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1Department of Neuroscience, Brown University, Providence, Rhode Island 02912; and 2Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Submitted 14 January 2004; accepted in final form 14 June 2004
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ABSTRACT |
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1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaV
and CaV2
subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaV2 to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but ON gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1 subunits. Auxiliary CaV and CaV2 subunits and additional associated proteins modulate surface expression efficiency, subcellular targeting, biophysical properties, and pharmacology of the CaV1 subunit (Birnbaumer et al. 1998
; Helton et al. 2002; Hering 2002; Walker and De Waard 1998).
Alternative splicing of CaV1 pre-mRNAs is extensive, endowing individual CaV1 genes with the capacity to generate multiple functionally specialized proteins (Lipscombe et al. 2002). The mammalian nervous system in particular uses alternative splicing to produce an array of functionally distinct and finely tuned proteins (Black 2000; Grabowski 1998). Sites of regulated alternative splicing occur in key functional domains of proteins (Garcia et al. 2004). The IVS3-IVS4 domain of CaV1 is a conserved site of alternative splicing. It is found in multiple Cav genes and different species from Drosophila to human (Barry et al. 1995; Bourinet et al. 1999; Hans et al. 1999; Ihara et al. 1995; Ligon et al. 1998; Lin et al. 1997, 1999; Lipscombe et al. 2002; Peixoto et al. 1997; Perez-Reyes et al. 1990; Smith et al. 1996, 1998; Snutch et al. 1991; Starr et al. 1991; Takimoto et al. 1997). Splice isoforms with different IVS3-IVS4 linkers can open at different rates and at different voltages and have different drug sensitivities (Bourinet et al. 1999; Hans et al. 1999; Krovetz et al. 2000; Lin et al. 1997, 1999; Lipscombe and Castiglioni 2004). The expression of exons in the IVS3-IVS4 region of at least two genes, CaV1.3 and CaV2.2, depends on tissue type (Lin et al. 1999; Takimoto et al. 1997). Evolutionary conservation, modification of channel function, and tissue-specific expression combine to suggest that alternative splicing in the IVS3-IVS4 region of CaV is physiologically important.
In the CaV2.2 gene, exon 24a (e24a) encodes a tetrapeptide sequence, SFMG, in domain IIIS3-IIIS4 and exon 31a (e31a), the dipeptide sequence ET in domain IVS3-IVS4 (Lipscombe et al. 2002). The expression pattern of these exons differs. In sympathetic neurons, all CaV2.2 mRNAs contain e31a, whereas this exon is absent in CaV2.2 mRNAs of brain and spinal cord. Exon e24a is present in an approximate reciprocal distribution. Most CaV2.2 mRNAs in brain and spinal cord contain e24a, whereas in sympathetic and dorsal ganglia a slight majority lack e24a (Lin et al. 1997). In previous studies, we characterized some aspects of alternative splicing in the S3–S4 linkers of the CaV2.2 channel in the Xenopus oocyte expression system. Our functional analyses in Xenopus oocytes showed that the peripheral-dominant CaV2.2e[
24a, 31a] splice form activated more slowly and at voltages slightly more depolarized compared with the central-dominant form, CaV2.2e[24a, 31a] (Lin et al. 1997, 1999). Subsequently, we showed that the presence of e31a in domain IVS3-IVS4 accounts fully for the slower activation time course of the CaV2.2e[24a, 31a] splice isoform, whereas both e24a and e31a contributed to the difference in activation thresholds (Lin et al. 1999).
In the present study, we generate mammalian cell lines (tsA201) stably expressing the N-type CaV2.2 splice forms with CaV3 alone or CaV3 and CaV2, the full complement of auxiliary subunits. We show that the presence of CaV2 is necessary to reconstitute native-like ionic currents and confirm that CaV2.2 splice isoforms activate at different rates but find no difference in ionic activation thresholds as reported in previous studies using the oocyte expression system. Gating currents measured from heterologously expressed CaV2.2 splice forms decay with significantly different rates, but activation thresholds of gating currents are identical. Our analysis of ionic and gating currents in these stable cell lines correlates with native currents recorded from sympathetic and hippocampal pyramidal neurons. Our findings support the conclusion that alternative splicing in the S3-S4 linkers of CaV2.2 primarily serves to modify channel gating kinetics.
