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Originally published In Press as doi:10.1074/jbc.M502223200 on April 6, 2005 Originally published In Press as doi:10.1074/jbc.M502223200 on March 28, 2005

J. Biol. Chem., Vol. 280, Issue 25, 24064-24071, June 24, 2005
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Gating Deficiency in a Familial Hemiplegic Migraine Type 1 Mutant P/Q-type Calcium Channel*

Curtis F. Barrett, Yu-Qing Cao, and Richard W. Tsien{ddagger}

From the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, February 28, 2005 , and in revised form, March 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding {alpha}1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM {alpha}1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM {alpha}1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Migraine is one of the most common neurological disorders, affecting ~15% of the general population. At least 20% of migraineurs suffer from migraine with aura, in which the headache is preceded by transient neurological symptoms, often including unilateral visual phenomena and occasionally also numbness, weakness, and speech deficits (for review see Refs. 1-3).

In 1996, Ophoff et al. (4) discovered a link between familial hemiplegic migraine type 1 (FHM1),1 a rare form of migraine with aura, and mutations in the human CACNA1A gene, which encodes the pore-forming {alpha}1A (CaV2.1) subunit of the P/Q-type voltage-gated calcium channel, the principal calcium channel type mediating neurotransmission at central synapses (5-8) and neuromuscular junctions (9, 10). Originally, four distinct missense mutations in CACNA1A were found to be associated with FHM1; 15 missense mutations have now been linked to FHM1 (3), with the majority located in regions of the protein known to affect channel function, such as within the pore or voltage sensor. To date, channelopathies in the CaV2 family of calcium channels have only been identified in P/Q-type channels (11, 12); no disease-linked mutations have been observed in N- or R-type calcium channels (CaV2.2 and CaV2.3, respectively).

CaV2.1 channels play a critical role in the mammalian nervous system. At synapses, presynaptic transmitter release is mediated predominantly by calcium influx through P/Q-type channels (13). In addition, postsynaptic (somatodendritic) P/Q-type calcium channels are important in neuronal excitability (14). Thus, the initial discovery of mutations in P/Q-type calcium channels linked to an inherited form of migraine led to the hope that the connection between altered calcium channel function and migraine would be rapidly established. Indeed, the functional consequences of various mutations have been intensely studied in several expression systems (15-19) as well as by a knock-in approach (20). However, the fundamental question of how the mutations affect channel properties still remains unanswered.

Our recent study of P/Q-type calcium channels in cultured hippocampal neurons demonstrated type-specific "slots" for presynaptic P/Q-type channels that contribute to synaptic transmission (21). Expressed in the context of these neurons, all four of the original FHM1 mutations led to reduced whole-cell calcium current density and a diminished contribution of P/Q-type channels to excitatory and inhibitory neurotransmission (22). Our findings suggested that the fundamental lesion targeted an intrinsic channel property rather than the global expression of the channel protein. However, the morphological limitations of cultured neurons precluded any detailed analysis of the nature of the channel defect.

Here, we examined more closely the expression and biophysics of FMH1 calcium channels, focusing on the most common missense mutation associated with FHM1 (23), a threonine to methionine substitution at position 666 (T666M). Channels were expressed in HEK293 cells, capitalizing on their suitability for fast voltage control and lack of endogenous voltage-gated channels. We directly compared the surface expression of mutant and wild-type channel protein and carried out a detailed analysis of their biophysical characteristics. Our findings are consistent with a model in which T666M P/Q type calcium channels undergo normal expression and trafficking to the cell membrane but exhibit an intrinsic defect that hampers their ability to undergo voltage-dependent gating and thus support Ca2+ influx. This study provides the first measurements of gating currents generated by P/Q-type calcium channels and presents novel evidence that defective gating of ion channels may ultimately give rise to neurological disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Human {alpha}1A Constructs—The cDNA of wild-type human {alpha}1A BI-1 (V1) was cloned into plasmid pCDNA3.1(+) (Invitrogen) downstream of the cytomegalovirus promoter. The splice variant content at seven key loci (24, 25) was as follows: {Delta}10A (-V+G), 16+17+, {Delta}17A (-VEA), -31* (-NP), 37a (EFa), 43+44+, and {Delta}47 (lacking exon 47). The generation of FLAG-tagged wild-type and T666M {alpha}1A cDNAs has been described previously (21). Briefly, the sequence encoding the FLAG epitope DYKDDDDK was inserted in-frame at the N terminus of the {alpha}1A cDNA by PCR. This construct was then used as the backbone to generate human FLAG-{alpha}1A cDNA with the T666M mutation using a QuikChange site-directed mutagenesis kit (Stratagene).

