WWW.JNEUROSCI.ORG
-
The Journal of Neuroscience MBF Stereo Investigator
 QUICK SEARCH:   [advanced]


     
-


HOME
  |  
SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (48)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, Z.
Right arrow Articles by Lipscombe, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, Z.
Right arrow Articles by Lipscombe, D.

 Previous Article  |  Next Article 

The Journal of Neuroscience, July 1, 1999, 19(13):5322-5331

Alternative Splicing of a Short Cassette Exon in alpha 1B Generates Functionally Distinct N-Type Calcium Channels in Central and Peripheral Neurons

Zhixin Lin, Yingxin Lin, Stephanie Schorge, Jennifer Qian Pan, Michael Beierlein, and Diane Lipscombe

Department of Neuroscience, Brown University, Providence, Rhode Island 02912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The N-type Ca channel alpha 1B subunit is localized to synapses throughout the nervous system and couples excitation to release of neurotransmitters. In a previous study, two functionally distinct variants of the alpha 1B subunit were identified, rnalpha 1B-b and rnalpha 1B-d, that differ at two loci;four amino acids [SerPheMetGly (SFMG)] in IIIS3-S4 and two amino acids [GluThr (ET)] in IVS3-S4. These variants are reciprocally expressed in rat brain and sympathetic ganglia (Lin et al., 1997a). We now show that the slower activation kinetics of rnalpha 1B-b (Delta SFMG/+ET) compared with rnalpha 1B-d (+SFMG/Delta ET) channels are fully accounted for by the insertion of ET in IVS3-S4 and not by the lack of SFMG in IIIS3-S4. We also show that the inactivation kinetics of these two variants are indistinguishable. Through genomic analysis we identify a six-base cassette exon that encodes the ET site and with ribonuclease protection assays demonstrate that the expression of this mini-exon is essentially restricted to alpha 1B RNAs of peripheral neurons. We also show evidence for regulated alternative splicing of a six-base exon encoding NP in the IVS3-S4 linker of the closely related alpha 1A gene and establish that residues NP can functionally substitute for ET in domain IVS3-S4 of alpha 1B. The selective expression of functionally distinct Ca channel splice variants of alpha 1B and alpha 1A subunits in different regions of the nervous system adds a new dimension of diversity to voltage-dependent Ca signaling in neurons that may be important for optimizing action potential-dependent transmitter release at different synapses.

Key words: N-type calcium channel; regulated alternative splicing; S3-S4 linker; genomic analysis; P/Q-type calcium channel; calcium channel alpha 1 subunits


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

N-type Ca channels, together with P/Q-type channels, control calcium-dependent neurotransmitter release at the majority of synapses in the mammalian nervous system (Hirning et al., 1988; Turner et al., 1992; Takahashi and Momiyama, 1993; Olivera et al., 1994; Wheeler et al., 1994; Dunlap et al., 1995). Neuronal Ca channels are also recognized as important targets for the treatment of chronic pain and neuronal degeneration after ischemic brain injury (Miljanich and Ramachandran, 1995). As the functional core of N-type Ca channels, the alpha 1B subunit contains domains critical for voltage-sensing, ion permeation (Dubel et al., 1992; Williams et al., 1992; Fujita et al., 1993), toxin binding (Ellinor et al., 1994), excitation-secretion coupling (Sheng et al., 1994), and second messenger-mediated modulation (Zamponi and Snutch, 1998). The alpha 1B subunit of the N-type Ca channel is subject to alternative splicing (Williams et al., 1992; Coppola et al., 1994; Lin et al., 1997a), but information regarding the functional significance of these splicing events is limited. We recently reported that variants of the alpha 1B-subunit that differ by four amino acids, SFMG (SerPheMetGly), in domain IIIS3-S4 and two amino acids, ET (GluThr), in domain IVS3-S4 (see Fig. 1) are differentially expressed in rat brain and sympathetic ganglia (Lin et al., 1997a). The sympathetic ganglia-dominant form of the Ca channel alpha 1B-subunit (rnalpha 1B-b, Delta /+; see Figs. 1, 2) activates 1.5-fold slower and at potentials 7 mV more depolarized relative to the brain-dominant form (rnalpha 1B-d, +/Delta ; see Figs. 1, 2) (Lin et al., 1997a). The relative functional impact of each of the spliced sequences (SFMG and ET) on the channel has not been determined. Furthermore, the genomic structures of these splice sites have not been characterized. It is also unclear whether alternative splicing in the functionally important S3-S4 linker region of alpha 1B is regulated similarly in all regions of the rat brain. In this study we examine these issues and also show that the functional differences between rnalpha 1B-b and rnalpha 1B-d channels may under certain conditions impact the magnitude of action potential-induced calcium transients as revealed in a model neuron. Variants of the closely related Ca channel alpha 1A subunit, which differ in the expression of two amino acids (NP) also in domain IVS3-S4, have been isolated (Yu et al., 1992; Zamponi et al., 1996; Ligon et al., 1998; Sutton et al., 1998; Hans et al., 1999). Recent studies show that these alpha 1A NP variants differ in their gating kinetics and sensitivity to block by omega -Aga IVA (Sutton et al., 1998; Hans et al., 1999), leading to the proposal that they account for P- and Q-type Ca channels described in mammalian neurons (Wheeler et al., 1994; Sutton et al., 1998). Little is known, however, about the relative abundance of alpha 1A splice variants of the NP locus in different regions of the nervous system. We begin to examine this issue by the use of the ribonuclease protection assay and establish that the NP exon in alpha 1A is also differentially expressed in different regions of the nervous system.

