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The Journal of Neuroscience, July 1, 1999, 19(13):5322-5331
Alternative Splicing of a Short Cassette Exon in
 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
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ABSTRACT |
The N-type Ca channel  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 1B subunit were identified,
rn1B-b and rn1B-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
rn1B-b ( SFMG/+ET) compared with rn1B-d
(+SFMG/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
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 1A gene and
establish that residues NP can functionally substitute for ET in domain IVS3-S4 of 1B. The selective expression of functionally
distinct Ca channel splice variants of 1B and
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 1 subunits
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INTRODUCTION |
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 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
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 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 1B-subunit
(rn1B-b, /+; see Figs. 1, 2) activates
1.5-fold slower and at potentials 7 mV more depolarized relative to the
brain-dominant form (rn1B-d, +/; 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 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 rn1B-b and rn1B-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 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 1A NP variants differ in their gating kinetics and sensitivity to block by  -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 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 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).
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MATERIALS AND METHODS |
Functional assessment of the Ca channel 1B
cDNA constructs
The functional properties of all Ca channel 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 1B cRNA without coexpression of
exogenous Ca channel  -subunit. Heterologous expression of the Ca
channel 3 subunit along with 1B increased
N-type Ca channel current expression levels but did not affect channel
gating kinetics nor did the presence of 3 affect the
relative differences in the time course and voltage dependence of
activation of the 1B splice variants (Lin et al., 1997a). Xenopus oocytes do express, however, an endogenous
Ca channel -subunit (3XO) (Tareilus et al.,
1997) that is highly homologous to the mammalian 3. It
is possible that the Xenopus 3XO associates
with and modulates heterologously expressed 1B in the
oocyte. This may explain why coexpression of heterologous 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 1B cDNA constructs subcloned into the
Xenopus -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 M 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 (ET 1B and +ET 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 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 rn1B-b
and NP (nt 4605-4930) in rb1A (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
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 rn1B-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 1B and
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 1B and 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 1B primers generated two
bands of ~11 kb and ~900 bases. The 11 kb band was derived from the
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 1E gene that contained a
relatively short ~700 bp intron and no intervening exon. The
1A primers generated a single 9 kb PCR product that was
confirmed to be derived from the 1A gene by DNA
sequencing (Yale University sequencing facility). Primers were as
follows: 1A: Aup4737 5'-tgcctggaacatcttcgactttgtga;
Adw4876 5'-cagaggagaatgcggatggtgtaacc; and 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 rn1B-b or rn1B-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 kcm2 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+] )/ R,
where [Ca2+] = 100 nM and 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: m, Na = 0.182 · (v + 25)/(1 e(v+25)/9); m, Na = 0.124 · (v + 25)/(1 e(v+25)/9);
h, Na = 0.024 · (v + 40)/(1 e(v+40)/5); h, Na = 0.0091
· (v + 65)/ (1 e(v+65)/5); h,
Na = 1/(1 + e(v+55)/6.2); delayed
recifier (IK(DR)),
m: m, K(DR) = 0.02 · (v 25)/(1 e(v+25)/9); m,
K(DR) = 0.002 · (v 25)/(1 e(v+25)/9); high threshold, N-type
rn1B-b calcium current (Ica),
m · h: m, Ca = 1/(1 + e(v3)/8); 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, rn1B-d,
was modeled by shifting the voltage dependence of the N-type Ca channel
conductance activation variable (m, Ca) by 7 mV and
decreasing the activation time constant by 1.5-fold (Lin et al., 1997a)
(see Fig. 2A).
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RESULTS |
Alternative splicing in the putative S3-S4 extracellular linkers
affects channel activation but not inactivation kinetics
In a previous study we showed that rn1B-b
(SFMG/+ET) and rn1B-d (+SFMG/ET) N-type currents
differ with respect to their gating kinetics when expressed in
Xenopus oocytes [compare /+ and +/ 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 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. rn1B-b (/+) (Fig.
1) and rn1B-d (+/)
(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 (fast 100-150 msec and slow
700-800 msec) (Fig. 3A). The
inactivation time constants of the cloned channels expressed in
Xenopus oocytes (rn1B-b, /+ and
rn1B-d, +/) (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 rn1B-b and rn1B-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 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 rn1B-b and rn1B-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 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.
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Figure 1.
The location of two alternatively spliced
sequences in the S3-S4 extracellular linkers of domains III and IV of
the Ca channel 1B subunit. Top, Putative
membrane topology of the Ca channel 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
1B clones differing in the presence of SFMG and ET
encoding sequences (/+, +/, +/+, /) used to evaluate their
relative effects on channel gating kinetics (Fig. 2).
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Figure 2.
The presence of ET in domain IVS3-S4 of
1B slows the rate of N-type Ca channel activation.
A, B, Ca channel 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 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 activ) at different
test potentials (between 20 and +10 mV) for clones +/+ ( ), /+
( ), +/ ( ), and / ( ). The presence of SFMG in domain
IIIS3-S4 did not affect the rate of channel activation. There was no
significant difference in activ between clones +/+ and
/+ or between clones +/ and / (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.
activ values for clones +/+ and /+ were significantly
slower compared with +/ and /, at all test potentials between
20 mV and +10 mV (p < 0.05).
