Alternative Splicing in the Cytoplasmic II–III Loop of the N-Type Ca Channel α_1B Subunit: Functional Differences Are β Subunit-Specific

Abstract

Structural diversity of voltage-gated Ca channels underlies much of the functional diversity in Ca signaling in neurons. Alternative splicing is an important mechanism for generating structural variants within a single gene family. In this paper, we show the expression pattern of an alternatively spliced 21 amino acid encoding exon in the II–III cytoplasmic loop region of the N-type Ca channel α_1B subunit and assess its functional impact. Exon-containing α_1B mRNA dominated in sympathetic ganglia and was present in ∼50% of α_1B mRNA in spinal cord and caudal regions of the brain and in the minority of α_1B mRNA in neocortex, hippocampus, and cerebellum (<20%). The II–III loop exon affected voltage-dependent inactivation of the N-type Ca channel. Steady-state inactivation curves were shifted to more depolarized potentials without affects on either the rate or voltage dependence of channel opening. Differences in voltage-dependent inactivation between α_1Bsplice variants were most clearly manifested in the presence of Ca channel β_1b or β₄, rather than β_2a or β₃, subunits. Our results suggest that exon-lacking α_1B splice variants that associate with β_1b and β₄ subunits will be susceptible to voltage-dependent inactivation at voltages in the range of neuronal resting membrane potentials (−60 to −80 mV). In contrast, α_1B splice variants that associate with either β_2a or β₃ subunits will be relatively resistant to inactivation at these voltages. The potential to mix and match multiple α_1B splice variants and β subunits probably represents a mechanism for controlling the plasticity of excitation–secretion coupling at different synapses.

Structural diversity of voltage-gated Ca channels is at the heart of the rich functional diversity in Ca signaling in mammalian neurons. Neurons can select from several distinct Ca channel α₁ genes that undergo extensive RNA processing, including alternative splicing (Perez-Reyes et al., 1990; Soldatov, 1994; Kollmar et al., 1997;Lin et al., 1997; Bourinet et al., 1999) and differential polyadenylation (Schorge et al., 1999). Each α₁subunit may also interact with multiple functionally distinct auxiliary subunits (Scott et al., 1996; Walker and De Waard, 1998), expanding the capacity for diversity within each Ca channel family. The α_1B subunit is the functional core of N-type Ca channels that localize to synapses and control calcium-dependent neurotransmitter release throughout the vertebrate nervous system (Hirning et al., 1988; Takahashi and Momiyama, 1993; Dunlap et al., 1995). Many neurotransmitters and neurohormones modulate excitation–secretion coupling by regulating the gating of N-type Ca channels via effects on the α_1B subunit (Dunlap et al., 1995). The α_1B gene is also subject to tissue-specific alternative splicing (Lin et al., 1997, 1999), which probably represents an important mechanism for optimizing neurotransmitter release in different regions of the nervous system. The extent of splicing in the α_1B gene is not known, but its large size (>100 kb in human genome), together with mismatches in α_1B cDNAs isolated from different tissues, supports the presence of multiple sites of alternative splicing.

In a previous study, we identified two short cassette exons in S3–S4 linkers of domains III and IV of the Ca channel α_1B subunit gene (see Fig.1A) (Lin et al., 1997, 1999). Alternative splicing of these exons was tissue-specific and influenced both the voltage-dependence and rate of Ca channel activation. A third variant site in loop I–II of the of α_1B gene involving one amino acid (A⁴¹⁵) originated from the random use of alternative 3′ acceptor–splice sites, but it is not associated with any obvious change in channel gating (Lin et al., 1997). In the present study, we characterize a larger alternatively spliced sequence encoding 21 amino acids in the cytoplasmic II–III loop of the α_1B gene (see Fig.1A) first identified in mouse neuroblastoma cells (Coppola et al., 1994) and more recently shown by PCR analysis to be present in rat brain (Ghasemzadeh et al., 1999). We report the genomic structure of this site in the rat α_1B gene, detail the differential expression of this exon in the rat nervous system, and assess its impact on channel function.

Fig. 1.