EGFP-FLAG-{alpha}1A constructs were generated by fusing various FLAG-{alpha}1A cDNAs in-frame at the C terminus of the pEGFP-C1 plasmid (Clontech). The HA-tagged EGFP-FLAG-{alpha}1A constructs (see Fig. 1A) were generated following a strategy similar to that described by Altier et al. (26). Briefly, an AscI site was first introduced at the S5-S6 loop of domain II in EGFP-FLAG-{alpha}1A. Complementary oligonucleotides encoding the HA epitope, flanked by additional amino acids to enlarge the loop, were annealed to form an AscI adaptor, which was then cloned into the AscI site of the plasmid. The resulting amino acid sequence reads DEGARHYPYDVPDYAVTFDEGTAPPT (inserted residues are underlined, and the HA epitope residues are shown in italic bold type). All PCR-generated cDNA fragments and linker regions were fully sequenced to verify the mutation and to confirm that no additional changes in the sequence had been introduced.

Cell Culture and Transfection—Cells were maintained in 5% CO2 at 37° in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 300 µg/ml G418-sulfate. For transfection, cells were plated into 35-mm culture dishes at sufficient density to reach >80% confluence at the time of transfection (typically 16-20 h after plating).

The various wild-type and mutant {alpha}1A cDNA constructs were transiently transfected into a stable HEK293 cell line expressing the calcium channel auxiliary {beta}1C and {alpha}2{delta}-1 subunits ({beta}1c/{alpha}2{delta}-293 cells (27)) using Lipofectamine 2000 (Invitrogen); for electrophysiology, pEGFP-N3 (Clontech) was co-transfected with the FLAG-{alpha}1A constructs. {beta} and {alpha}2{delta} subunits were not a limiting factor because including {beta}2a and {alpha}2{delta} constructs in the transfections failed to increase whole-cell calcium currents (data not shown). Mock transfections contained no plasmid DNA. Forty-eight hours posttransfection, cells were either lysed for Western blot analysis or plated on poly-L-lysine-coated glass coverslips at low density and either fixed for immunostaining or used for whole-cell patch clamp recording.

Western Blotting—Transfected cells from confluent 35-mm culture dishes were lysed on ice in 200 µl of CelLytic-M lysis buffer (Sigma) with 1% (v/v) mammalian protease inhibitor mixture (Sigma) and then spun at 10,000 rpm to separate the soluble (supernatant) and insoluble (pellet) fractions. Pellet fractions were dissolved by boiling for 10 min in 50 µl of 1% SDS, 10 mM Tris, pH 7.5, 10 mM EDTA and then adjusted to 200 µl with CelLytic-M. Fractions (10 µg of total protein/lane) were separated on a 4-12% Nu-Page bis-Tris polyacrylamide gel (Invitrogen) under denaturing conditions. The proteins were transferred to 0.45-µm polyvinylidene difluoride membrane (Millipore), which was then probed with horseradish peroxidase-conjugated anti-FLAG(M2) antibody (Sigma) for 1 h. A signal was detected by enhanced chemiluminescence and exposure to autoradiograph film.

Immunostaining—For the data shown in Fig. 2, B and C, FLAG-{alpha}1A-transfected cells were fixed in 4% paraformaldehyde at room temperature, then permeabilized with 0.5% Triton X-100 for 10 min at room temperature, and stained with a monoclonal anti-FLAG(M2) antibody (1:1000, Sigma) at 4 °C overnight followed by rhodamine-conjugated goat anti-mouse antibody (1:200, Jackson ImmunoResearch) for 30 min at room temperature. The coverslips were then mounted on glass slides using VectaShield Hard-Set with 4',6-diamidino-2-phenylindole mounting medium (Vector Laboratories). Images were captured on a Zeiss epifluorescent microscope.



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  		FIG. 1.
Cells expressing TM have reduced whole-cell calcium currents. A, schematic of the CaV2.1 ({alpha}1A) constructs used in this study, showing the locations of the N-terminal (intracellular) EGFP and FLAG epitopes. The extracellular HA epitope and surrounding linker regions were inserted in the P-loop between S5 and S6 in the second domain (see "Materials and Methods"); the location of the threonine-to-methionine substitution at position 666 (T666M) is also shown. B, exemplar traces of {beta}1c/{alpha}2{delta}-293 cells transfected with either WT or TM FLAG-{alpha}1A subunits together with EGFP. Whole-cell currents were elicited by applying 300-ms test pulses to +10 mV from a holding potential of -90 mV with 10 mM Ca2+ in the bath solution. C, calcium currents (ICa) in WT- and TM-transfected cells (n = 15 and 11 cells, respectively) were elicited at +10 mV, and peak current amplitude was plotted. *, p < 0.0001. D, membrane capacitance (CM) was not significantly different between WT- and TM-transfected cells (p = 0.3).