Preliminary reports of these findings have been presented previously in abstract form (Lin et al., 1997b; Schorge et al., 1998).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Functional assessment of the Ca channel alpha 1B cDNA constructs

The functional properties of all Ca channel alpha 1B cDNA constructs described in this paper were assessed in the Xenopus oocyte expression system. All methods and procedures were essentially the same as described in Lin et al. (1997a). Robust N-type Ca channel currents were expressed in Xenopus oocytes after injection of alpha 1B cRNA without coexpression of exogenous Ca channel beta -subunit. Heterologous expression of the Ca channel beta 3 subunit along with alpha 1B increased N-type Ca channel current expression levels but did not affect channel gating kinetics nor did the presence of beta 3 affect the relative differences in the time course and voltage dependence of activation of the alpha 1B splice variants (Lin et al., 1997a). Xenopus oocytes do express, however, an endogenous Ca channel beta -subunit (beta 3XO) (Tareilus et al., 1997) that is highly homologous to the mammalian beta 3. It is possible that the Xenopus beta 3XO associates with and modulates heterologously expressed alpha 1B in the oocyte. This may explain why coexpression of heterologous beta 3 is not required for functional expression of N-type Ca channels in this system (Tareilus et al., 1997). cRNAs were in vitro transcribed using the mMESSAGE mMACHINE kit (Ambion) from the various alpha 1B cDNA constructs subcloned into the Xenopus beta -globin expression vector (pBSTA) (Goldin and Sumikawa et al., 1992). A cRNA solution (46 nl of 750 ng/µl) was injected into defolliculated oocytes using a precision nanoinjector (Drummond). N-type Ca channel currents were recorded 6-7 d after injection. At least 15 min before recording, oocytes were injected with 46 nl of a 50 mM solution of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA). This we have found critical to minimize activation of an endogenous Ca-activated Cl- current, even when Ba2+ is the charge carrier (Lin et al., 1997a). Cells exhibiting slowly deactivating tail currents, indicative of the presence of Ba2+-dependent activation of the Ca-activated Cl- current, were excluded from the analyses.

N-type Ca channel currents were recorded from oocytes using the two-microelectrode voltage-clamp recording technique (Warner amplifier; OC-725b). Micropipettes of 0.8-1.5 and 0.3-0.5 MOmega resistance when filled with 3 M KCl were used for the voltage and current recording electrodes, respectively. Oocytes expressing Ca channel currents usually had resting membrane potentials between -40 and -50 mV when impaled with two electrodes. A grounded metal shield was placed between the two electrodes to increase the settling time of the clamp. Recording solutions contained 5 mM BaCl2, 85 mM tetraethylammonium, 5 mM KCl, and 5 mM HEPES, pH adjusted to 7.4 with methanesulfonic acid. The recording temperature was between 19° and 22°C.

The properties of each mutant construct were assessed by expressing it together with appropriate controls (Delta ET alpha 1B and +ET alpha 1B). Each mutant was tested in three separate batches of oocytes, and within each batch recordings were made from at least six oocytes for each mutant construct and control. Recordings from the oocytes expressing the various Ca channel alpha 1B constructs were randomized throughout the data collection period.

Data analysis. Data were acquired on-line and leak-subtracted using a P/4 protocol (PClamp V6.0; Axon Instruments). Voltage steps were applied every 10-30 sec depending on the duration of the step, from a holding potential of -80 mV. Ca channel currents recorded under these conditions showed little run-down over the duration of the recordings. Three sets of current-voltage relationships were obtained from each cell using step depolarizations of 26.3 msec, 650 msec, and 2.6 sec in duration and digitized at 25 kHz, 10 kHz, and 250 Hz, respectively. Exponential curves (activation and inactivation) were fit to the data using curve-fitting routines in PClamp (Axon Instruments) and Origin (Microcal). Inactivation time constants in the range of 70-800 msec were estimated from currents evoked by the longest depolarizations (2.6 sec). Activation time constants were best resolved from currents evoked by the shortest depolarizations (26.3 msec; sampled at 25 kHz).

Ribonuclease protection assay

The procedures are essentially the same as those described in Lin et al. (1997a). Total RNA was purified from various neuronal tissues of adult rats using a guanidium thiocyanate and phenol-chloroform extraction protocol [adapted from Chomczynski and Sacchi (1987)]. 32P-labeled antisense RNA probes overlapping ET [nucleotide (nt) 4379-4836] in rnalpha 1B-b and NP (nt 4605-4930) in rbalpha 1A (Starr et al., 1991) were constructed from linearized plasmids (pGEM-T vector) containing appropriate RT-PCR-derived subclones using the Maxi-script kit (Ambion). Probes were gel-purified and stored as ethanol precipitates. RNA (1 µg) purified from sympathetic or sensory ganglia or RNA (5 µg) isolated from various CNS tissues was precipitated with 2 × 105 cpm of probe and resuspended in 30 µl hybridization buffer containing 60% formamide, 0.4 M NaCl, 10 mM EDTA, and 40 mM PIPES at pH 6.4. Samples were denatured at 85°C and allowed to hybridize overnight at 60°C. The samples were then digested in a 350 µl reaction mix containing 0.3 M NaCl, 5 mM EDTA, 3.5 µl of the RNase mixture (Ambion), and 10 mM Tris at pH 7.5, then treated with proteinase K, extracted, and precipitated with 10 µg of tRNA as carrier. After resuspension in 30 µl formamide loading buffer, the samples were denatured and separated on a 5% polyacrylamide gel. After exposure to a phosphorimaging plate to quantify relative band intensities (Fuji BAS 1000), the gel was subsequently exposed to film with an intensifying screen for 4-5 d at -80°C.