Note on nomenclature
In this paper, we use CaV2.2 for the alpha1 subunit of the N-type calcium channel (Ertel et al. 2000). There is no accepted nomenclature to identify splice isoforms. We know the structure of the human CaV2.2 gene (Lipscombe et al. 2002) and therefore use specific exon numbers to distinguish splice isoforms. The SFMG splice site in domain IIIS3-IIIS4 of CaV2.2 is encoded by exon 24a and the ET site in domain IVS3-IVS4 by exon 31a. The peripheral-dominant splice isoform studied here is CaV2.2 e[24a, 31a], formerly named rn1B-a, and the central-dominant splice isoform is CaV2.2 e[24a, 31a], formerly rn1B-c (Lin et al. 1997).
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METHODS |
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We generated four tsA201 cell lines that stably express the following calcium channel subunits. 1) CaV2.2 e[24a, 31a] and CaV3; 2) CaV2.2 e[24a, 31a] and CaV3; 3) CaV2.2 e[24a, 31a], CaV3, and CaV21; and 4) CaV2.2 e[24a, 31a], CaV3, and CaV21. All clones were generated in our laboratory. CaV2.2 subunits were subcloned into pcDNA6, blasticidin selection (Invitrogen). The two clones CaV2.2 e[24a, 31a] and CaV2.2 e[24a, 31a], originally called rn1B-a and rn1B-c (Lin et al. 1997) (GenBank No. AF055477), were linearized with SpeI. CaV3 was PCR-amplified from rat brain (Pan and Lipscombe 2000), subcloned into pcDNA3.1 (zeocin), and linearized by PvuI. CaV21 was isolated from a rat superior cervical ganglia cDNA library (GenBank No. AF286488), subcloned into pcDNA3 (hygromycin), and linearized with SpeI. (see http://neuroscience.brown.edu/LipscombeLab/HOMEPAGE/home2.htm for additional information on clones).
Two cell lines were initially generated that stably expressed one of the two CaV2.2 splice forms together with CaV3. tsA201 cells were transfected by electroporation with a 10 µg mix of linearized CaV2.2 and CaV3 cDNAs. Cells were diluted and grown without antibiotics for 2 days; 7.5 µg/ml blasticidin and 250 µg/ml zeocin were then added and single colonies isolated 10 days later. Selection with blasticidin and zeocin was very efficient. For example, 20 of 20 colonies contained CaV2.2 e[24a, 31a] and 18 of 20 contained CaV3 based on PCR screening. For each condition, six cell lines were chosen randomly for functional screening by whole cell recording, and two cell lines that contained high current density were selected. There was significant variability in current density among cells in each line. A second round of dilution cloning, PCR screening, and functional assessment was carried out, and 20 new colonies were isolated for each subunit combination. Cell lines 2017 (CaV2.2 e[24a, 31a], CaV3) and 1911 (CaV2.2 e[24a, 31a], CaV3) were selected for further study because they had particularly high current density and homogenous expression levels among cells. Cells were maintained in DMEM supplemented with 10% fetal bovine serum, 5 µg/ml blasticidin, and 250 µg/ml zeocin. Expression levels of the two splice forms were approximately equal as measured by current density. Peak current densities were –31.2 ± 4.4 (SE) pA/pF (n = 9) and –28.1 ± 6.6 pA/pF (n = 9) for CaV2.2 e[24a, 31a] and CaV2.2 e[24a, 31a], respectively. Lines 2017 and 1911 were then used to generate CaV21-expressing cells as described in the preceding text. After a further round of dilution cloning, lines 201719 (CaV2.2 e[24a, 31a], CaV3, CaV21) and 191105 (CaV2.2 e[24a, 31a], CaV3, CaV21) were used for further study. We fully sequenced all CaV2.2 cDNAs from these cell lines to ensure that no random mutations were introduced.