 
For the data shown in Fig. 3, wild-type or T666M EGFP-FLAG-HA-{alpha}1A constructs were transfected into {beta}1c/{alpha}2{delta}-293 cells. Two days posttransfection, cells were fixed with 4% paraformaldehyde at 4 °C for 10 min. Cells were incubated with a monoclonal anti-HA antibody (1:500, Covance) at 4 °C overnight, followed by staining with Cy3-conjugated goat anti-mouse secondary antibody (1:800, Jackson ImmunoResearch). Images were collected using a Leica laser-scanning confocal microscope. Midsection cell images (0.5-µm thickness) were used to assess the intensity of EGFP fluorescence and HA immunoreactivity. For permeabilized cells, all solutions contained 0.1% Triton X-100 and 10% normal goat serum; Triton X-100 was omitted in the nonpermeabilized (surface labeling) experiment. The specificity of the anti-HA antibody was tested in permeabilized cells expressing EGFP-tagged channels lacking the HA epitope; as expected, no HA immunoreactivity was detected (supplemental Fig. S1C).

Electrophysiology—Whole-cell currents were recorded at room temperature (20-22 °C) using an Axopatch 200B patch clamp amplifier (Axon Instruments). Borosilicate glass capillaries were pulled in a model P-87 puller (Sutter Instruments) and heat-polished prior to use with a model MF-9 microforge (Narishige). Pipette resistance was ~2-3 M{Omega} when filled with an internal solution consisting of (in mM) 122 Cs-Asp, 10 HEPES, 10 EGTA, 5 MgCl2, 4 ATP, 0.4 GTP, pH 7.5. For recording calcium currents, the bath solution contained (in mM) 155 N-methyl-D-glucamine-Asp, 0.1 EGTA, 10 HEPES, 10 CaCl2, pH 7.4; to record gating currents, the CaCl2 was replaced with 5.2 mM LaCl3 and 2 mM MgCl2 (28). Solution exchanges were performed via a gravity-fed perfusion system. Series resistance (8.95 ± 1.1 M{Omega}) was compensated electronically by ≥90%, and membrane capacitance (16.4 ± 0.6 pF) was corrected on line; residual linear capacitive and leak currents were subtracted by the -P/4 method.



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  		FIG. 2.
WT and TM P/Q-type calcium channels are globally expressed at similar levels. A, Western blot of cell lysates from {beta}1c/{alpha}2{delta}-293 cells either mock-transfected or transfected with WT or TM FLAG-{alpha}1A. Total cell lysates were separated into insoluble pellet (P) and soluble (S) fractions (see "Materials and Methods") and resolved by SDS-PAGE under denaturing conditions. Proteins were transferred to polyvinylidene difluoride and probed with anti-FLAG antibody. No signal was detected in mock-transfected cells nor in the soluble fractions of cells transfected with either WT or TM {alpha}1A. B, representative images of cells either mock-transfected or transfected with WT or TM FLAG-tagged {alpha}1A subunits. Cells were permeabilized with Triton X-100 and incubated with anti-FLAG and rhodamine-conjugated secondary antibodies; nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). C, summary of mean anti-FLAG immunoreactivity in permeabilized cells either mock transfected (n = 39 cells) or transfected with WT or TM FLAG-{alpha}1A (n = 43 and 37 cells, respectively). anti-FLAG immunoreactivity in TM-expressing cells was not significantly different than WT (p = 0.8). In contrast, both WT- and TM-expressing cells displayed significantly more anti-FLAG immunoreactivity than mock-transfected cells (p < 0.0001).

 
EGFP-positive cells were visualized by epifluorescence and selected for recording; 100% of EGFP-positive cells recorded expressed whole-cell voltage-dependent calcium currents, whereas mock-transfected cells had no detectable current (data not shown). Cells were voltage-clamped at -90 mV, and pulse depolarizations were applied at 10-s intervals. Data were passed through a 4-pole low pass Bessel filter, digitized using a Digidata 1320A (Axon Instruments), and stored on a personal computer for off-line analysis. Currents were filtered at 1-10 kHz and digitized at 5 kHz (ionic currents) or 100 kHz (gating currents).