Site-directed mutagenesis

A recombinant PCR-based technique was used to introduce mutations (QT, EA, AT, AA, NP) at the ET site in the IVS3-S4 linker of alpha 1B-b. A pair of primers 5'-attcttgtggtcatcgccttgag (Bup 3460) and 5'-gacaggcctccaggagcttggtg (Bdw 5623) flanked a region of the clone that contained two restriction sites, RsrII (nt 3510) and BglII (nt 5465), located either side of ET (nt 4674). A second primer pair contained the desired mutation and directly overlapped the ET site (Bdwmut and Bupmut; see below). Two separate PCRs were performed with Bup 3460 and Bdwmut, and Bupmut and Bdw 5623. The PCR product then served as template for a second round of PCR using Bup 3460 and Bdw 5623, generating the final mutant PCR fragment that was subsequently subcloned into rnalpha 1B-b at the Rsr II and BglII sites. Mutants were screened by restriction digest and confirmed by DNA sequencing. All PCR was performed using Expand High Fidelity (Boehringer Mannheim, Indianapolis, IN). The mutagenesis primers used were as follows: ET/AT: Bupmut 5'-gagattgcgGCAACGaacaacttcatc-3'; Bdwmut 5'-aagttgttCGTTTCcgcaatctccg-3'; ET/QT: Bupmut 5'-gagattgcgCAGACGaacaacttcatc-3'; Bdwmut 5'-aagttgttCGTCTGcgcaatctccg-3'; ET/EA: Bupmut 5'-gagattgcgGAAGCTaacaacttcatc-3'; Bdwmut 5'-aagttgttAGCTTCcgcaatctccg-3'; ET/AA: Bupmut 5'-gagattgcgGCAGCTaacaacttcatc-3'; Bdwmut 5'-aagttgttAGCTGCcgcaatctccg-3'; ET/NP: Bupmut 5'-gagattgcgAACCCTaacaacttcatc-3'; Bdwmut 5'-aagttgttAGGGTTcgcaatctccg-3'.

Genomic analysis

The IVS3-S4 region of the rat alpha 1B and alpha 1A genes were analyzed by genomic PCR. Primer pairs were directed to the IVS3 and IVS4 membrane-spanning regions that were presumed to reside in the 5' and 3' exons flanking the ET and NP insertions of the alpha 1B and alpha 1A genes, respectively. PCR was performed in a 50 µl reactions mix containing 250 ng rat liver genomic DNA, 250 µM each nucleotide, and 0.4 µM each primer. After a preincubation for 15 min at 92°C, 0.75 µl enzyme mix was added to start the amplification. The resultant gDNA products were gel-purified, cloned into pGEM-T (Promega), and sequenced. The alpha 1B primers generated two bands of ~11 kb and ~900 bases. The 11 kb band was derived from the alpha 1B gene and contained the desired ET encoding exon in IVS3-S4. The 900 base product resulted from amplification of the equivalent site in the alpha 1E gene that contained a relatively short ~700 bp intron and no intervening exon. The alpha 1A primers generated a single 9 kb PCR product that was confirmed to be derived from the alpha 1A gene by DNA sequencing (Yale University sequencing facility). Primers were as follows: alpha 1A: Aup4737 5'-tgcctggaacatcttcgactttgtga; Adw4876 5'-cagaggagaatgcggatggtgtaacc; and alpha 1B: Bup4599 5'-cagagatgcctggaacgtctttgac; Bdw4744 5'-ataacaagatgcggatggtgtagcc.

Modeling Ca entry

A one-compartment cell model using standard compartmental modeling techniques in NEURON (Hines and Carnevale, 1997) was used to predict the amount of Ca entering a neuron expressing either rnalpha 1B-b or rnalpha 1B-d N-type Ca channel currents. The cell had a total membrane area of 1250 µm2, 0.75 µF/cm2 specific membrane capacitance, and 30 kOmega cm2 specific membrane resistance. For action potential simulation a fast sodium conductance (gNa) and a delayed rectifying potassium conductance (gK,DR) were included (Mainen and Sejnowski, 1995), each with densities of 300 pS/µm2. Ca2+ influx was mediated by a fast calcium conductance (gCa) (Yamada et al., 1989) with a density of 1 pS/µm2. Action potentials were evoked by a 5 msec, 400 pA current step. Resultant currents were calculated using conventional Hodgkin-Huxley kinetic schemes according to the formulae given below. The resting membrane potential was set at -70 mV, and Na and K current reversal potentials were set at +50 mV and -75 mV, respectively. The calcium current was computed using the Goldman-Hodgkin-Katz equation. Extracellular Ca concentration was 2.5 mM, and the intracellular Ca concentration was computed using entry via ICa and removal via a first order pump d[Ca2+]i/dt = (-1 · 105 · ICa/2F) - ([Ca2+]i - [Ca2+]infinity )/tau R, where [Ca2+]infinity  = 100 nM and tau R = 80 msec. The simulation does not incorporate mobile or immobile Ca buffers. The time constants and maximal Ca channel conductances used in this model were measured at room temperature. These values were adjusted for simulation at 37°C based on Q10 values of 1.5 and 3.0 for the conductance and activation kinetics, respectively (Cota et al., 1983). Formulae used for calculation of various currents were as follows: sodium current (INa), m3 · h: alpha m, Na = 0.182 · (v + 25)/(1 - e-(v+25)/9); beta m, Na = -0.124 · (v + 25)/(1 - e(v+25)/9); alpha h, Na = 0.024 · (v + 40)/(1 - e-(v+40)/5); beta h, Na = -0.0091 · (v + 65)/ (1 - e(v+65)/5); hinfinity , Na = 1/(1 + e(v+55)/6.2); delayed recifier (IK(DR)), m: alpha m, K(DR) = 0.02 · (v - 25)/(1 - e-(v+25)/9); beta m, K(DR) = -0.002 · (v - 25)/(1 - e(v+25)/9); high threshold, N-type rnalpha 1B-b calcium current (Ica), m · h: minfinity , Ca = 1/(1 + e-(v-3)/8); tau m, Ca = 7.8/(e(v+6)/16 + e-(v+6)/16); hCa = K/(K + [Ca2+]i) with K = 0.01 mM. The brain-dominant form, rnalpha 1B-d, was modeled by shifting the voltage dependence of the N-type Ca channel conductance activation variable (minfinity , Ca) by -7 mV and decreasing the activation time constant by 1.5-fold (Lin et al., 1997a) (see Fig. 2A).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alternative splicing in the putative S3-S4 extracellular linkers affects channel activation but not inactivation kinetics

