 4) is in direct contrast to the findings of a previous study showing that R1281x cRNA coinjection into Xenopus oocytes failed to exhibit significant suppression effects on the current amplitude of human WT-Cav2.1 channels (56). One difference in the methodology between the two studies involves different splice isoforms of human {alpha}1A-subunits. The {alpha}1A-subunit we have used in the present study is the so-called long isoform of CACNA1A (GenBank accession no. AF004884), which is ~234 amino acids longer than the short isoform used by Wappl et al. (Ref. 56; GenBank accession no. AF004883). The major structural difference between the two variants is that the long isoform contains a long COOH terminus with 11 consecutive glutamine residues (15). Incidentally, another EA2 nonsense mutant, R1824x, which predicts a premature stop at the homologous domain 4 S6 segment, resulting in complete loss of the COOH terminus, displayed pronounced dominant-negative effects when it was introduced into the rabbit {alpha}1A-subunit for heterologous expression (25) but was shown not to possess significant dominant-negative effects upon being introduced into the short isoform of the human {alpha}1A-subunit (18). Therefore, these discrepancies raise the possibility that splice variants of CACNA1A may display different interactions between the {alpha}1A-WT and EA2 mutants.

To address this possibility, we repeated the coexpression experiment for {alpha}1A-WT and R1281x using the short isoform of the {alpha}1A-subunit for both WT and mutant. Upon coexpressing {alpha}1A-WT and R1281x cRNA in 1:1 molar ratio in Xenopus oocytes, there was a small (~24%), statistically insignificant (P > 0.05) reduction in the mean amplitude of Ba2+ currents relative to that of expressing {alpha}1A-WT alone (Fig. 9). Similarly, no significant suppression effects were observed for coinjection ratios of 1: 3 or 1:5. A prominent dominant-negative effect, however, was discerned when we increased the coinjection ratio to 1:10, at which the mean current amplitude at +20 mV decreased by ~67%, which was quantitatively similar to the extent of current reduction conferred by equimolar coexpression of the {alpha}1A-WT and R1281x long isoforms (Fig. 4). These data indicate that the dominant-negative effect of the R1281x mutation is dramatically reduced for short-isoform human {alpha}1A-subunits, consistent with the idea that the two splice variants of CACNA1A display different interactions between {alpha}1A-WT and EA2 mutants.


Figure 9
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  		Fig. 9. Dominant-negative effect of R1281x is considerably diminished for short {alpha}1A-subunit isoform. Short isoform of {alpha}1A-subunits was used for both {alpha}1A-WT and R1281x. Mean Ba2+ current amplitudes of WT-Cav2.1 channels in presence of different molar ratios of R1281x were normalized to those for WT-Cav2.1 channels in absence of mutant (1:0) using procedure described in Fig. 2B. Mean current amplitudes of coexpressing {alpha}1A-WT and R1281x in the molar ratios 1:0, 1:1, 1:3, and 1:5 were not significantly different (P > 0.05; 1-way ANOVA). In contrast, mean current amplitude of coexpressing {alpha}1A-WT and R1281x in molar ratio of 1:10 were significantly smaller (*P < 0.05; t-test) than that for expressing {alpha}1A-WT alone (1:0), indicating significant dominant-negative effect was present only if relative molar ratio of R1281 cRNA was notably high. As a negative control, also shown is mean current amplitude for coexpression of {alpha}1A-WT and ROLF in ratio of 1:10. Number of oocytes measured for each coexpression paradigm is shown in parentheses.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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Both missense and nonsense EA2 mutants exhibit dominant-negative effects on human WT-Cav2.1 channels comprising the long {alpha}1A-subunit isoform. EA2 is an autosomal dominant neurological disorder associated with mutations in the pore-forming {alpha}1A-subunits of human Cav2.1 channels. Because the majority of EA2 mutations result in a loss-of-function phenotype, both haploinsufficiency and dominant-negative effects may contribute to the mechanism leading to the cerebellar dysfunctions observed in patients with EA2. In theory, both haploinsufficiency and dominant-negative effects may result in the functional dominance of a loss-of-function mutation. Haploinsufficiency-conferred dominance usually is not a direct result of the reduced gene expression level per se; instead, it typically arises from an ineffective feedback system for the regulatory mechanism to compensate the reduced expression as exemplified by many human diseases caused by null mutations of transcription factors (42, 54). On the other hand, dominant-negative effects involve the interference of the function of a normal protein by the mutant one. Interestingly, different mutations of the same gene may lead to either haploinsufficiency or dominant-negative effects (31, 43).