Data Analysis—Data were acquired and analyzed using Clampex 8.2 and Clampfit 8.2, respectively (Axon Instruments). Fluorescence intensity was quantified using ImageJ 1.32j, downloaded from the National Institutes of Health website (rsb.info.nih.gov/). Plots were generated with Origin 7 (OriginLab), and figures were rendered with Illustrator 10 (Adobe Systems).

Total gating charge movement, Q, was measured by integrating the area under the gating current and is expressed in |pA|·ms or simply femtocoulombs (fC). The single Boltzmann fits of the activation data presented in Fig. 6 were calculated using the equation

(Eq. 1)

where I/Imax is the normalized OFF gating current amplitude, V is the membrane potential in mV, V0.5 is the voltage at half-maximal I/Imax, k is the slope factor of activation at V0.5 in mV/e-fold change in I/Imax, and I1 and I2 are the minimum and maximum values of I/Imax, respectively.

When applicable, data are presented as mean ± S.E. Statistical significance was tested using a two-tailed Student's unpaired t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Early studies of FHM1 P/Q-type calcium channels were performed using recombinant expression systems, including Xenopus oocytes (15, 29) and HEK293 cells (16), whereas more recent studies have examined these channels in the more physiological setting of neurons lacking endogenous {alpha}1A subunits (17, 21, 22). For this study we chose to examine whole-cell channel properties using a recombinant expression system rather than neurons for several reasons. First, to accurately record gating currents, it was imperative to have fast voltage control and space clamp, conditions easily achieved with spherical cells but much less so with cultured neurons. Second, neurons have endogenous voltage-dependent sodium, potassium, and calcium channels, and gating currents from these channels would have interfered with our measure of exogenous P/Q-type channels. The HEK293 cells used in this study, however, express no endogenous voltage-dependent channels to speak of and no detectable gating currents (data not shown). Finally, the HEK293 expression system allowed us to control the identity of the {beta} subunit, avoiding complications arising from the co-existence of multiple endogenous {beta} subunit isoforms in brain neurons (30) and their uncertain and possibly variable influence on the gating of FHM1 mutant channels (16, 18).

T666M Channels Have Reduced Whole-cell Current Density—Previous experiments using {alpha}1A knock-out neurons transfected with either wild-type or T666M {alpha}1A subunits showed that TM channels give rise to significantly smaller whole-cell calcium currents (17, 21). Our first step was to compare TM and wild-type whole-cell calcium currents in our expression system. HEK293 cells stably expressing the calcium channel auxiliary subunits {beta}1c and {alpha}2{delta}-1 ({beta}1c/{alpha}2{delta}-293 cells) were transfected with either wild-type or TM FLAG-tagged {alpha}1A channels. Two days posttransfection, EGFP-positive cells were voltage-clamped and whole-cell calcium currents were elicited by pulse depolarizations from -90 mV to +10 mV, using 10 mM Ca2+ as the charge carrier. Under these conditions, cells expressing WT P/Q-type channels displayed inward currents ~850 pA in amplitude (Fig. 1, B and C); no calcium currents were detected in untransfected {beta}1c/{alpha}2{delta}-293 cells (data not shown). Cells transfected with TM {alpha}1A displayed whole-cell currents ~250 pA in amplitude, significantly less than WT (Fig. 1, B and C). In contrast, membrane capacitance (a measure of cell surface area) did not differ significantly between wild-type- and TM-transfected cells (Fig. 1D). Accordingly, whole-cell current density of TM-transfected cells was 17.4 ± 2.8 pA/pF, compared with 58.4 ± 6.0 pA/pF for WT-transfected cells (n = 11 and 15 cells, respectively; p < 0.0005). A comparable decrease in whole-cell current density was observed by Hans et al. (16) using HEK293 cells transfected with TM {alpha}1A together with {alpha}2{delta} and either {beta}2e or {beta}3a, as well as by Tottene et al. (17).



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  		FIG. 3.
WT and TM P/Q-type calcium channels are expressed at similar levels at the plasma membrane. A, representative images of {beta}1c/{alpha}2{delta}-293 cells transfected with either WT or TM EGFP-FLAG-HA-{alpha}1A P/Q-type calcium channels. To selectively stain surface proteins, cells were not permeabilized and were incubated with anti-HA and Cy3-conjugated secondary antibodies. B, summary of mean surface HA immunoreactivity and total EGFP fluorescence in nonpermeabilized cells transfected with either WT or TM EGFP-FLAG-HA-{alpha}1A (n = 45 and 33 cells, respectively). Neither HA nor EGFP intensity differed significantly between the two groups (p = 0.3 and 0.9, respectively). C, summary of the ratio of total HA signal intensity to EGFP intensity in Triton X-100-permeabilized cells transfected with either WT or TM EGFP-FLAG-HA-{alpha}1A (n = 32 cells for each group). The two groups did not differ significantly (p = 0.5).