Recording methods
All recordings were carried out at room temperature (22–23.5°C). Currents were recorded using the whole cell recording method (Axopatch 200A), digitized at 10 kHz, and low-pass filtered at frequencies between 2 and 5 kHz. Analysis was performed with pCLAMP7 (Axon Instruments), software was custom-written in Quick Basic or MatLab, and Origin (Microcal). The bath solution contained (in mM) 160 TEA-Cl, 10 HEPES, and 2 BaCl2, pH 7.4 with TEA-OH. The pipette solution contained (in mM) 56 CsCl, 68 CsF, 2.2 MgCl2, 4.5 EGTA, 9 HEPES, 4 MgATP, 14 creatine phosphate (Tris salt), and 0.3 GTP (Tris salt), pH 7.4 with CsOH. Electrode resistances were 1–2 M
when filled with pipette solution. The series resistance was always <10 M and compensated 
90%. Currents evoked by voltage steps were subtracted for leak and capacitance using appropriately scaled currents evoked by a hyperpolarization from the holding potential. 
-conotoxin GVIA (Peptides International or Bachem) was used to isolated N-type currents from whole cell calcium currents in sympathetic and hippocampal neurons. Except as noted, statistics are given as means ± SE and significance was tested by unpaired Student's t-test.
Gating currents
Gating currents were resolved in cells expressing CaV2.2 together with CaV3 and CaV2. Currents were sampled at 10 kHz and filtered at 5 kHz; twice the digitization frequency to avoid aliasing. At 5 kHz (–3 dB), the rise time of events is 66.42 µs. For event amplitudes to be measured reliably, durations should be at least two times tr or 132 µs (Colquhoun 1994). It is possible that our fastest signals (160 µs for ON gating of central dominant isoform) were slightly undersampled and amplitudes underestimated. However, these were clearly distinguishable from the slower gating currents of the peripheral dominant isoform (
= 280 µs) that were well resolved. Nonlinear charge movements were recorded in barium with steps directly to the ionic reversal potential. The reversal potential was determined for each cell and was between +55 and +65 mV. Currents were induced by step depolarizations from a holding potential of between –100 and –120 mV. Leak subtraction was carried out off-line with appropriately scaled currents evoked by a 10 or 20 mV hyperpolarization from the holding potential. Recordings were only made from cells with small leak currents, in the range of 10–50 pA at the holding potential (–100 or –120 mV). Small transient outward currents remained after leak subtraction of size in relation to ionic currents appropriate for gating currents (Jones et al. 1997–1999; Noceti et al. 1996). No nonlinear charge movements were recorded from cells expressing no or very few functional channels. Similar nonlinear charge movements were recorded at the onset and offset of test depolarizations when ionic currents were blocked by replacing 2 mM Ba2+ with 2 mM Co2+ and 0.2 mM Cd2+.