Sites of alternative splicing in the N-type calcium channel α_1B subunit. A, Putative membrane topology of the α_1B subunit and location of four alternatively spliced sequences encoding Ala⁴¹⁵ in intracellular loop I–II (A), 21 amino acids in intracellular loop II–III (FVKQTRGTVSRSSSVSSVNSP), four amino acids in IIIS3–IIIS4 (SFMG), and two amino acids in IVS3–IVS4 (ET). With the exception of Ala⁴¹⁵ whose expression depends on the use of alternative 3′ splice–acceptors, expression of the other three sites is regulated by alternative splicing of isolated exon cassettes (Lin et al., 1997, 1999). B, Genomic sequence derived from analysis of the α_1B gene in the region of the intracellular II–III loop (middle) together with the amino acid sequences of two cDNAs derived by RT-PCR from rat neurons(top, bottom). The location of exons (uppercase letters, shaded), introns (lowercase), the 63 base exon cassette (boxed), and splice junction consensusag-gt dinucleotide sequences (underlined) are indicated. Nucleotides 2270 and 2271and amino acids A⁷⁵⁷ (757) and R⁷⁵⁸ (758) denote the splice junction (numbering according to GenBank sequence M92905) (Dubel et al., 1992).Dashed lines indicate the two patterns of alternative splicing that give rise to +21α_1B (top) and Δ21α_1B (bottom)_. The genomic sequence of this region is available under GenBank accession number AF222338.

MATERIALS AND METHODS

Genomic analysis. A 6.3 kb region of the rat α_1B gene was amplified from liver genomic DNA using primers directed to 5′ and 3′ exons flanking the splice junction of interest in the II–III loop region. Primer sequences were as follows: Bup2170, (5′-GAG GAG ATG GAA GAG GCA GCC AAT-3′); and Bdw2361, (5′-CTC CGG GTC CAT CTC ACT GTA CAG T-3′). The 50 μl PCR reaction mix contained 250 ng of rat liver genomic DNA, 350 μm each deoxynucleotide and 0.4 μm each primer. After a 15 min preincubation at 92°C, 0.75 μl of enzyme mix (Expand Long Template; Boehringer Mannheim, Indianapolis, IN) was added to “hot start” the reaction. After 30 amplification cycles, a single 6.3 kb product was generated and subsequently gel-purified, subcloned, and sequenced by primer walking (Yale University Sequencing Facility, New Haven, CT). The sequence is available under GenBank accession number AF222338.

Reverse transcription-PCR. Total RNA was isolated from different regions of the adult rat nervous system using the guanidium–thiocyanate method and used for first strand cDNA synthesis; 4–6 μg of RNA from CNS tissue or 1–2 μg of RNA from sensory and sympathetic ganglia was used as template in each 20 μl reaction mix containing 0.5 mm each deoxynucleotide, 50 ng/μl random hexamers, 10 mm DTT, and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, NY), incubated for 1 hr at 37°C. One microliter of cDNA from each reverse transcription (RT) reaction (or 3 μl from pituitary RT reaction) was used for PCR amplification in a 50 μl standard reaction mix using the following protocol: 1 cycle at 94°C for 2 min, 30 cycles at 94°C for 10 sec, 59°C for 35 sec, 72°C for 50 sec, and 1 cycle at 72°C for 8 min. Primers Bup2104 (5′-TTG AAC GTT TTC TTG GCC ATT GCT GT) and Bdw2363 (5′- CTC CTC CGG GTC CAT CTC ACT GTA CA) were located in 5′ and 3′ exons flanking the splice junction. Negative controls that lacked template confirmed that reagents were not contaminated by α_1B cDNA clones. The primers flanked a 6.1 kb intron so contamination by genomic DNA could be ruled out, and reverse transcriptase-lacking controls were also performed. Thirty rounds of amplification were used routinely in our analysis. To control for the possibility of saturation, the number of cycles was increased stepwise from 10, 15, 20, 25, to 30 in one experiment. Products were first observed after 25 cycles, and relative band intensities were identical to those observed after 30 cycles. Band intensities were quantified and normalized to the size of the DNA fragments using a gel documentation system (Alpha Inotech).