To the best of our knowledge, we have presented herein the first evidence of human Cav2.1 channels that EA2 mutants, including three nonsense mutations (R1281x, R1549x, R1669x) and two missense mutations (F1406C, E1761K), display prominent dominant-negative effects. Our studies suggest that the intense suppression effects conferred by both the missense and nonsense EA2 mutants are well beyond the expectation of simple haploinsufficiency effects. Previously, two other EA2 nonsense mutants, when introduced into rabbit or rat {alpha}1A-subunits, have also been shown to exhibit significant dominant-negative effects (25, 37). The inheritance modes of these seven EA2 mutations include both de novo and familial (autosomal dominant) mutations, suggesting that the dominant-negative effect may be a general feature of EA2 mutations. We thus propose that dominant-negative effects may serve as the major molecular mechanism underlying EA2 phenotypes.

Our proposal is consistent with findings from previous studies involving genetic elimination of the {alpha}1A-gene in mice. Homozygous null ({alpha}1A–/–) mice developed progressive neurological deficits such as ataxia and dystonia and died within 4 wk after birth (13, 26). In contrast, heterozygous {alpha}1A+/– mice were phenotypically normal (13, 26), suggesting that the decrease in {alpha}1A-WT conferred by the single-allele knockout fails to produce detectable neurological dysfunction in mice. Our coexpression results clearly indicate that the reduction of the amount of {alpha}1A-WT bestowed by the dominant-negative effects of EA2 mutants is significantly more than that in the heterozygous {alpha}1A+/– condition but also significantly less than that of the homozygous {alpha}1A–/– condition. This phenomenon may explain why patients with EA2 do not display normal motor function that {alpha}1A+/– mice do and why these patients do not develop the severe, lethal neurological deficits found in {alpha}1A–/– mice.

Interestingly, the present study demonstrates that the dominant-negative effect of EA2 mutants is notably more potent in human Cav2.1 channels comprising the long {alpha}1A-subunit isoform. The long and short splice variants are generated by the differential use of the splice acceptor at the boundary of intron 46 and exon 47 of CACNA1A (44, 64). The short {alpha}1A-subunit isoform contains a stop codon immediately after exon 46, whereas the long isoform contains a longer COOH tail encoded by exon 47. In human cerebellum, approximately two-thirds of the transcripts of CACNA1A are composed of the long isoform (44). One potential functional distinction between these two isoforms has been implicated in a recent report showing that alternative splicing at the COOH terminus confers different modes of Ca2+/CaM-dependent facilitation of Cav2.1 channels (7). Our demonstration that the two splice variants of CACNA1A display differential interactions between {alpha}1A-WT and EA2 mutants further highlights the physiological significance of splice-related functional diversity. We have shown in the present study that for the short splice variant, the suppression effect of R1281x was not significant until the relative molar ratio was enhanced to 1:10. On the basis of the assumption that the genes for {alpha}1A-WT and the EA2 mutant share comparable transcription efficiency in vivo, an important implication of our data is that the dominant-negative effect of EA2 mutants is physiologically negligible for the short {alpha}1A-subunit isoform. Because the major structural distinction between the two {alpha}1A-subunit isoforms lies in the length of the COOH terminus, the identification of the molecular mechanism underlying this differential EA2 suppression effect emerges as a critical challenge for the future.

That the suppression effect of EA2 mutants varies considerably between the two splice variants may constitute a plausible rationale for reconciling our findings with those of the two previous reports (25, 56) showing that EA2 mutants failed to exhibit detectable dominant-negative effects on human WT-Cav2.1 channels comprising the short {alpha}1A-subunit isoform (see last subsection under RESULTS). Our conclusion that the short {alpha}1A-subunit isoform of R1281x exhibits a considerable suppression effect when coinjected at a 1:10 molar ratio, however, contrasts with the observation by Wappl et al. (56) that a 20-fold excess of R1281x coinjection into Xenopus oocytes failed to exhibit any detectable effect. One difference in the methodology between the two studies lies in the auxiliary beta-subunit subtype. Wappl et al. used the beta1-subunit, whereas we chose the beta4-subunit. A recent report demonstrated that a rat EA2-like {alpha}1A-truncation mutation that also predicts a premature stop at the S1 segment of homologous domain 3 displayed marked dominant-negative effects on rat WT-Cav2.1 channels comprising beta4-subunits (37). Similarly, a rabbit 95-kDa, two-domain form of {alpha}1A-subunit also showed significant dominant-negative effects on rabbit WT-Cav2.1 channels comprising beta1-subunits (3). Thus whether the difference in beta-subunit subtype could explain the discrepancy between the two studies remains to be clarified.