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RESULTS |
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2 subunit modifies channel gating kinetics
N-type Ca channel currents were recorded from mammalian tsA201 cells stably expressing CaV2.2e[24a, 31a] together with CaV3 and CaV21 (Fig. 1). For both splice forms, gating kinetics of the channels in tsA201 cells were significantly faster than those of the same clones expressed in Xenopus oocytes with CaV3 (Lin et al. 1997) and resembled N-type currents of neurons (see following text; Table 1). At least part of the explanation for faster and more native-like gating kinetics of CaV2.2 clones in tsA201 cells is the presence of exogenous CaV21 (Fig. 1). The CaV21 auxiliary subunit was not included in our earlier studies of the same clones in Xenopus oocytes, although it is known to be part of the in vivo N-type channel complex (Witcher et al. 1993) and to speed N-type channel gating kinetics in oocytes (Wakamori et al. 1999). Another factor could be species-dependent posttranslational modifications between the two expression systems. To test this, currents were recorded from tsA201 cell lines expressing the same subunit combination used in our earlier oocyte studies: CaV2.2 splice form together with CaV3 without exogenous CaV21. Current densities were significantly smaller in the absence of exogenous CaV21 (Fig. 1, legend). N-type currents in tsA201 cells without exogenous CaV21 activate and deactivate significantly more slowly than those with exogenous CaV21 (Fig. 1, C and D; Table 1), more like currents recorded in oocytes. Macroscopic CaV2.2e[24a, 31a] currents activated with time constants of 
2 ms at +10 mV in Xenopus oocytes (Lin et al. 1997), compared with 1.6 and 0.7 ms in tsA201 cells in the absence and presence of CaV21, respectively (Table 1). Activation curves generated from tail current analysis in cells expressing exogenous CaV21 were also steeper at voltages between –30 and +30 mV compared with those generated from cells lacking CaV21 and generally contained two Boltzmann components rather than one (Figs. 1B, 2F, and 5A). We conclude that the presence of CaV21 is an important determinant of channel kinetics, accounting for the faster, more native-like kinetics of currents in tsA201 cells that express exogenous CaV21.
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Current densities and therefore channel expression levels were sufficiently high in these stable cell lines to resolve gating charge movement: about nine elementary charges per channel (Noceti et al. 1996). We recorded gating currents using identical ionic conditions as those used to record ionic currents (Figs. 2, 4, and 5). We applied test pulse depolarizations to precisely the ionic reversal potential where net current is zero. After leak subtraction only small, transient currents remained that decayed with time constants in the range of 0.2–0.3 ms, consistent with the time course of ON gating currents (Fig. 2, A, B, and D) (Jones et al. 1997, 1999; Noceti et al. 1996). Figure 2 shows that the kinetics of these gating currents were the same when measured in barium at the ionic reversal potential (+60 mV) and when measured in the same cell with ionic currents blocked by a combination of cobalt (2 mM) and cadmium (0.2 mM) (compare Fig. 2, A and B, and see D). The time course of the gating current during the test pulse, representing the speed of ON movement of the charge sensor, was fit well by a single exponential function ( = 0.36 ms for this cell expressing CaV2.2e[24a, 31a]; Fig. 2D). As a control, we also show that the total charge during ON gating currents equals that of OFF gating currents, over a range of potentials (Fig. 2, B and E). The size of ON gating currents at the reversal potential diminished as the magnitude of the prepulse depolarization immediately preceding the step to +60 mV was increased (Fig. 2, A and C). This is expected for current originating from the movement of putative voltage sensors. As progressively more channels activate and as more gating charge moves during the prepulse, a proportionally smaller number of channels is available for activation and less gating charge moves during the subsequent test pulse. The total charge left to move at +60 mV as a function of prepulse voltage is shown in Fig. 2C. The inverse of this plot is the ON gating current activation curve (Figs. 2F and 5B). As expected, gating currents activate at voltages more hyperpolarized than those that elicit ionic currents (40 mV; Figs. 2F, and 5, C and D, also see Table 1). However, we found that gating currents in the presence of cobalt and cadmium required much stronger depolarizations to activate (25 mV more depolarized) compared with those recorded in barium (Fig. 2F). Differences in surface charge screening between barium and cobalt may account for the differential shifts in the voltage dependence of channel gating (Hille et al. 1975). To avoid the need to correct for surface charge screening and to facilitate direct comparisons between gating and ionic currents, we measured gating currents at the ionic reversal potential in the presence of 2 mM barium.