 
T666M Channels Are Not Defective in Expression or Trafficking to the Cell Surface—A reduction in whole-cell current density can be most simply explained by a reduction in channels at the plasma membrane, resulting from either a decrease in protein expression or a defect in protein trafficking to the cell surface. Previous studies using cell-attached patch recordings suggested that functional TM channels were present on the cell surface at decreased density (16); however, overall expression of channel protein was not directly compared. We therefore used several approaches to look for differences in expression and trafficking to the cell surface between WT and TM channels.

First, lysates prepared from cells transfected with FLAG-tagged WT or TM {alpha}1A were analyzed by Western blot and probed for the N-terminal FLAG epitope. No signal was detected in mock-transfected cells, whereas a clear band was detected in the insoluble pellet fractions of cells transfected with WT or TM {alpha}1A (Fig. 2A). The signal intensities of WT and TM bands were similar, suggesting that the proteins were expressed at similar levels. Moreover, WT and TM {alpha}1A were both expressed as full-length proteins, running as a single band at the expected size (>250 kDa); similar results were obtained by Müllner et al. (18) using tsA-201 cells expressing other FHM1 mutant channels together with {beta}3 and {alpha}2{delta}. Consistent with our Western blot results, cells expressing FLAG-tagged WT or TM {alpha}1A displayed similar FLAG immunoreactivity (Fig. 2, B and C), again indicating that the TM mutation did not affect the level of protein expression.

We went on to test whether or not the TM mutation affected the ability of the protein to traffic to the cell surface, using an approach similar to the strategy of Altier et al. (26) for measuring surface expression of {alpha}1C. We inserted an extracellular HA epitope within the S5-S6 loop in domain II of FLAG-{alpha}1A, and also fused EGFP to the N terminus, resulting in the EGFP-FLAG-HA-{alpha}1A construct (Fig. 1A). EGFP-FLAG-HA-tagged WT and TM channels were then expressed in {beta}1c/{alpha}2{delta}-293 cells. In both permeabilized (supplemental Fig. S1, A and B) and nonpermeabilized (Fig. 3, A and B) cells, the EGFP intensity was comparable between WT and TM groups, indicating that the insertion of the EGFP and HA tags did not differentially affect the expression of WT and TM proteins. In addition, the intensity of HA immunoreactivity was not significantly different between permeabilized cells expressing WT or TM channels (supplemental Fig. S1B). More importantly, the ratio of anti-HA immunoreactivity to EGFP intensity did not differ between WT- and TM-expressing cells (Fig. 3C), indicating that the TM mutation did not affect the accessibility of the HA epitope.



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  		FIG. 4.
Cells expressing TM {alpha}1A have reduced whole-cell gating currents. A and B, whole-cell currents were recorded from cells transfected with either WT (A) or TM (B) {alpha}1A subunits. Upper panels, ionic Ca2+ currents were elicited as described for Fig. 1B. Ionic currents were completely blocked by substitution of Ca2+ with the impermeable cation La3+ (5.2 mM). Lower panels, gating currents were elicited by stepping to +40 mV for 24 ms in the presence of 5.2 mM La3+. C, summary of ON gating current amplitude (IgON) for WT- and TM-transfected cells (n = 15 and 7 cells, respectively; p < 0.002). D, ratio of peak Ca2+ current amplitude (ICa) to peak ON gating current amplitude for WT- and TM-transfected cells. The two groups were not significantly different (p = 0.5).

 
The crucial experiment was to measure HA immunoreactivity in nonpermeabilized cells expressing WT or TM EGFP-FLAG-HA-{alpha}1A to detect selectively channels at the cell surface (see supplemental Fig. S2 for a control experiment). EGFP signals were observed in both the plasma membrane and intracellular compartments (Fig. 3A). In contrast, HA immunoreactivity was restricted to the cell surface (Fig. 3A), on which WT- and TM-expressing cells displayed similar immunoreactivity (Fig. 3B), thus providing clear evidence that the TM mutation does not affect the ability of the channel to traffic to the plasma membrane.