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Figure 3.
Alternative splicing in the S3-S4 linkers of
domains III and IV of 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 rn1B-b
(SFMG/+ET, thick line) and rn1B-d
(+SFMG/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 rn1B-b ()
and rn1B-d () at test potentials between 0 mV and +30
mV (p > 0.1). Each point is the average
value ± SE (n > 5).
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Splicing of ET in domain IVS3-S4 underlies the major functional
difference between rn1B-b and rn1B-d
rn1B-b and rn1B-d differ in
composition by six amino acids located in two distinct regions of the
Ca channel 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 rn1B-b (SFMG/+ET)
and rn1B-d (+SFMG/ET) we constructed two additional
clones, +/+ and / (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
rn1B-b currents compared with rn1B-d.
Activation time constants measured from N-type Ca channel currents in
oocytes expressing clones /+ (rn1B-b) and +/+
were indistinguishable and 1.5-fold slower on average than those
induced by the expression of clones +/
(rn1B-d) and / (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 /+ (rn1B-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, +/
(rn1B-d; 15.4 ± 0.4 mV,
n = 7) and / (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 /+ 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 (+/ and +/+) activated at potentials that
were 2 mV hyperpolarized compared with those that lacked SFMG (/+
and /). 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 /+ and +/+, but did
reach significance in a comparison of +/ and / (p < 0.025, Student's t test).
The pattern of expression of ET-containing Ca channel
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 1B subunit accounts for the
major functional differences between rn1B-b and
rn1B-d. This prompted us to systematically analyze the
expression pattern of the six bases in 1B mRNA that
encoded ET. We had shown previously that ET-containing
1B (+ET 1B) mRNA was in very low
abundance in total rat brain extracts (Lin et al., 1997a). To determine
whether ET-lacking 1B (ET 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 1B mRNA expressed in the CNS lacked
the ET encoding sequence. In contrast, in sympathetic and sensory
ganglia the majority of 1B mRNA contained the ET
encoding sequence (Fig. 4). Together these findings suggest that +ET
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 +ET1B mRNA in
brain (~10%) and high levels (~70%) in ganglia (S. Schorge and D. Lipscombe, unpublished data).
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Figure 4.
The expression pattern of ET1B
splice variants in various regions of the rat nervous system. RNase
protection analysis of 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 +ET1B 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
1B RNA. Bottom, Average levels of
+ET1B RNA calculated from at least three separate
experiments. In all regions of the brain tested, >90% of the
1B RNA lacked the ET encoding sequence, whereas in
ganglia ~80% 1B RNA contained the ET sequence. RNA
products were separated on a 5% acrylamide gel, and relative band
intensities were calculated using a phosphorimager.
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Site-directed mutagenesis within IVS3-S4
Having shown that alternative splicing of the ET encoding sequence
in the IVS3-S4 linker of 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 /+
(rn1B-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 ET (100% fast; Fig. 5). All
mutants expressed equally well in the Xenopus oocyte
expression system.
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Figure 5.
Functional analysis of site-directed mutagenesis
of the ET splice site in domain IVS3-S4 of the 1B
subunit. Activation time constants were estimated from currents induced
by the expression of the various mutant 1B constructs
(QT, AT, EA, AA, NP) in oocytes and compared with clones
ET and ET (A). Shifts in the activation time
constants of the mutant channels, relative to clones ET (100% slow)
and 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).
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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 1B (Fig. 5, QT). Substituting alanine for glutamate (AT) decreased 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 +ET1B variant. Similarly, alanine substitution of
either threonine alone (EA) or together with glutamate (AA) generated channels with activation kinetics that were intermediate between +ET
1B and ET 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
1B-subunits that dominate in sensory and sympathetic ganglia.
Evidence for alternative splicing in the IVS3-S4 linker regions of
the 1B and 1A genes
The existence of an alternatively spliced exon in the IVS3-S4
region of the rat Ca channel 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 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
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,
1B). A six-base cassette exon encoding ET was
located 8 kb into the 5' intron and establishes that
ET-1B variants are generated by alternative splicing.
Sequence comparisons of several cDNAs encoding 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
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 1B-subunit. A
comparison of the IVS3-S4 region of various mammalian
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
1A gene was therefore also determined (Fig.
6A). The rat 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 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
1A and 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 1E gene that encodes a
pharmacologically and functionally distinct class of Ca channel (Soong
et al., 1993). The 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 1E gene is
consistent with RNase protection analysis of 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 1B and 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
rn1B-b with NP. Figure 5 shows that the
+NP1B mutant gives rise to N-type Ca channel currents in
oocytes with gating kinetics indistinguishable from wild type (i.e.,
+ET1B).
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Figure 6.
Alternative splicing in the IVS3-S4 linker region
of different Ca channel 1 genes. A, An
alignment of amino acid sequences IVS3-S4 linkers of six different Ca
channel 1 subunits derived from rat tissue.