Differences among heterologous expression systems used by investigators at various laboratories may also contribute to the foregoing conflicting results. One important issue that needs to be addressed in future studies, therefore, is whether the dominant-negative effects that we observed in oocytes also exist in humans. The dominant-negative effect of the rat EA2-like {alpha}1A-truncation mutant was present in both oocyte and COS-7 expression systems (37), and the suppression effect of the rabbit two-domain isoform was demonstrated in a mammalian cell line (3). Hence, our present results are unlikely to represent an oocyte-specific phenomenon.

Potential molecular mechanisms underlying the dominant-negative effects of EA2 mutants. What is the mechanism underlying the dominant-negative effects of the five EA2 mutants? At least two scenarios might account for a reduction of the macroscopic current amplitudes of Ca2+ channels: 1) altered biophysical properties and 2) defective biosynthetic processes. We have shown that the reduction of the current amplitude of WT-Cav2.1 currents in the presence of EA2 mutants is not due to an alteration of the voltage-dependent gating property of the channel (Fig. 3). Previous single-channel analyses revealed that the gating kinetics of an EA2 missense mutant were significantly different from those for the WT (56). Still unknown, however, is whether this EA2 mutant possesses any dominant-negative effect. A nonfunctional two-domain isoform of rabbit {alpha}1B-subunit (N-type) was previously shown to display a dominant-negative effect on rabbit WT-Cav2.2 channels. When these channels were functionally examined at the single-channel level, the biophysical properties of WT-Cav2.2 channels were found to remain virtually the same in the absence or presence of the truncated construct (38). Therefore, whether single-channel properties such as gating kinetics and single-channel conductance of human WT-Cav2.1 channels may be modified in the presence of EA2 mutants is still an open question.

In addition to an alteration of biophysical property, the mechanism of dominant-negative effect may also be explained by a decreased number of normal Cav2.1 channels in the plasma membrane, i.e., a faulty biosynthesis of WT-Cav2.1 channels in the presence of EA2 mutants. The biosynthetic process of ion channels can be divided into two general steps (9): biogenesis (e.g., protein folding and coassembly of multiple subunits) and membrane trafficking (e.g., clustering and localization).

It is still not clear whether the truncated {alpha}1A-subunits encoded by the nonsense mutations R1281x, R1549x, and R1669x can properly be inserted into the plasma membrane. Depending on the location of premature termination codons within an mRNA, mRNA derived from nonsense mutations may trigger nonsense-mediated mRNA decay, thereby preventing the production of the encoded truncated proteins (33). Consequently, some nonsense EA2 mutants may not be expressed at significant levels as a result of this posttranscriptional mRNA quality control mechanism. Alternatively, granted that normal transcriptional and translational processes do take place, a misfolded protein such as truncated channels may fail to pass protein quality control mechanisms taking place in the endoplasmic reticulum (ER), resulting in defective membrane trafficking and increased protein degradation (10, 32). Similar to the dominant-negative effects of mutations in cardiac human ether-à-go-go-related gene K+ channels (12, 27, 63), through yet to be identified mechanisms, truncated EA2 mutants may cause {alpha}1A-WT to misfold, thereby preventing proper trafficking and increasing protein degradation of the WT-Cav2.1 channel. Furthermore, an accumulation of misfolded protein in the ER may trigger an ER-mediated translation inhibition mechanism known as the unfolded protein response (17). A recent study of the mechanism of the dominant-negative effect of a rat EA2-like {alpha}1A-subunit nonsense mutant suggests that the truncated construct may interact with the cognate full-length {alpha}1A-WT to activate an unfolded protein response, leading to the suppression of the translation of {alpha}1A-WT (37). More experiments are needed to determine whether the human EA2 truncation mutants that we have studied can also activate a similar unfolded protein response.