In a previous study we showed that rnalpha 1B-b (Delta SFMG/+ET) and rnalpha 1B-d (+SFMG/Delta ET) N-type currents differ with respect to their gating kinetics when expressed in Xenopus oocytes [compare Delta /+ and +/Delta in Fig. 2A,B; see also Lin et al. (1997a)]. We reported apparent differences in both the macroscopic rates of channel activation and the inactivation between the two splice variants, but because relatively short duration depolarizations were used, the inactivation kinetics were not fully resolved. Consequently, and as discussed in Lin et al. (1997a), it was difficult to ascribe the differences in gating kinetics between the N-type Ca channel alpha 1B splice variants exclusively to differences in either channel activation or inactivation mechanisms. In the present study we have used both short (26 msec) and long (2.6 sec) depolarizations to resolve the time course of Ca channel activation and inactivation. rnalpha 1B-b (Delta /+) (Fig. 1) and rnalpha 1B-d (+/Delta ) (Fig. 1) subunits were expressed in Xenopus oocytes, and the resulting N-type Ca channel currents were recorded using 5 mM Ba as the charge carrier (Figs. 2, 3). N-type Ca channel currents evoked by depolarizations to 0 mV or higher inactivated with a bi-exponential time course (tau fast 100-150 msec and tau slow 700-800 msec) (Fig. 3A). The inactivation time constants of the cloned channels expressed in Xenopus oocytes (rnalpha 1B-b, Delta /+ and rnalpha 1B-d, +/Delta ) (Fig. 3A,B) were weakly voltage dependent, consistent with studies of native N-type Ca channels of bullfrog sympathetic neurons (Jones and Marks, 1989). Figure 3A,B shows that the fast and slow inactivation time constants of rnalpha 1B-b and rnalpha 1B-d currents evoked by relatively long duration step depolarizations to between 0 mV and +30 mV were not significantly different. In contrast, the rates of channel activation of the two variants in the same cells were significantly different (Fig. 2A,B). On the basis of these observations we conclude that alternative splicing in domains IIIS3-S4 and IVS3-S4 of the alpha 1B subunit alters the time course of N-type Ca channel activation but has no direct effect on inactivation kinetics. The apparent differences in the time courses of inactivation previously reported for rnalpha 1B-b and rnalpha 1B-d using relatively short duration depolarizing pulses (Lin et al., 1997a) can thus be ascribed to differences in their rates of channel activation, not inactivation. A selective effect on channel activation kinetics is consistent with the close proximity of the spliced sites, S3-S4 linkers, to their respective S4 helices that are the putative voltage sensors of the six transmembrane family of voltage-gated ion channels (Hille, 1992). In contrast, the domains of the Ca channel alpha 1B subunit implicated in voltage-dependent inactivation of N-type Ca channels (IS6 and flanking putative extracellular and intracellular linkers) (Zhang et al., 1994) are likely to be more distant from the S3-S4 linker splice sites.



View larger version (55K):
[in this window]
[in a new window]
 
Figure 1.   The location of two alternatively spliced sequences in the S3-S4 extracellular linkers of domains III and IV of the Ca channel alpha 1B subunit. Top, Putative membrane topology of the Ca channel alpha 1B subunit and location of two alternatively spliced sequences encoding SerPheMetGly (SFMG) and GluThr (ET) in the S3-S4 linkers of domains III and IV. Bottom, Four alpha 1B clones differing in the presence of SFMG and ET encoding sequences (Delta /+, +/Delta , +/+, Delta /Delta ) used to evaluate their relative effects on channel gating kinetics (Fig. 2).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 2.   The presence of ET in domain IVS3-S4 of alpha 1B slows the rate of N-type Ca channel activation. A, B, Ca channel alpha 1B subunits that differ in the expression of ET in the IVS3-S4 linker activate at different rates. A, Averaged, normalized Ca channel current induced by the expression in Xenopus oocytes of four different alpha 1B constructs (see Fig. 1). Currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. Each trace represents the average, normalized current calculated from at least six oocytes. SFMG-containing clones are distinguished from SFMG-lacking clones by thin and thick lines, respectively. B, Plot of average activation time constants (ln tau activ) at different test potentials (between -20 and +10 mV) for clones +/+ (), Delta /+ (), +/Delta (open circle ), and Delta /Delta (black-square). The presence of SFMG in domain IIIS3-S4 did not affect the rate of channel activation. There was no significant difference in tau activ between clones +/+ and Delta /+ or between clones +/Delta and Delta /Delta (p > 0.1 at all test potentials between -20 mV and +10 mV). The presence of ET in domain IVS3-S4 slowed channel activation kinetics. tau activ values for clones +/+ and Delta /+ were significantly slower compared with +/Delta and Delta /Delta , at all test potentials between -20 mV and +10 mV (p < 0.05).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 3.   Alternative splicing in the S3-S4 linkers of domains III and IV of alpha 1B does not affect channel inactivation kinetics. A, B, The rate of N-type Ca channel inactivation is not affected by the presence of SFMG or ET in the S3-S4 linkers of domains III and IV. A, Ca channel currents recorded from oocytes expressing rnalpha 1B-b (Delta SFMG/+ET, thick line) and rnalpha 1B-d (+SFMG/Delta ET, thin line). Currents were evoked by 2.6 sec depolarizations to 0 mV from a holding potential of -80 mV. Each trace represents the average, normalized current calculated from at least six oocytes. B, Plot of the fast and slow inactivation time constants of currents at different test potentials. There is no significant difference between the fast and slow inactivation time constants calculated for rnalpha 1B-b () and rnalpha 1B-d (open circle ) at test potentials between 0 mV and +30 mV (p > 0.1). Each point is the average value ± SE (n > 5).