Functional assessment of the Ca channel α_1B cDNA constructs. The N-type Ca channel α_1B-b splice variant (Lin et al., 1997) (GenBank accession number AF055477) referred to here as Δ21α_1B was used as template for constructing the +21α_1B splice variant (GenBank accession number AF222337) by standard cloning methods. Δ21α_1B and +21α_1Bsplice variants were cloned into the Xenopus β-globin vector pBSTA to facilitate functional expression (Goldin and Sumikawa, 1992). Functional properties of Δ21α_1B and +21α_1B were assessed in the Xenopusoocyte expression system using methods and procedures essentially as described previously (Lin et al., 1997, 1999). Forty-six nanoliters of α_1B (67–400 ng/μl) and β (22–133 ng/μl) cRNA mix (α_1B/β is 3:1 μg/μg) were injected into defolliculated Xenopus oocytes, and currents were recorded 3–5 d later. Before recording, oocytes were injected with 46 nl of a 50 mm BAPTA solution to reduce activation of the endogenous Ca-activated Cl⁻ current (Lin et al., 1997). N-type Ca²⁺ channel currents were recorded with the two microelectrode voltage-clamp technique using electrodes of 0.8–1.5 and 0.3–0.5 MΩ resistance (3 m KCl) for voltage and current electrodes, respectively. Recording solutions contained either 5 mm BaCl₂ or 2 mmCaCl₂, 85 mm tetraethylammonium, 5 mm KCl, and 5 mm HEPES, pH adjusted to 7.4 with methanesulfonic acid. The β_1b subunit used in this study was provided by K. P. Campbell (University of Iowa, Iowa City, IA) (Pragnell et al., 1992); β_2a and β₄ was provided by E. Perez-Reyes (Loyola University, Maywood, IL) (Perez-Reyes et al., 1992; Castellano et al., 1993b), and β₃ was cloned in our lab from rat brain and is almost identical to the published rat brain sequence (Castellano et al., 1993a). Both Ca channel α_1Bsplice variants expressed equally well in Xenopus oocytes [e.g., in the presence of β₃, the average N channel current amplitude was 2.68 ± 0.26 μA (n= 8) compared with 2.46 ± 0.11 μA (n = 7) for +21α_1B and Δ21α_1B, respectively]. With the exception of β₄, Ca currents were twofold to threefold larger in oocytes expressing α_1B together with a β subunit. β₄ did not increase current amplitude, but it did modulate N channel gating, confirming that it was expressed (see Results). Data were acquired on-line and leak subtracted using a P/4 protocol (pClamp V6.0; Axon Instruments, Foster City, CA). Voltage steps were applied every 10–30 sec depending on the duration of the step, from various holding potentials. Current-voltage and steady-state inactivation relationships and activation and inactivation rates were measured.

RESULTS

We analyzed the α_1B gene to determine whether alternative splicing could explain the presence of α_1B cDNA variants containing an extra 21 amino acid encoding sequence in the II–III intracellular loop region. The salient features of this region of the α_1B gene are presented in Figure1B. A single 6.3 kb product was amplified from rat genomic DNA using PCR primers directed to cDNA sequences flanking the putative alternatively spliced exon. We sequenced this product and showed that codon A757 is interrupted by a 6125 bp intronic sequence that contains consensus gt andag splice junctions (Sharp and Burge, 1997). A 63 base cassette exon was identified midway through the intronic sequence flanked by ag-gt splice junction motifs (Fig.1B) and a polypyrimidine track 5′ to the exon (data not shown). The sequence of the cassette exon corresponds to the 63 base insert, confirming that +21α_1B and Δ21α_1B variants that we (Fig.2) (Pan et al., 1999) and others have observed in the II–III loop region of α_1B mRNA (Coppola et al., 1994; Ghasemzadeh et al., 1999) arise from alternative splicing. In their study, Coppola et al. (1994) showed that the alternatively expressed sequence contained 66, rather than 63, bases. This is because all α_1B cDNAs isolated from mouse neuroblastoma cells that lacked the 63 base exon sequence also lacked three bases encoding R758. Figure 1B shows that the R758 codon is not part of the isolated 63 base exon but rather is contiguous with the 3′ flanking exon, and its expression depends on the use of alternative dinucleotide, ag, splice–acceptors at this distal 3′ intron–exon boundary (flanking nucleotide 2271) (Fig. 1B). Use of the first splice–acceptor site would result in α_1B mRNA containing the R758 codon, whereas R758 would be skipped if the second splice–acceptor site is used. All α_1B clones that we have so far isolated from rat contain R758, but the presence of R758-lacking α_1B clones in mouse neuroblastoma cells (Coppola et al., 1994) and bovine chromaffin cells (Cahill et al., 2000) confirms that both splice–acceptor sites are used. We have not investigated the expression pattern or the functional consequences of R758 but rather focused on the 63 base cassette exon.