Gating Currents from Recombinant P/Q-type Calcium Channels—Having shown that the decrease in whole-cell calcium currents in TM-expressing cells was not due to differences in surface expression relative to WT, we compared the number of functional channels at the cell surface using nonlinear gating charge movement (gating currents, for review see Ref. 31); decreased whole-cell gating currents would indicate a decrease in the number of surface channels capable of undergoing voltage-dependent gating. Gating currents were measured by replacing the Ca2+ in the bath solution with the nonpermeant cation La3+ (28). Bath application of 5.2 mM La3+ caused a complete block of inward ionic current and revealed gating currents that could be easily resolved (Fig. 4); nontransfected {beta}1c/{alpha}2{delta}-293 cells gave no detectable gating currents (data not shown).

The gating currents that we observed for wild-type P/Q-type calcium channels had properties similar to those seen with other, previously examined calcium channel types, including N- (28, 32), L- (33-36) and R-type (37). For example, ON gating currents (seen at the onset of the test pulse) were typically smaller in amplitude with slower kinetics than OFF gating currents (seen at the offset of the test pulse). Moreover, with a relatively brief depolarization (~24 ms), total charge movement was conserved (QOFF/QON was 1.02 ± 0.07, n = 15 cells), consistent with little or no inactivation during the depolarization pulse (38).

One striking feature of P/Q-type gating currents was their small magnitude relative to other calcium channel types. In our hands, cells transfected with WT P/Q-type calcium channels had ON gating currents (Fig. 4C, IgON) of ~40 pA and total charge movement (Fig. 5B, QON) of ~75 fC. Cells transfected with similar molar amounts of a plasmid encoding wild-type L-type calcium channels ({alpha}1C, under the same promoter as {alpha}1A) displayed calcium current densities of ~25 pA/pF (compared with ~58 pA/pF for P/Q-type currents), but L-type ON gating currents were significantly larger than for P/Q-type channels, with amplitude and total charge movement of ~130 pA and 200 fC, respectively (data not shown), consistent with previous reports (34, 39).



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  		FIG. 5.
WT and TM P/Q-type calcium channels have similar gating current kinetics and show gating charge conservation. A, shown is an exemplar WT gating current trace elicited by stepping to +40 mV from a holding potential of -90 mV. The solid gray line is a single exponential fit of ON gating current decay. At the right is the summary of ON gating current decay ({tau}ON) for WT (n = 15 cells) and TM (n = 7 cells) channels. The two groups were not significantly different (p = 0.1). B, total gating charge movement (Q) was measured at the onset (QON) and offset (QOFF) of the test pulse by integrating the area of the gating current (indicated by the shaded areas) and is expressed in femtocoulombs (fC). At the right is the summary of QON and QOFF for WT and TM P/Q-type calcium channels (n = 7-15 cells). QON and QOFF were not significantly different for either WT (p = 0.2) or TM (p = 0.2) channels. In contrast, TM QON and QOFF were both significantly reduced compared with WT (p < 0.01).

 
T666M Channels Have Reduced Gating Currents—We then used whole-cell gating currents to test whether the T666M mutation affected the number of functional channels (i.e. channels capable of undergoing gating conformational changes) at the cell surface. In the exemplar traces shown in Fig. 4, the cell expressing TM channels had considerably smaller gating currents than the cell expressing wild-type channels. Fig. 4C shows the summary of ON gating current amplitude (IgON) for cells transfected with WT or TM {alpha}1A. As with whole-cell calcium currents, cells transfected with TM channels had significantly reduced whole-cell gating currents. TM OFF gating current amplitude was similarly reduced (35 ± 5 pA versus 125 ± 13 pA for WT; p < 0.0001). TM gating currents were reduced to the same extent as ionic currents (by ~65-70%); the ratio of calcium current to gating current, determined in individual cells, was not significantly different between wild-type and TM channels (Fig. 4D). Together with our immunostaining data, these findings suggest that mutant channels were expressed on the cell surface at normal levels but were deficient in some way in their ability to gate.

T666M Channels Display Normal Gating Kinetics and Voltage-dependent Activation—Because the reduction in whole-cell gating currents was sufficient to account for the reduction in calcium currents, we hypothesized that, at any given time, TM channels consist of two pools of channels at the cell surface: 1) those that are deficient to gate (and therefore upon depolarization fail to give rise to gating currents and subsequent calcium currents); and 2) those that are able to undergo normal gating. If true, then the pool of TM channels that are capable of gating might do so with WT properties. We therefore examined the biophysical properties of the TM channels that can undergo gating (those in group 2). We first examined the kinetics of ON gating currents. The time to peak for TM ON gating currents was 1.16 ± 0.12 ms, not significantly different than for wild-type currents (1.00 ± 0.06 ms, p = 0.2). Thus, TM and wild-type channels activated with a similar time course. In addition, we assessed the decay kinetics of wild-type and TM ON gating currents by fitting the decay with a single exponential (Fig. 5A). TM gating current decay was not significantly different from wild-type, suggesting that TM channels that were able to gate did so with wild-type kinetics.