Splicing of ET in domain IVS3-S4 underlies the major functional difference between rnalpha 1B-b and rnalpha 1B-d

rnalpha 1B-b and rnalpha 1B-d differ in composition by six amino acids located in two distinct regions of the Ca channel alpha 1B subunit (SFMG in domain IIIS3-S4 and ET in domain IVS3-S4) (Fig. 1). To separate the relative contribution of SFMG in domain IIIS3-S4 and ET in domain IVS3-S4 to the different gating kinetics observed between rnalpha 1B-b (Delta SFMG/+ET) and rnalpha 1B-d (+SFMG/Delta ET) we constructed two additional clones, +/+ and Delta /Delta (Fig. 1) and compared the functional properties of all four clones. Figure 2A,B demonstrates that the presence of the dipeptide sequence ET in domain IVS3-S4 is directly correlated with the altered activation kinetics of rnalpha 1B-b currents compared with rnalpha 1B-d. Activation time constants measured from N-type Ca channel currents in oocytes expressing clones Delta /+ (rnalpha 1B-b) and +/+ were indistinguishable and 1.5-fold slower on average than those induced by the expression of clones +/Delta (rnalpha 1B-d) and Delta /Delta (Fig. 2A,B). The presence of ET in domain IVS3-S4 also influenced the voltage-dependence of channel activation. A comparison of the midpoints of the rising phase of the peak current-voltage plots (V1/2) generated for the two ET containing clones Delta /+ (rnalpha 1B-b; -7.8 ± 0.6 mV, n = 6) and +/+ (-9.7 ± 1.0 mV, n = 6) shows that they are not significantly different from each other (p > 0.05, Student's t test). Likewise, V1/2 values estimated from two ET-lacking constructs, +/Delta (rnalpha 1B-d; -15.4 ± 0.4 mV, n = 7) and Delta /Delta (-13.4 ± 0.7, n = 6), were not significantly different from each other (p > 0.05) and activated at potentials that were, on average, 6 mV more negative compared with ET-containing clones Delta /+ and +/+ (data not shown). Although the presence of ET in domain IVS3-S4 dominates in regulating the voltage dependence of activation, the analysis does reveal a small contribution of SFMG. SFMG-containing clones (+/Delta and +/+) activated at potentials that were 2 mV hyperpolarized compared with those that lacked SFMG (Delta /+ and Delta /Delta ). A 2 mV shift in the voltage dependence of activation was not significant at the 5% level, in a comparison of V1/2 values from clones Delta /+ and +/+, but did reach significance in a comparison of +/Delta and Delta /Delta (p < 0.025, Student's t test).

The pattern of expression of ET-containing Ca channel alpha 1B mRNA in different regions of the nervous system

Figure 2 indicates that alternative splicing of ET within domain IVS3-S4 of the Ca channel alpha 1B subunit accounts for the major functional differences between rnalpha 1B-b and rnalpha 1B-d. This prompted us to systematically analyze the expression pattern of the six bases in alpha 1B mRNA that encoded ET. We had shown previously that ET-containing alpha 1B (+ET alpha 1B) mRNA was in very low abundance in total rat brain extracts (Lin et al., 1997a). To determine whether ET-lacking alpha 1B (Delta ET alpha 1B) mRNA dominated throughout the CNS we used the ribonuclease protection assay and analyzed RNA isolated from spinal cord, cerebellum, cortex, hippocampus, hypothalamus, medulla, and thalamus of adult rats (Fig. 4). In all regions tested >90% of the alpha 1B mRNA expressed in the CNS lacked the ET encoding sequence. In contrast, in sympathetic and sensory ganglia the majority of alpha 1B mRNA contained the ET encoding sequence (Fig. 4). Together these findings suggest that +ET alpha 1B subunits are primarily restricted to neurons of the peripheral nervous system. Consistent with this we have analyzed RNA isolated from human brain and trigeminal ganglia and observed analogous patterns of expression: low levels of +ETalpha 1B mRNA in brain (~10%) and high levels (~70%) in ganglia (S. Schorge and D. Lipscombe, unpublished data).



View larger version (71K):
[in this window]
[in a new window]
 
Figure 4.   The expression pattern of ETalpha 1B splice variants in various regions of the rat nervous system. RNase protection analysis of alpha 1B RNA isolated from dorsal root ganglia (DRG), superior cervical ganglia (SCG), and various brain regions of adult rats using a complimentary probe that extends from nt 4379 to nt 4836 and contains the ET insert at nt 4675. Top, Gel separation of RNase digested 32P-labeled probe hybridized to the various RNA samples. The strong signal corresponding to fully protected probe (+ET, 463 bases) in the DRG and SCG lanes indicates a predominance of +ETalpha 1B RNA. The two shorter bands (-ET, 296 and 161 bases) most prominent in the CNS lanes correspond to cleaved probe, indicating high levels of ET-lacking alpha 1B RNA. Bottom, Average levels of +ETalpha 1B RNA calculated from at least three separate experiments. In all regions of the brain tested, >90% of the alpha 1B RNA lacked the ET encoding sequence, whereas in ganglia ~80% alpha 1B RNA contained the ET sequence. RNA products were separated on a 5% acrylamide gel, and relative band intensities were calculated using a phosphorimager.

Site-directed mutagenesis within IVS3-S4

Having shown that alternative splicing of the ET encoding sequence in the IVS3-S4 linker of alpha 1B has a significant effect on the kinetics and voltage dependence of N-type Ca channel gating, we next used site-directed mutagenesis to determine the relative importance of each amino acid, glutamate and threonine. A series of mutants in which ET was replaced with either QT, AT, EA, AA, or NP were constructed (Fig. 5) from clone Delta /+ (rnalpha 1B-b), which served as the background structure. The mutant constructs were then expressed in Xenopus oocytes, and their properties were compared with clones +ET (100% slow; Fig. 5) and Delta ET (100% fast; Fig. 5). All mutants expressed equally well in the Xenopus oocyte expression system.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 5.   Functional analysis of site-directed mutagenesis of the ET splice site in domain IVS3-S4 of the alpha 1B subunit. Activation time constants were estimated from currents induced by the expression of the various mutant alpha 1B constructs (QT, AT, EA, AA, NP) in oocytes and compared with clones ET and Delta ET (A). Shifts in the activation time constants of the mutant channels, relative to clones ET (100% slow) and Delta ET (100% fast) are plotted (B). Each point represents data collected from at least 18 oocytes per mutant (each mutant was tested in three separate batches of oocytes, and within each experiment at least six oocytes per mutant were analyzed). Values plotted are means ± SEs from the three data sets. The asterisk indicates a significant slowing of the activation time constant compared with clone ET (p < 0.05).