Fig. 2.

Expression pattern of +21α_1B and Δ21α_1B mRNAs in various regions of the nervous system of the adult rat. Top, Summary of RT-PCR analysis showing the relative abundance of the +21α_1B mRNA variant expressed as a fraction of total α_1B mRNA derived from brain (Brain), superior cervical ganglia (SCG), dorsal root ganglia (DRG), spinal cord (SpC), pons and medulla (Pn/Med), midbrain (MdBr), thalamus (Thal), hypothalamus (Hypo), pituitary (Pituit), hippocampus (Hippo), cerebellum (Cereb), neocortex (NCtx), and template negative PCR controls (−). Data were averaged from analysis of at least three different RNA samples for each tissue isolated from multiple rats (mean ± SE).Bottom, Example of PCR-derived cDNAs separated by electrophoresis in 2% agarose. Two cDNA products were amplified from each sample (285 and 348 bp) corresponding to Δ21α_1Band +21α_1B mRNA. The first lane shows 300 and 400 bp size markers. One microliter of RT reaction, except for pituitary (3 μl), was used as template for PCR amplification for all RNA samples. Relative band intensities were estimated using Alpha Inotech gel documentation software and normalized for size differences.

To determine the expression pattern of the 63 base cassette exon, we analyzed RNA isolated from different regions of the nervous system of the adult rat by RT-PCR (Fig. 2). PCR primers that flanked the splice junction were chosen to generate two readily separable size cDNA products (348 and 285 bp) corresponding to +21α_1B and Δ21α_1BmRNAs, respectively. Contamination by genomic DNA could be excluded because the primers flanked a 6.1 kb intron. By measuring relative band intensities, we show that exon expression is differentially regulated in different regions of the nervous system. +21α_1B mRNA dominates in sympathetic ganglia (80.8 ± 0.2% of total α_1B,n = 3), whereas exon expression is suppressed in α_1B mRNA isolated from whole brain (15.3 ± 0.8% of total α_1B, n = 3). Exon expression was not, however, suppressed uniformly throughout the CNS. Although relatively low levels of +21α_1B mRNA (<20% total α_1B) were present in neocortex, hippocampus, and pituitary, reflecting the pattern in whole brain, significant amounts of both splice variants (+21α_1B and Δ21α_1B) were detected in more caudal regions of the CNS, including spinal cord and brainstem.

Having established that alternative splicing in the II–III intracellular loop region of α_1B is strongly region-specific, we next determined whether exon expression affected channel function. The kinetics and voltage dependence of N-type Ca channel activation and inactivation were studied in oocytes expressing Δ21α_1B and +21α_1Bsplice variants. At least three different β subunits copurify with α_1B in native membranes (Scott et al., 1996), and heterologous expression studies have shown that multiple β subunits modulate N-type Ca channel function (Walker and De Waard, 1998). We therefore thought it pertinent to assess the functional impact of splicing in the presence of four different Ca channel β subunits known to be expressed in rat brain (β_1b, β_2a, β₃, and β₄) (Pragnell et al., 1991; Perez-Reyes et al., 1992; Castellano et al., 1993a,b).

Figure 3 compares N-type currents induced by expressing Δ21α_1B and +21α_1B splice variants in Xenopusoocytes in the presence of each β subunit. Δ21α_1B and +21α_1Bsplice variants expressed equally well in oocytes, and their normalized, peak current–voltage curves and channel activation kinetics were indistinguishable. Peak current–voltage curves for Ca channels recorded from oocytes expressing β₃were shifted by ∼10 mV in the depolarizing direction relative to β_1b, β_2a, and β₄ similarly for both α_1B splice variants (see also Fig.5A). We therefore conclude that the presence of an additional 21 amino acids in the cytoplasmic II–III loop of the α_1B subunit does not affect the kinetics or voltage dependence of N-type Ca channel opening.