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We compared N-type currents recorded from tsA201 cells stably expressing each CaV2.2 splice isoform. Macroscopic CaV2.2e[24a, 31a] currents activated and deactivated with time constants that were significantly longer compared with CaV2.2e[24a, 31a] (Fig. 3, C and D; P < 0.05; Table 1). That is, the predicted peripheral-dominant clone activated and deactivated more slowly than the predicted central-dominant clone. The difference in activation speed was 1.5-fold over a range of voltages. This difference in macroscopic activation kinetics was also independent of the presence of CaV21 (Table 1) and matches almost exactly our previous studies of the same splice isoforms in Xenopus oocytes (Lin et al. 1997, 1999).
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We compared gating currents from cells expressing CaV2.2 splice isoforms as described earlier. Gating currents measured in cells expressing CaV2.2 e[24a, 31a] decayed with time courses that were significantly slower (1.7-fold) compared with CaV2.2 e[24a, 31a] (P < 0.05; Fig. 4; Table 1). The 1.7-fold difference parallels the different gating kinetics of ionic currents between splice forms (Figs. 3C; Table 1). Although gating currents were measured at +60 mV, ionic currents were measured at voltages between –10 and +30 mV. Our results suggest that alternative splicing in IVS3-IVS4 linker in CaV2.2 modifies the rate of movement of the voltage sensor that precedes channel opening.
Steady-state voltage dependence of activation does not differ between isoforms
Next we analyzed the steady-state voltage dependence of activation of ionic and gating currents. In our previous studies, we reported a small (7 mV) but significant difference in the voltage dependence of channel activation between splice isoforms expressed in oocytes. This difference was not recapitulated in mammalian cells (Table 1; Fig. 5A). Most ionic current activation curves (8 of 10 recordings) were best fit by two Boltzmann functions with activation mid-points close to 0 and +45 mV, respectively (Figs. 2F and 5A; Table 1). There was no consistent difference in tail current activation curves between splice isoforms. In contrast to ionic currents, activation curves of gating currents were well fit by single Boltzmann functions with activation mid-points close to –40 mV (Figs. 2F and 5B; Table 1). Gating current activation curves were also indistinguishable between splice isoforms (Fig. 5B; Table 1; P > 0.05 for all parameters). Gating currents activate at voltages 40 mV more hyperpolarized than those that activate ionic currents (Fig. 5, C and D, also see Table 1). Interestingly, the gating charge appears to move almost completely before any ionic current is measured, a comparison made possible by recording ionic and gating currents in the same ionic conditions.
Inhibition by peptide toxins
There is considerable interest in pharmacological tools to distinguish between CaV2.2 splice isoforms in peripheral and central neurons, as potential tools to treat CNS disorders while minimizing blood pressure liability. We tested four Conus toxins (GVIA, MVIIA, MVIIC, and SVIB) that are likely pore blockers (McDonough 2004) and found that they inhibited both CaV2.2 splice isoforms equally well (Fig. 6). We also found equal inhibition by -grammotoxin-SIA (GTx), which inhibits inward current by altering the speed and voltage dependence of channel opening (McDonough et al. 1997). Nonetheless, by demonstrating that Conus GVIA in particular inhibits both splice isoforms equally well, we used this toxin to isolate and compare native N-type currents from sympathetic and hippocampal neurons.
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Several studies have demonstrated biophysical differences between alternatively spliced calcium channel clones studied in heterologous expression systems (Lipscombe et al. 2002), but few connect them to the neurons in which they are expressed. Many factors that affect the behavior of N-type channels including CaV subunits and G protein modulation are likely to differ in different types of neurons (Ikeda and Dunlap 1999; Walker and De Waard 1998). However, our analyses of cloned splice isoforms, together with earlier studies reporting tissue-specific expression of alternatively spliced exons in CaV2.2, make predictions about how native N-type currents in different regions of the nervous system might differ (Lipscombe et al. 2002). The vast majority of N-type currents of sympathetic neurons contain the e31a-containing CaV2.2 isoform. These N-type currents should activate and deactivate with slower kinetics compared with central neurons that only express CaV2.2 isoforms lacking e31a. In addition, our analyses in tsA201 cells suggest no significant difference in the voltage-dependence of N-type channel activation between sympathetic (peripheral) and hippocampal (central) neurons.