The role of the glutamate in domain IVS3-S4 was of major interest because it should be negatively charged at neutral pH and consequently might influence the gating machinery of the channel via electrostatic interactions. Figure 5, however, shows that replacing glutamate with glutamine resulted in a channel that activated only slightly faster than +ET alpha 1B (Fig. 5, QT). Substituting alanine for glutamate (AT) decreased tau act, but consistent with the QT mutant, suggests that the presence of a negative charge in IVS3-S4 (glu) does not underlie the slow gating kinetics of the +ETalpha 1B variant. Similarly, alanine substitution of either threonine alone (EA) or together with glutamate (AA) generated channels with activation kinetics that were intermediate between +ET alpha 1B and Delta ET alpha 1B clones. Together, these results suggest that the presence of both glutamate and threonine in the IVS3-S4 linker is necessary to reconstitute the relatively slow channel opening rates characteristic of N-type Ca channel alpha 1B-subunits that dominate in sensory and sympathetic ganglia.

Evidence for alternative splicing in the IVS3-S4 linker regions of the alpha 1B and alpha 1A genes

The existence of an alternatively spliced exon in the IVS3-S4 region of the rat Ca channel alpha 1B gene has been hypothesized (Lin et al., 1997a) but not yet confirmed. Genomic analysis was therefore undertaken to locate the splice junctions in the IVS3-S4 region of the alpha 1B gene and to pinpoint the precise location of the putative six-base, ET-encoding exon. PCR amplification from rat genomic DNA using primers designed to hybridize to the transmembrane spanning S3 and S4 helices flanking IVS3-S4 in alpha 1B revealed the presence of a long ~10 kb stretch of intron sequence. DNA sequencing established the location of exon/intron and intron/exon boundaries and conserved ag-gt splice junction signature sequences immediately 5' and 3' to the putative ET insertion site (Fig. 6A, alpha 1B). A six-base cassette exon encoding ET was located 8 kb into the 5' intron and establishes that ET-alpha 1B variants are generated by alternative splicing. Sequence comparisons of several cDNAs encoding alpha 1 subunits of other voltage-gated Ca channels suggests that alternative splicing in the IVS3-S4 linker could be a general mechanism for regulating voltage-dependent Ca channel gating (Fig. 6B). This has recently been demonstrated for alpha 1A (Sutton et al., 1998; Hans et al., 1999), a Ca channel subunit that is closely related both structurally and functionally to the N-type Ca channel alpha 1B-subunit. A comparison of the IVS3-S4 region of various mammalian alpha 1A cDNAs derived from kidney, pancreas, and brain (Fig. 6B) (Yu et al., 1992; Ligon et al., 1998; Sutton et al., 1998; Hans et al., 1999) is consistent with alternative splicing of six bases encoding asp, pro (NP) in this region. The exon/intron structure in the IVS3-S4 linker region of the closely related rat alpha 1A gene was therefore also determined (Fig. 6A). The rat alpha 1A gene contained a long stretch of intron sequence (~8 kb) and ag-gt splice junctions at the 5' (gt) and 3' (at) ends of the intronic segment (Fig. 6A). We have not yet determined the precise location of the NP encoding cassette exon in the rat alpha 1A gene but conclude that it must reside within the 8 kb of intron sequence in the IVS3-S4 linker region. Tissue-specific alternative splicing of six-base cassette exons in the IVS3-S4 linkers of both alpha 1A and alpha 1B explains the presence of splice variants of these subunits in the mammalian brain and underscores the high level of conservation between these two functionally related genes. We have also analyzed the genomic structure of the more distantly related rat alpha 1E gene that encodes a pharmacologically and functionally distinct class of Ca channel (Soong et al., 1993). The alpha 1E gene contains a ~700 bp intron in the IVS3-S4 linker region and no obvious intervening exon (Fig. 6A). The absence of an alternatively spliced cassette exon in the IVS3-S4 linker region of the alpha 1E gene is consistent with RNase protection analysis of alpha 1E mRNA from rat brain, which revealed no evidence of sequence variations in this IVS3-S4 linker (data not shown). The high degree of sequence homology between alpha 1B and alpha 1A in the IVS3-S4 linker region (Fig. 6B) together with the finding that a six-base sequence is alternatively spliced at both of these sites (Fig. 6A) suggested that ET and NP share a common functional role. To test this hypothesis we studied the functional impact on N-type Ca channel currents of replacing ET in rnalpha 1B-b with NP. Figure 5 shows that the +NPalpha 1B mutant gives rise to N-type Ca channel currents in oocytes with gating kinetics indistinguishable from wild type (i.e., +ETalpha 1B).