Fig. 3.

+21α_1B and Δ21α_1Bsplice variants have similar voltage- and time-dependent activation properties. Normalized, averaged peak current–voltage plots were calculated from Xenopus oocytes expressing Δ21α_1B (●) and +21α_1B (○) subunits together with Ca channel β_1b (A), β_2a (B), β₃(C), and β₄(D) subunits. Currents were activated by brief depolarizations to various test potentials from a holding potential of −80 mV. Barium (5 mm) was the charge carrier. Normalized, averaged current traces for Δ21α_1B (thick line) and +21α_1B (thin line) are shown superimposed as insets in A–D. Currents shown were activated by depolarization to −10 mV for β_1b, β_2a, and β₄ and, to compensate for its different voltage-dependent activation, to 0 mV for β₃. Activation midpoints for currents induced by the expression were as follows: +21α_1B/β_1b, −12.7 ± 0.8 mV (n = 5); +21α_1B/β_2a, −12.1 ± 0.7 mV (n = 6); +21α_1B/β₃, −5.5 ± 0.7 mV (n = 5); and +21α_1B/β₄, −13.2 ± 0.5 mV (n = 7). These values were not significantly different from Δ21α_1B (see legend to Fig. 5 for Δ21α_1B values).

Fig. 5.

Functional differences in α_1Bcoexpressed with different Ca channel β subunits. N-type Ca channel currents induced by coexpressing Δ21α_1B with β_1b(●), β_2a (○), β₃(▴), and β₄ (▵) subunits in Xenopus oocytes. Barium (5 mm) was the charge carrier. A, Currents activated by step depolarizations to various test potentials from a holding potential of −80 mV. Normalized, averaged peak current–voltage plots for each β subunit are shown. Each point is mean ± SE. Activation midpoints were as follows: Δ21α_1B/β_1b, −13.6 ± 0.6 mV (n = 6); Δ21α_1B/β_2a, −11.7 ± 1.1 mV (n = 6); Δ21α_1B/β₃, −4.6 ± 1.0 mV (n = 4); and Δ21α_1B/β₄, −13.2 ± 0.5 mV (n = 5). In the presence of β₃, currents activated at voltages ∼10 mV more depolarized compared with β_1b, β_2a, and β₄. B, Average, steady-state inactivation curves of Δ21α_1Bchannels expressed with different β subunits. Currents were activated by depolarizations to 0 mV from different holding potentials. Peak currents were measured and expressed relative to maximum current (holding potential, −120 to −100 mV). Inactivation midpoints were as follows: Δ21α_1B/β_1b, −68.5 ± 0.8 mV (n = 6); Δ21α_1B/β_2a, −34.1 ± 0.7 mV (n = 5); Δ21α_1B/β₃, −43.8 ± 1.0 mV (n = 5); and Δ21α_1B/β₄, −71.2 ± 0.7 mV (n = 6).

N-type Ca channels recorded from native cells vary considerably in their voltage dependence and kinetics of inactivation (Bean, 1989). We therefore compared both the time course of inactivation and steady-state inactivation curves of Δ21α_1Band +21α_1B splice variants in the presence of different β subunits. Normalized, averaged currents recorded from oocytes expressing each α_1B/β combination are shown as insets in Figure4A–D. Inactivation kinetics of the splice variants are indistinguishable in the presence of a given β subunit, and superimposed averaged currents overlap almost perfectly. The kinetics of N-type Ca channel inactivation did, however, vary depending on the type of β subunit expressed. For example, N-type Ca channel currents recorded in β_1b- and β₃-expressing oocytes inactivate with relatively fast time courses compared with β_2a similarly for both α_1B splice variants (Fig. 4). These findings are consistent with other studies that associate the presence of β_2a with relatively slowly inactivating Ca channels (Olcese et al., 1994; Walker and De Waard, 1998). Alternative splicing in the II–III loop of the α_1B subunit does not, therefore, affect channel inactivation kinetics during step depolarizations to relatively positive voltages. However, a significant difference in the N-type Ca channel availability curve was observed between Δ21α_1B and +21α_1B when the holding potential was varied. N-type Ca channels induced by expressing +21α_1Bsplice variant with β_1b and β₄ inactivated at membrane potentials that were ∼10 mV more depolarized relative to Δ21α_1B(Fig. 4A,D; see also Fig.6B). These differences were only revealed in the presence of specific α_1B/β subunit combinations because steady-state inactivation curves were similar between α_1B splice variants when coexpressed with β_2a and only slightly different in the presence of β₃ (Fig.4B,C; see also Fig.6B).