As mentioned previously, WT gating charge movement was not reduced during the test pulse, consistent with gating charge conservation. To quantify this conservation for both WT and TM channels, total gating charge movement (Q) was measured by integrating the gating current (Fig. 5B). QON and QOFF were not significantly different for either wild-type or TM channels, and both QON and QOFF were reduced to a similar extent by the mutation. Thus, the TM mutation did not affect gating charge conservation, suggesting that the mutation did not lead to charge immobilization during the test pulse. This was consistent with our finding that neither WT nor TM channels have any appreciable inactivation during a 24-ms depolarization (data not shown).



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  		FIG. 6.
TM P/Q-type calcium channels have WT voltage-dependent activation. A, exemplar gating currents from cells transfected with either WT or TM {alpha}1A. Whole-cell gating currents were elicited by 24-ms pulse depolarizations to the voltage indicated from a holding potential of -90 mV. B, mean, normalized activation curves were generated by plotting normalized OFF gating current amplitude (IgOFF) against voltage. The solid lines are Boltzmann fits of the data (see "Materials and Methods"). V0.5 (the voltage that elicited half-maximal gating current amplitude) was -4.6 ± 2.0 mV for WT ({circ}) (n = 15 cells) and -4.2 ± 1.8 mV for TM ({blacksquare}) (n = 8 cells, p = 0.9 compared with WT). The slope factor, k, was 9.2 ± 0.5 for WT and 6.6 ± 1.2 for TM channels (p = 0.08).

 
In principle, a significant positive shift in the voltage dependence of activation could account for the observed reduction in gating currents and hence the subsequent reduction in calcium current. Tottene et al. (17) reported previously a negative shift in activation; despite a significant decrease in whole-cell current density and unitary channel conductance (16), the authors reported that this shift was sufficient to cause increased ion flow and a net gain of function at negative test potentials. In contrast, we recently reported no shift in TM activation when measured with ionic tail currents in transfected neurons (22). This apparent discrepancy may be explained in part by the splice variants and/or accessory {beta} subunits used. Here, we measured voltage-dependent activation using OFF gating current amplitude as a measure of channel activation. With La3+ in the bath solution, test pulses were applied at 10-mV increments ranging from -60 mV through +50 mV (Fig. 6A), and OFF tail current amplitude was normalized and plotted against voltage (Fig. 6B). Activation of both wild-type and TM channels began between -20 and -10 mV, had similar steepness, and saturated at around +30 mV. Similar results were obtained when OFF gating charge movement, QOFF, was plotted against voltage (data not shown). Therefore, the TM channels that were capable of undergoing voltage-dependent gating did so with wild-type properties.



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  		FIG. 7.
Schematic summary of the finding that T666M P/Q-type calcium channels have defective voltage-dependent gating. At rest (top panel), both WT and TM channels are closed, with their positively charged S4 segments (+) in proximity to the intracellular face of the bilayer, and calcium (depicted as ) cannot permeate the channels. Upon depolarization (middle panel), the majority of TM channels are incapable of gating; as a result, ON and OFF gating currents (Ig) are significantly reduced relative to WT. Subsequently, only the gating-competent TM channels are able to open and allow calcium to enter the cell (ICa, bottom panel). For simplicity, all three WT channels are depicted as competent for gating, whereas only about one-third as many TM channels are capable of gating. For schematic purposes only, rearrangement of gating machinery is represented as a displacement of positively charged S4 residues along the direction of the electric field.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our experiments demonstrated that the most common {alpha}1A missense mutation linked to FHM1, T666M, did not diminish expression of {alpha}1A protein or its trafficking to the cell surface but still caused a dramatic reduction in P/Q calcium current. By measuring P/Q channel gating currents, we pinpointed the reduction in Ca2+ current to a corresponding deficiency in the ability of mutant channels to undergo voltage-dependent transitions required for channel opening. The defect in the pore-forming {alpha}1A subunit kept the majority of the P/Q channels in a nonconducting state, in which they were unable to support calcium influx (Fig. 7). However, the mutation did not abolish all gating because cells expressing mutant channels exhibited both gating and Ca2+ currents at ~30% of wild type levels, indicating that a minority of channels could undergo gating with normal properties.