View larger version (37K):
[in this window]
[in a new window]
 
Figure 6.   Alternative splicing in the IVS3-S4 linker region of different Ca channel alpha 1 genes. A, An alignment of amino acid sequences IVS3-S4 linkers of six different Ca channel alpha 1 subunits derived from rat tissue. Shaded regions indicate probable sites of alternative splicing based on the identification of alpha 1 cDNAs variants in the IVS3-S4 region for all but the alpha 1E subunit (alpha 1A: Starr et al., 1991; Yu et al., 1992; Zamponi et al., 1996; Ligon et al., 1998; Sutton et al., 1998; Hans et al., 1999; alpha 1B: Dubel et al., 1992; Williams et al., 1992; Lin et al., 1997a; alpha 1C: Perez-Reyes et al., 1990; Snutch et al., 1991; Barry et al., 1995; alpha 1D: Barry et al., 1995; Ihara et al., 1995; alpha 1E: Soong et al., 1993; alpha 1S: Perez-Reyes et al., 1990; Barry et al., 1995). The location of a splice junction identified in the IVS3-S4 region of the alpha 1E gene is indicated (arrow, see below). B, A comparison of the rat Ca channel alpha 1A, alpha 1B, and alpha 1E genes in the region of the IVS3-S4 linker splice junction. Relevant exon (upper case) and intron (lower case) sequences are shown together with the splice junction consensus sequences ag-gt (underlined). The six base exon encoding ET resides ~8 kb into the 5' intron of the IVS3-S4 region of the alpha 1B gene (upper case, shaded). The putative exon encoding NP in the alpha 1A gene is displayed (*), but its precise location not yet determined. The absence of an intervening exon in the alpha 1E intron is denoted by a continuous dotted line. GenBank accession numbers AF146632, AF146633, AF146634.

+NPalpha 1A and Delta NPalpha 1A mRNAs are expressed in different regions of the rat nervous system

Although the presence of variants of the Ca channel alpha 1A that differ in the expression of the NP site has been reported (Yu et al., 1992; Ligon et al., 1998; Sutton et al., 1998; Hans et al., 1999), the distribution of +NPalpha 1A and Delta NPalpha 1A mRNAs in different regions of the rat nervous system have not been quantified. We therefore used the RNase protection analysis to determine the expression pattern of the IVS3-S4 splice variants of alpha 1A (Fig. 7). Low levels of +NP alpha 1A mRNA were found in rat, spinal cord, striatum, and thalamus, a pattern that parallels the low levels of +ET alpha 1B mRNA in the CNS (Fig. 4). However, the pattern of NP expression in the cerebellum, cortex, and hippocampus did not conform to this picture because mRNA isolated from these tissues contained a significant proportion of +NPalpha 1A mRNAs. In fact, in the hippocampus +NPalpha 1A mRNAs dominated (~60%). Consistent with the abundance of +ETalpha 1B mRNAs in peripheral tissue, the majority of alpha 1AmRNA in superior cervical and dorsal root ganglia contained the six bases encoding NP in domain IVS3-S4 of alpha 1A. The absolute level of alpha 1A mRNA expressed in sympathetic neurons was very low as expected from the absence of P-type currents in recordings from rat sympathetic neurons (Mintz et al., 1992).



View larger version (90K):
[in this window]
[in a new window]
 
Figure 7.   The expression pattern of NPalpha 1A splice variants in various regions of the rat nervous system. RNase protection analysis of alpha 1A RNA isolated from dorsal root ganglia (DRG), superior cervical ganglia (SCG), and various regions of the brain of adult rats using a probe complimentary to the region of alpha 1A from nt 4605 to nt 4930 and containing the NP insertion at nt 4805. Top, Gel separation of RNase-digested 32P-labeled probe hybridized to the various RNA samples. The predominance of fully protected probe (331 nt) in DRG indicates an abundance of +NPalpha 1A RNA, and the presence of two shorter bands (-NP, 200 nt and 125 nt, corresponding to cleaved probe) most prominent in the spinal cord, striatum, and thalamus preps after digestion, indicates that most of alpha 1A RNA in these tissues lacks the NP site. The histogram shows the average levels of +NP containing alpha 1A RNA in at least three experiments. In ganglionic tissue ~80% of the alpha 1A RNA pool contained the NP site. The weak signal in SCG reflects low expression levels of the alpha 1A gene in this tissue. Cerebellum, cortex, and hippocampal RNA contained a mix of +NPalpha 1A and Delta NPalpha 1A RNAs, and in spinal cord, striatum, and thalamic tissue Delta NPalpha 1A RNAs dominated. RNA products were separated on a 5% acrylamide gel, and relative band intensities were calculated using a phosphorimager.

The differences in the properties of rnalpha 1B-b (Delta SFMG/+ET) and rnalpha 1B-d (+SFMG/Delta ET) currents may influence action potential-induced Ca entry