Fig. 4.

+21α_1B and Δ21α_1Bsplice variants have different steady-state inactivation curves depending on which β subunit is coexpressed. Normalized, averaged steady-state inactivation curves were calculated from currents activated by brief depolarizations to 0 mV from various holding potentials in oocytes expressing Δ21α_1B (●) and +21α_1B (○) subunits together with Ca channel β_1b (A), β_2a(B), β₃ (C), and β₄ (D) subunits. Normalized, averaged currents activated by long depolarizations to +20 mV are also shown for Δ21α_1B (thick line) and +21α_1B (thin line) to compare inactivation kinetics (insets, A–D). Barium (5 mm) was the charge carrier. Midpoints of steady-state inactivation curves for currents induced by the expression were as follows: +21α_1B/β_1b, −58.1 ± 0.8 mV (n = 6); +21α_1B/β_2a, −33.0 ± 0.8 mV (n = 5); +21α_1B/β₃,=−41.5 ± 0.9 mV (n = 5); and +21α_1B/β₄, −63.3 ± 0.8 mV (n = 6). Values for Δ21α_1Bcurrents are in the legend to Figure 5.

Fig. 6.

Functional differences between α_1Bsplice variants are β subunit-specific. Average shifts in channel activation (A) and steady-state inactivation (B) midpoints between Δ21α_1B and +21α_1B splice variants in the presence of different Ca channel β subunits. Currents were measured using 2 mm Ca (dark shading) and 5 mm Ba (light shading) as the permeant ions. Midpoints of activation calculated from current–voltage plots using 2 mm Ca were as follows: Δ21α_1B/β_1b, −13.9 ± 1.2 mV (n = 7); Δ21α_1B/β_2a, −7.9 ± 0.8 mV (n = 6); Δ21α_1B/β₃, −5.3 ± 0.6 mV (n = 4); and Δ21α_1B/β₄, −14.9 ± 1.1 mV (n = 5); and compared with +21α_1B/β_1b, −16.1 ± 1.2 mV (n = 5); +21α_1B/β_2a, −8.6 ± 0.6 mV (n = 6); +21α_1B/β₃, −7.0 ± 0.6 mV (n = 5); and +21α_1B/β₄, −15.0 ± 0.5 mV (n = 5). Midpoints from steady-state inactivation curves using 2 mm Ca as charge carrier were as follows: Δ21α_1B/β_1b, −72.1 ± 0.6 mV (n = 6); Δ21α_1B/β_2a, −37.0 ± 1.6 mV (n = 5); Δ21α_1B/β₃, −41.9 ± 0.6 mV (n = 5); and Δ21α_1B/β₄, −72.7 ± 1.3 mV (n = 7); and compared with +21α_1B/β_1b, −64.4 ± 0.5 mV (n = 6); +21α_1B/β_2a, −38.0 ± 1.0 mV (n = 5); +21α_1B/β₃, −40.4 ± 0.7 mV (n = 5); and +21α_1B/β₄, −64.1 ± 0.7 mV (n = 5). Values are mean ± SE. See legends to Figures 3-5 for values with 5 mm Ba as charge carrier.