To compare N-type currents in sympathetic and pyramidal hippocampal neurons, we isolated currents using -conotoxin GVIA-subtraction because -conotoxin GVIA inhibits both CaV2.2 splice isoforms equally well. The N-type current represents 80% of the whole cell calcium channel current in sympathetic neurons and 20% in hippocampal neurons (Fig. 7). When we compared the voltage dependencies of activation, we found no significant difference between native N-type currents in sympathetic and hippocampal neurons, consistent with our studies in tsA201 cells (Fig. 8, A and B; Table 1). There was not obvious difference in the time course of inactivation of N-type currents between these cell types, at least during the 20-ms test depolarizations used here (Lin et al. 1999). However, N-type currents isolated from recordings of sympathetic neurons both activated and deactivated with kinetics that were consistently slower than N-type currents recorded from hippocampal neurons (Fig. 8, C and D; Table 1; P < 0.05). In fact, deactivation kinetics of native N-type currents in sympathetic and hippocampal neurons were indistinguishable from CaV2.2 e[24a, 31a] and CaV2.2e[24a, 31a] splice isoforms expressed in tsA201 cells, respectively (Fig. 7D; Table 1). We found that activation kinetics of native N-type currents were somewhat slower than those of CaV2.2 e[24a, 31a] expressed in tsA201 cells (Table 1). Although not a perfect match, overall, the time course and voltage dependence of channel opening of native N-type channel currents are most similar to the properties of CaV2.2 splice isoforms expressed in tsA201 cells that also express CaV21 (Figs. 3–5; Table 1). Our results from recordings of cloned and native N-type currents suggest that the major consequence of alternative splicing in the S3-S4 linkers of CaV2.2 is modulation of channel gating kinetics and not steady-state voltage dependence.
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DISCUSSION |
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). This form of alternative splicing is characterized by inverse insertions of short cassette exons in domains IIIS3-IIS4 and IVS3-IVS4. Results show that the CaV2.2e[24a, 31a] splice isoform gates more rapidly compared with CaV2.2e[24a, 31a] over a range of voltages, independent of expression system and of the presence of the CaV2 auxiliary subunit. Steady-state voltage dependencies of channel opening and of charge movement were not different between splice forms when expressed in mammalian cells. Data from gating currents provide direct evidence that the putative voltage sensor of the CaV2.2e[24a, 31a] splice isoform moves faster compared with CaV2.2e[24a, 31a].
Biophysical differences between these splice forms are likely to be physiologically significant because expression of the two exons is under tight cellular control. More generally, alternative splicing, particularly in the IVS3-IVS4 linker, is conserved among CaV1 genes (Lipscombe et al. 2002). In support of this view we show that native N-type currents recorded in hippocampal neurons reflect the faster kinetics of the CaV2.2e[24a, 31a] splice isoform, whereas N-type currents in sympathetic neurons consistently gate more slowly similar to CaV2.2e[24a, 31a]. Clearly, other factors will contribute to setting the time course of calcium channel activation and deactivation, including G protein activation and association with different CaV subunits (Ikeda and Dunlap 1999; Walker and De Waard 1998), and these may differ between sympathetic and hippocampal neurons. CaV2.2 has been shown to associate with different CaV subunits in vivo (Scott et al. 1996). However, we only observed significant differences in activating and deactivation kinetics of native N-type currents in sympathetic and hippocampal neurons consistent with the expression pattern of CaV2.2 splice isoforms. Exon e31a of CaV2.2 is particularly notable because it is expressed in peripheral neurons but suppressed throughout the CNS, including the hippocampus. Further, in a previous study we showed that exon 31a can fully confer the slower activation kinetics of the CaV2.2e[24a, 31a] splice isoform (Lin et al. 1999). The different kinetics of ionic and gating currents of CaV2.2 splice isoforms studied here most likely originate from the splice site in the S3-S4 linker of the fourth domain.