These findings were made possible by the favorable properties of the HEK293 cell expression system: excellent voltage control and no interference from other voltage-gated channels. Using the same expression system, Hans et al. (16) carried out extensive whole-cell and cell-attached patch recordings and reported that the density of functional TM channels was ~40% of that of WT channels, whereas the voltage dependence of activation was not significantly different (but see also Ref. 17). These observations and our own experiments are thus in good agreement. Hans et al. (16) also found that TM channels generally exhibited a smaller unitary current, which also contributed to the greatly diminished whole-cell current.

The reduced functional channel density seen in both studies was cast in a new light by our finding that the TM mutation neither diminished the total level of {alpha}1A protein seen by Western blot and immunostaining nor decreased the abundance of surface channel proteins measured by surface staining of {alpha}1A. These experiments allowed us to rule out defects in transcription, translation, and protein trafficking to the surface and to focus on an intrinsic deficiency in the ability of surface-expressed {alpha}1A subunits to support Ca2+ influx; the presence of nonfunctional channels would not have been evident from electrophysiology alone. Such an intrinsic defect has important implications for synaptic transmission because P/Q-type channels compete for and occupy type-selective slots that are ultimately positioned at the presynaptic active zone near sites of transmitter release (21). If a majority of TM channels were functionally inoperative at any given moment, defective channels would take up slots normally occupied by functional channels, and the fractional contribution of P/Q channels to synaptic transmission would be correspondingly attenuated. This scenario might explain how products of a mutant allele could interfere with those of a wild-type allele and provides a working hypothesis for the dominant inheritance of FHM.

How might the defective gating in the majority of TM channels be explained? Candidate biophysical mechanisms can be derived from previous studies of voltage-gated channels. One scenario that can be readily eliminated is a channel that undergoes voltage-dependent gating transitions but is blocked before the final opening step, as reported for the Shaker potassium channel mutant W434F (40). On the contrary, the majority of the TM mutant channels simply fail to gate. An additional hypothesis is that the TM channels are reluctant to open because of tonic inhibition by G protein binding (41); such inhibition has been shown to block gating charge movement in N-type calcium channels (28). This was also unlikely because Ca2+ currents through TM channels failed to show any classic hallmarks of G protein-mediated inhibition such as slowed activation, facilitation by prepulses, and a positive shift in the voltage dependence of activation (42, 43). Yet another scenario is that TM channels are more susceptible to gating charge immobilization because of inactivation (28, 36). We tried to remove any such inactivation by prolonged hyperpolarizations (-150 mV for ~5 min) but found no increase in TM calcium currents.

A remaining hypothesis is that the mutant P/Q channels can exist in two distinct states, either locked (unavailable for opening) or unlocked (ready to be activated). This has been demonstrated for mutant forms of the inwardly rectifying potassium channel (Kir) 1.1a (ROMK) linked to Bartter's syndrome, which suffer no deficit in channel protein expression or trafficking but yield diminished whole-cell currents because they enter and leave a locked state (44). Similarly, TM channels may transition reversibly between locked and unlocked states. On the other hand, a large majority of TM channels might undergo an irreversible modification such as misfolding or proteolysis (45, 46) that renders them unavailable for gating. Our model (Fig. 7) allows for both alternatives.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NS24067 (to R. W. T.), the George D. Smith Professorship (to R. W. T.), a Stanford Dean's Fellowship and an individual National Research Service Award (to Y.-Q. C.), and a NHLBI National Institutes of Health training grant (to C. F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org/) contains supplemental Figs. S1 and S2. Back

{ddagger} To whom correspondence should be addressed: Stanford University School of Medicine, Beckman Center, Rm. B105, Stanford, CA 94305-5345. Tel.: 650-725-7557; Fax: 650-725-8021; E-mail: rwtsien{at}stanford.edu.

1 The abbreviations used are: FHM1, familial hemiplegic migraine type 1; EGFP, enhanced green fluorescent protein; fC, femtocoulombs; HEK293, human embryonic kidney 293; TM, T666M {alpha}1A; WT, wild type; HA, hemagglutinin; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; pF, picofarad; M{Omega}, megohm(s). Back


   ACKNOWLEDGMENTS
 
We thank Maureen McEnery, Charles Harata, Kirsten Canté-Barrett, and Henry Lam for technical assistance and helpful discussions and Emmanuel Bourinet for providing an HA-tagged {alpha}1C construct, sharing unpublished data, and participating in helpful discussions.



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