In Figure 5 we compare normalized current–voltage and steady-state inactivation curves of the four different β subunits expressed with one α_1Bsplice variant (Δ21α_1B). With the exception of β₃, which is associated with Ca channel currents activating at more depolarized voltages (V_1/2 of approximately −5 mV), current–voltage relationships were similar between β subunits (Fig.5A). However, a comparison of steady-state inactivation curves highlights how similar inactivation profiles of N-type Ca channels recorded from β_1b- and β₄-expressing oocytes are to each other on the one hand and different from β_2a and β₃ on the other. This correlation could be significant in light of the fact that differences between α_1B splice variants are only significant when coexpressed with β_1b and β₄ but not β_2a and β₃. Figure 5B shows that N-type Ca channels in β_1b- and β₄-expressing oocytes inactivate at more hyperpolarized voltages (V_1/2 of −60 to −80 mV) relative to N-type Ca channels associated with β_2a and β₃, which inactivate at significantly more depolarized membrane potentials (V_1/2 of −35 to −45 mV) (Fig.5B). Similar results were also obtained using 2 mm extracellular calcium rather than 5 mm barium as the permeant ion (Fig.6).

DISCUSSION

Alternative splicing is region-specific

Alternative splicing in intracellular loop II–III of the Ca channel α_1B subunit mRNA is differentially regulated in distinct regions of the nervous system. Sympathetic ganglia express the highest levels of exon-containing α_1B mRNA (>80% of total), and in the CNS, +21α_1B mRNA levels decrease in an approximately caudal to rostral pattern, from ∼50% in brainstem to <20% in neocortex. Overall, exon skipping at this splice site prevails in whole brain, consistent with the absence of the exon in α_1B cDNAs isolated previously from rat brain-derived cDNA libraries (Dubel et al., 1992; Fujita et al., 1993). It has been suggested recently that the II–III intracellular loop exon of α_1B is preferentially expressed in brain regions of the rat enriched in monoaminergic neurons, based on in situ hybridization and RT-PCR analysis (Ghasemzadeh et al., 1999). Our results do not test this hypothesis directly but nonetheless do favor more widespread distribution. For example, there is a relatively high representation of +21α_1B mRNA in regions of the CNS not particularly rich in monoaminergic neurons, such as spinal cord and hypothalamus (Fig. 2). The expression pattern of the II–III intracellular loop exon differs from other splice sites that we have studied previously in the IIIS3–IIIS4 and IVS3–IVS4 extracellular linkers of the α_1B subunit, suggesting that the α_1B gene contains multiple, independently regulated sites of alternative splicing.

Functional consequences of alternative splicing

The functional effect of lengthening the II–III intracellular loop of the α_1B subunit by 21 amino acids is summarized in Figure 6. Exon inclusion did not affect the voltage dependence of channel activation but did induce a shift in the channel availability curve to more depolarized membrane voltages similarly with calcium or barium as permeant ion. These results contrast nicely with our analysis of alternative splicing in the IVS3–IVS4 extracellular linker of the α_1B subunit, a domain close to the putative voltage sensor (S4). Splicing in IVS3–IVS4 affected both the voltage dependence and kinetics of channel activation but not inactivation (Lin et al., 1997, 1999).

Functional differences between +21α_1B and Δ21α_1B splice variants were only observed in the presence of Ca channel β_1b or β₄ subunits that also shift channel steady-state inactivation curves into the physiologically interesting range of potentials between −60 and −90 mV. In contrast, small or no shifts were observed between the splice variants coexpressed with either β_2a or β₃ that form N channels that inactivate at significantly more depolarized voltages (−50 to −20 mV). Xenopus oocytes express an endogenous Ca channel β subunit that is highly homologous to mammalian β₃ (β_3XO) (Tareilus et al., 1997). If present at high enough levels, endogenous β_3XO could partially mask the actions of exogenously expressed β subunits; consequently, hyperpolarizing shifts in N-type Ca channel steady-state inactivation curves associated with β_1b and β₄ might be slightly underestimated. Nonetheless, our results demonstrate that functional differences between α_1B splice variants depend on their interactions with specific β subunits.