Effects on gating kinetics
The slower gating kinetics of CaV2.2e[24a, 31a] reported here and in earlier studies (Lin et al. 1997, 1999) are consistent with studies of alternative splicing in the equivalent IVS3-IVS4 linker of the closely related CaV2.1 P/Q-type channel (Hans et al. 1999; Krovetz et al. 2000). The different phenotypes of S3-S4 splice isoforms of CaV1 are interesting in light of the proposed central role this region plays in forming the putative voltage-sensing paddles recently predicted from crystallographic studies of the voltage-gated potassium channel (Jiang et al. 2003; see also Bezanilla 2000; Nakai et al. 1994; Stotz and Zamponi 2001). Apparent rates of activation of macroscopic currents, deactivation rates of macroscopic tail currents, and the speed of ON gating currents all differ between splice forms, with the CNS-dominant CaV2.2e[+24a, 31a] channels faster by 1.5-fold in all cases (Figs. 3 and 4; Table 1). We note, however, that gating currents were monitored at or close to +60 mV, whereas ionic currents were recorded at voltages between –100 and +30 mV where signals are measurable (Fig. 3). Several schemes modeling the kinetics of activation of voltage-gated channels, including calcium channels, invoke a final voltage-independent conformational closed-open step that is rate limiting at sufficiently strong depolarizations (Chen and Hess 1990; Frazier et al. 2001; Zagotta et al. 1988). We observed kinetic differences between S3 and S4 splice isoforms over a range of voltages while steady-state activation curves for both gating and ionic currents were not distinguishable between isoforms. These observations are consistent with the involvement of a gating transition closely linked to the open state perhaps in the final voltage-independent closed-open transition.
Summary
The mammalian Cav2.2 gene is encoded by 50 exons, 10 of which can be alternatively spliced. In addition to exons 24a and 31a, alternative splicing involves exon 10 in the I–II intracellular loop, exons 18a, 19, 20, and 21 in the II–III intracellular loop, and exons 37a/37b, and 46 in the C terminus (Lipscombe and Castiglioni 2004). It is therefore important to note that there is no one peripheral or central form of the CaV2.2 gene. Indeed, our recent single-cell RT-PCR analysis shows that even individual neurons of the dorsal root ganglia express multiple CaV2.2 splice isoforms (Bell et al. 2004). Significantly, however, of the 10 sites of alternative splicing that have been identified so far in mammalian CaV2.2 genes (Lipscombe and Castiglioni 2004), only exon 31a is restricted to peripheral neurons; based on our recent single-cell RT-PCR analysis in dorsal root ganglia, exon 31a is present in every CaV2.2 mRNA (Bell et al. 2004). Collectively, our studies are therefore consistent with the conclusion that tissue-specific expression of exon 31a of CaV2.2 underlies the comparatively slow kinetics of N-type calcium channels in peripheral neurons. Pharmacological tools to discriminate between these splice isoforms would help establish their relative contribution to synaptic events. The Conus peptides tested here did not differentiate between splice isoforms, although others have reported differential effects of Conus catus, peptides under certain conditions (Lewis et al. 2000). The voltage dependence of the two splice forms in mammalian cells are equal, therefore pharmacological strategies to distinguish between them would probably need to exploit the different binding epitopes in the S3-S4 linkers directly. The cell lines developed here provide useful tools for studying splice isoforms of the N-type Ca channel that exhibit properties similar to those of native currents (Barrett et al. 2001) and with current densities sufficiently high to monitor gating currents.
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ACKNOWLEDGMENTS |
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Present address of S. I. McDonough: Amgen, Inc., 1 Amgen Center Dr., mailstop 29-2-B, Thousand Oaks, CA 91320-1799.
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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.
Address for reprint requests and other correspondence: D. Lipscombe, Dept. of Neuroscience, Brown University, 192 Thayer St., Providence, RI 02912 (E-mail: Diane_Lipscombe{at}Brown.Edu).
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