We have demonstrated functional differences between splice variants of the N-type Ca channel alpha 1B subunit and shown that they are differentially expressed in peripheral and central neurons. We do not as yet know whether these functional differences are sufficient to influence action potential-dependent Ca entry in neurons. As a first step toward addressing this question we have used standard modeling techniques to predict whether rnalpha 1B-b (Delta SFMG,+ET) and rnalpha 1B-d (+SFMG, Delta ET) N-type currents can, under certain conditions, differentially affect action potential-induced Ca influx in a model neuron. A more direct comparison of the effectiveness of the two Ca channel splice variants for supporting action potential-induced Ca entry in Xenopus oocytes is not feasible using conventional two-microelectrode voltage-clamp methods because of the limited temporal resolution associated with cells of this size. Simulated action potentials similar to those recorded in native sympathetic neurons (Yamada et al., 1989) (Fig. 8) were therefore used to trigger voltage-dependent Ca influx in model neurons (Na, K, and Ca current densities of 300, 300, and 1.0 pS/µm2, respectively) expressing either rnalpha 1B-b or rnalpha 1B-d N-type Ca channel currents. Ca channel current densities and peak intracellular Ca concentrations were modeled at room temperature (22°C) (Fig. 8A) and at 37°C (Fig. 8B). Under these conditions increases in both the total charge transfer and peak intracellular Ca concentration of 45% (at 22°C) and 35% (at 37°C) were observed after an action potential in a neuron expressing rnalpha 1B-d-type Ca channels (Fig. 8, dashed line) relative to rnalpha 1B-b (Fig. 8, solid line). The model also predicts a slightly faster rate of rise of the intracellular calcium signal (~1.3-fold) in a neuron expressing rnalpha 1B-d-type Ca channels compared with rnalpha 1B-b.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 8.   Predicting the impact of alternative splicing in the S3-S4 linkers of the alpha 1B subunit on action potential-dependent Ca influx in a model neuron. A one-compartment model was used to predict the time course and magnitude of calcium entry in a neuron during action potential-induced depolarization at 22°C (A) and 37°C (B). A simulated action potential evoked by a 5 msec, 400 pA current step (top) together with a comparison of the resultant N-type channel current (middle) and time course of intracellular calcium concentration (bottom) expected in a model neuron expressing either rnalpha 1B-b (Delta /+, solid line) or rnalpha 1B-d (+/Delta , dashed line)-type channels. A shift in the voltage-dependence of the N-type Ca channel conductance activation variable (minfinity , Ca) by -7 mV and a 1.5-fold decrease in the activation time constant expected for rnalpha 1B-d compared with rnalpha 1B-b [Lin et al. (1997a); and see our Fig. 2A] results in both an increase in total charge transfer and peak intracellular Ca concentration, respectively, of 46 and 44% at 22°C (A) and of 36 and 35% at 37°C (B). An accompanying increase in the rate of increase of the intracellular Ca concentration of 1.3-fold at 22° and 37°C was also observed. Channel gating parameters were obtained from measurements at room temperature and were modified for the 37°C simulation based on a Q10 of 2.3 for Na and K channels and a Q10 of 3.0 for the Ca channel (Cota et al., 1983). We assume that the relative differences in the gating properties of the alpha 1B splice variants observed at room temperature are maintained at 37°C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mammalian genes encoding voltage-gated Ca channel alpha 1 subunits are large and complex, containing approximately 50 exons, several of which are alternatively spliced (Soldatov, 1994; Yamada et al., 1995; Hogan et al., 1996; Ophoff et al., 1996). With few exceptions (Lin et al., 1997a,b; Sutton et al., 1998; Hans et al., 1999), studies that address the functional consequences of these splicing events have been limited to non-neuronal-derived L-type Ca channel subunits (Klockner et al., 1997; Soldatov et al., 1997; Welling et al., 1997; Zuhlke et al., 1998). Building on our previous studies (Lin et al., 1997a), we now focus on the importance of regulated alternative splicing in the IVS3-S4 linkers of the alpha 1B and closely related alpha 1A subunits.

Two amino acids in the IVS3-S4 extracellular linker of alpha 1B slow N-type Ca channel activation

The S3-S4 linkers of a number of voltage-gated and structurally related ion channels are important in determining the time course and voltage dependence of channel activation (Perez-Reyes et al., 1990; Nakai et al., 1994; Lin et al., 1997a; Mathur et al., 1997; Tang and Papazian, 1997; Hans et al., 1999). We now demonstrate that alternative splicing of just two amino acids, ET, in domain IVS3-S4 of the Ca channel alpha 1B subunit underlies the different gating kinetics of two previously identified variants, rnalpha 1B-b and rnalpha 1B-d (Lin et al., 1997a). We also demonstrate that a chemically dissimilar but functionally homologous dipeptide sequence NP (see below) can fully replace and substitute for ET in domain IVS3-S4 of alpha 1B, showing that the side chains of ET are not unique in contributing to the relatively slow activation kinetics of +ETalpha 1B. The importance of the IVS3-S4 linker in influencing activation of the Ca channel is supported by the high level of conservation of alternative splicing in this region of other alpha 1 subunits (Fig. 6) (Snutch et al., 1991; Barry et al., 1995; Ihara et al., 1995; Lin et al., 1997a; Ligon et al., 1998; Sutton et al., 1998; Hans et al., 1999). Moreover, in a recent study Hans et al. (1999) compared the functional properties of two variants of the Ca channel alpha 1A subunit that also differed by two amino acids (NP) in the IVS3-S4 linker and showed that the Delta NP variant of alpha 1A activated at a rate 1.7-fold faster compared with +NPalpha 1A. Remarkably, this difference corresponds precisely to that observed between Delta ET alpha 1B and +ET alpha 1B (1.5-fold) (Fig. 2). The similar functional consequences of splicing at the ET and NP loci, combined with the high degree of conservation between the IVS3-S4 splice junctions of the alpha 1B and alpha 1A genes, imply a common functional role. alpha 1A and alpha 1B subunits are both critically important for coupling excitation to transmitter release at the majority of synapses throughout the nervous system (Dunlap et al., 1995); thus one consequence of tissue-specific expression of functionally distinct IVS3-S4 splice variants of these proteins might be to optimize the release of neurotransmitters in different regions. +ETalpha 1B and +NPalpha 1A mRNAs that encode relatively slow activating channels (alpha 1B; Fig. 2) (alpha 1A; Hans et al., 1999) dominate in peripheral neurons (Figs. 4, 7), implying that excitation-secretion coupling might be less efficient at postganglionic synapses in comparison with many synapses in the CNS. +NPalpha 1A mRNAs, however, are not solely restricted to peripheral neurons (Fig. 7), implying that different regions within the CNS may contain functionally distinct alpha 1A-containing Ca channels. On the basis of their different omega -aga-IVA sensitivities, Sutton et al. (1988) have proposed that the expression of Delta NPalpha 1A and +NPalpha 1A splice variants correlates with the presence of high (P-type) and low (Q-type) affinity omega -aga-IVA-sensitive Ca channels (Sather et al., 1993; Stea et al., 1994; Wheeler et al., 1994; Randall and Tsien, 1995). Additional studies are needed to test this hypothesis, but our ribonuclease protection analysis (Fig. 7) suggests a reasonable, although not perfect, correlation between the presence of Delta NPalpha 1A mRNA and P-type channels on the one hand and +NPalpha 1A mRNA and Q-type on the other. For example, in DRG neurons the majority of the alpha 1A mRNA pool contains the NP exon (Fig. 7), and in these neurons