How does this β subunit specificity arise? One possibility is that β_1b and β₄ subunits, but not β_2a or β₃, specifically interact with the 21 amino acid insert in the II–III intracellular loop of α_1B. Although the primary β subunit binding site on α₁ is in the intracellular loop between domains I and II (Pragnell et al., 1993; De Waard et al., 1994), secondary β-interaction sites in the C-terminal region of α₁ have been identified recently (Walker et al., 1998, 1999), leaving open the possibility that additional β-interaction sites in α₁, such as in the II–III loop, might exist. Alternatively, β subunit specificity might not depend on direct interactions between β subunits and the II–III intracellular loop of α_1B. For example, modification of N-type Ca channel inactivation might be most permissible at membrane potentials between −60 and −90 mV; consequently, differences between splice variants would surface in the presence of β_1bor β₄ but be less obvious with β_2a or β₃ subunits. In this case, functional differences between α_1Bsplice variants in native cells might also depend on interactions with other Ca channel subunits (e.g., α₂δ) and modulators (e.g., G-proteins) that, like β subunits, affect channel inactivation.

Our studies suggest that all α_1B/β combinations are permissible and form functional channels in theXenopus oocyte expression system, but which combinations occur in native cells? There is significant overlap in the distribution of α_1B and β₃ mRNA and protein in mammalian brain but the correlation is not perfect (Ludwig et al., 1997), and biochemical studies have demonstrated significant levels of α_1B/β₃ and α_1B/β₄ complexes in native N-type Ca channel proteins isolated from brain (Scott et al., 1996). The study by Scott et al. (1996) does not, however, distinguish between different α_1B splice variants. Although α_1B, β₃, and β₄ mRNAs are broadly distributed in brain, in brainstem β_1b and β₄mRNAs are present to the exclusion of β_2a and β₃ (Ludwig et al., 1997). Because significant levels of Δ21α_1B and +21α_1B mRNAs (Fig. 2) are also present in brainstem, we suggest that both α_1B splice variants probably form native N-type Ca channels with β subunits other than β₃ (i.e., β_1b or β₄). In light of our results, it will now be important to establish which β subunits associate with which specific α_1B splice variant, Δ21α_1B and +21α_1B, in different regions of the nervous system.

The potential to mix and match multiple α_1Bsplice variants and β subunits may represent a mechanism for fine-tuning excitation–secretion coupling at different synapses. We speculate that the availability of N-type Ca channels at synapses dominated by Δ21α_1B/β_1b or Δ21α_1B/β₄ complexes may be more sensitive to modulation by agents or stimuli that induce relatively prolonged changes in the resting membrane potential between −60 and −80 mV compared with β_2a- or β₃-dominant synapses containing either Δ21α_1B or +21α_1B. Heterogeneity in α_1B/β complexes (Scott et al., 1996) probably account for native N-type Ca channel currents in neurons that differ in their sensitivity to inactivation as the membrane potential is depolarized (Bean, 1989; Kongsamut et al., 1989;Plummer et al., 1989). Our results also highlight the II–III intracellular loop of the Ca channel α_1Bsubunit as a domain important for regulating N-type Ca channel availability at voltages close to the resting membrane potential. The demonstration that syntaxin, a SNARE [SNAP (solubleN-ethylmaleimide-sensitive factor attachment protein) receptor] that binds to a region in the II–III intracellular loop overlapping the splice junction, also affects the position of the steady-state inactivation curve is consistent with this hypothesis (Bezprozvanny et al., 1995).

Future directions

Our findings provide the framework to begin to address other questions related to the functional significance of splicing in the II–III intracellular loop of the Ca channel α_1B subunit. For example, the alternatively spliced exon is unusually enriched in serine and threonine residues (9 of 21), suggesting that Δ21α_1B and +21α_1B splice variants might be differentially modulated by protein kinase. Furthermore, because the exon overlaps the synaptic protein interaction site on α_1B(synprint) (Catterall, 1999), it would be interesting to determine how its presence influences SNARE binding (Sheng et al., 1994; Charvin et al., 1997; Catterall, 1999). Along these lines, there is evidence for isoform-specific interactions of SNAREs with II–III intracellular loop variants of the closely related α_1A subunit (Rettig et al., 1996; Catterall, 1999), although the isoforms of α_1A reported by Catterall and colleagues do not correspond to the Δ21α_1B and +21α_1B splice variants described here. Finally, we know very little about the mechanisms that regulate expression of this exon and other alternative spliced exons of α_1B in different regions of the nervous system. The identification of regulatory elements presumably in introns upstream of the exon might provide clues as to the nature of the proteins that direct these functionally significant splicing events (Grabowski, 1998).