The auxiliary {alpha}2{delta}- and beta4-subunits play an essential role in facilitating the membrane targeting of {alpha}1A-subunits (2, 50). Accordingly, EA2 mutants may suppress the functional expression of Cav2.1 channels by competing for the availability of auxiliary subunits. Increasing the amount of auxiliary {alpha}2{delta}- and beta4-subunits fourfold resulted in a small reduction of the dominant-negative effects of the three truncation mutants, whereas the same treatment did not significantly affect the suppression effects of the two missense mutants (Fig. 8), suggesting that competition for the availability of auxiliary {alpha}2{delta}- and beta4-subunits may contribute to the dominant-negative effects of nonsense EA2 mutants. Further studies are required to determine whether competition for other chaperone proteins may also be involved in the suppression effect of EA2 mutants.

Our observation that competition for auxiliary subunits can partially account for the dominant-negative effects of nonsense but not missense mutants implies that the dominant-negative effects conferred by missense and nonsense mutants may not necessarily share the same molecular mechanism. In fact, the missense mutants F1406C and E1761K may not necessarily encode misfolded proteins. The loss-of-function phenotype of F1406C may reflect a significant alteration of the biophysical property of the channel conferred by the mutation located at the outer pore region (the S5-S6 linker) of homologous domain 3 (Fig. 1A). Similarly, with E1761K, the mutation site corresponds to one of the four conserved glutamate residues within the pore that dictate the divalent cation selectivity of the channel (11, 61). In rabbit cardiac L-type Ca2+ channels, individual replacements of each of the four conserved glutamate residues with lysine resulted in functional channels that were significantly more permeable to monovalent cations (61). Although there is no evidence yet, we speculate that both F1406C and E1761K mutants are likely to form properly folded {alpha}1A-subunits that, upon coassembly with the auxiliary subunits, are targeted for the plasma membrane. Whether (or how) the translation inhibition mechanism is applicable to the dominant-negative effects of missense mutants requires further investigation. In addition, it is of great interest to determine whether other identified EA2 missense mutants also possess dominant-negative effects.

For missense (and perhaps nonsense) EA2 mutants that retain proper membrane-trafficking properties, their loss-of-function phenotypes imply that they behave like virtually silent channels in the plasma membrane. The dominant-negative effects of these EA2 mutants can then be explained by competition between WT-Cav2.1 and silent Cav2.1 channels for a limited number of channel slots in the membrane. In other words, in the presence of EA2 mutants, the whole cell Ca2+ current amplitude decreases significantly because considerably fewer functional Cav2.1 channels gain access to a common membrane-trafficking pathway that does not distinguish between WT and functionally silent channels. Such a slot theory was recently put forward by Cao et al. (4), who elegantly demonstrated that human WT-Cav2.1 and mutant Cav2.1 channels could compete for specific channel type-preferring slots located at presynaptic terminals and that the mutant channels' impairment in contributing to neurotransmission matched precisely their deficiency in supporting whole cell Ca2+ current density. Notably, one of the mutants tested by Cao et al. involved a quadruple-mutation (E4A) of the four glutamate residues at the selectivity filter, similarly to the missense mutant E1761K used in the present study.

One remarkable implication of the slot theory is that the dominant-negative effects of EA2 mutants may also affect the clustering and subcellular localization of WT-Cav2.1 channels. In human cerebellum, in which the long {alpha}1A-subunit splice variant is the dominant isoform (44), quantitative and qualitative alterations in the expression pattern of Cav2.1 channels lead to dire consequences, such as erroneous wiring of synaptic inputs from parallel fibers and climbing fibers during synaptogenesis (35, 40) as well as aberrant burst firing patterns of Purkinje cells (46, 59). Because Purkinje cells are the only output neurons of the cerebellar cortex, the foregoing neuropathological scenarios may in turn contribute to the development of EA2-related ataxic symptoms.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Council of Taiwan Research Grants NSC92-2320-B-002-085 (to C.-J. Jeng) and NSC91-2320-B-002-117 (to C. Y. Tang).


   ACKNOWLEDGMENTS
 
We are indebted to Drs. Joanna C. Jen and Jijun Wan for providing WT and mutant cDNA clones, for subcloning the constructs into pGEMHE vectors, and for valuable discussions throughout the whole process. We thank Drs. Jörg Striessnig and Martina Sinnegger-Brauns for preparing and providing the short isoform of {alpha}1A-subunit cDNA. We also thank Dr. Ting-Chi Tang for critically reviewing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C.-Y. Tang, Dept. of Physiology, College of Medicine, National Taiwan Univ., Taipei, Taiwan (e-mail: cytang{at}ntumc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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7. Chaudhuri D, Chang SY, DeMaria CD, Alvania RS, Soong TW, and Yue DT. Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+<