Shaded regions indicate probable sites of alternative
splicing based on the identification of 1 cDNAs variants
in the IVS3-S4 region for all but the 1E subunit
(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; 1B: Dubel et al., 1992; Williams et al., 1992; Lin
et al., 1997a; 1C: Perez-Reyes et al., 1990; Snutch et
al., 1991; Barry et al., 1995; 1D: Barry et al., 1995;
Ihara et al., 1995; 1E: Soong et al., 1993;
1S: Perez-Reyes et al., 1990; Barry et al., 1995). The
location of a splice junction identified in the IVS3-S4 region of the
1E gene is indicated (arrow, see
below). B, A comparison of the rat Ca channel
1A, 1B, and
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 1B gene (upper case,
shaded). The putative exon encoding NP in the 1A
gene is displayed (*), but its precise location not yet determined. The
absence of an intervening exon in the 1E intron is
denoted by a continuous dotted line. GenBank accession
numbers AF146632, AF146633, AF146634.
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+NP1A and NP1A mRNAs are expressed
in different regions of the rat nervous system
Although the presence of variants of the Ca channel
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 +NP1A and
NP1A 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 1A (Fig. 7).
Low levels of +NP 1A mRNA were found in rat, spinal
cord, striatum, and thalamus, a pattern that parallels the low levels of +ET 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 +NP1A mRNAs. In
fact, in the hippocampus +NP1A mRNAs dominated
(~60%). Consistent with the abundance of +ET1B mRNAs
in peripheral tissue, the majority of 1AmRNA in superior
cervical and dorsal root ganglia contained the six bases encoding NP in
domain IVS3-S4 of 1A. The absolute level of
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).
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Figure 7.
The expression pattern of NP1A
splice variants in various regions of the rat nervous system. RNase
protection analysis of 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 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 +NP1A 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 1A RNA in these
tissues lacks the NP site. The histogram shows the average levels of
+NP containing 1A RNA in at least three experiments. In
ganglionic tissue ~80% of the 1A RNA pool contained
the NP site. The weak signal in SCG reflects low expression levels of
the 1A gene in this tissue. Cerebellum, cortex, and
hippocampal RNA contained a mix of +NP1A and
NP1A RNAs, and in spinal cord, striatum, and thalamic
tissue NP1A RNAs dominated. RNA products were
separated on a 5% acrylamide gel, and relative band intensities were
calculated using a phosphorimager.
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The differences in the properties of rn1B-b
(SFMG/+ET) and rn1B-d (+SFMG/ET) currents may
influence action potential-induced Ca entry
We have demonstrated functional differences between splice
variants of the N-type Ca channel 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 rn1B-b
(SFMG,+ET) and rn1B-d (+SFMG, 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
rn1B-b or rn1B-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
rn1B-d-type Ca channels (Fig. 8, dashed line)
relative to rn1B-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
rn1B-d-type Ca channels compared with
rn1B-b.
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Figure 8.
Predicting the impact of alternative splicing in
the S3-S4 linkers of the 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 rn1B-b (/+,
solid line) or rn1B-d (+/,
dashed line)-type channels. A shift in the
voltage-dependence of the N-type Ca channel conductance activation
variable (m, Ca) by 7 mV and a 1.5-fold
decrease in the activation time constant expected for
rn1B-d compared with rn1B-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 1B splice variants observed
at room temperature are maintained at 37°C.
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DISCUSSION |
The mammalian genes encoding voltage-gated Ca channel
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 1B and closely related 1A subunits.
Two amino acids in the IVS3-S4 extracellular linker of
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 1B subunit underlies the different gating
kinetics of two previously identified variants, rn1B-b
and rn1B-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 1B, showing that the side chains of
ET are not unique in contributing to the relatively slow activation
kinetics of +ET1B. 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
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 1A subunit that also differed by two
amino acids (NP) in the IVS3-S4 linker and showed that the NP
variant of 1A activated at a rate 1.7-fold faster
compared with +NP1A. Remarkably, this difference
corresponds precisely to that observed between ET 1B
and +ET 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
1B and 1A genes, imply a common
functional role. 1A and 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. +ET1B and +NP1A mRNAs that encode relatively slow activating channels
(1B; Fig. 2) (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.
+NP1A mRNAs, however, are not solely restricted to peripheral neurons (Fig. 7), implying that different regions within the
CNS may contain functionally distinct 1A-containing Ca
channels. On the basis of their different -aga-IVA sensitivities,
Sutton et al. (1988) have proposed that the expression of
NP1A and +NP1A splice variants
correlates with the presence of high (P-type) and low (Q-type) affinity
-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 NP1A mRNA and P-type channels on the one hand and +NP1A mRNA
and Q-type on the other. For example, in DRG neurons the majority of
the 1A mRNA pool contains the NP exon (Fig. 7), and in
these neurons -aga-IVA inhibits the Ca current with relatively slow
kinetics (Mintz et al., 1992), not inconsistent with the presence of a Q-type current. In spinal cord, NP1A mRNA dominates
(Fig. 7), and block by -aga-IVA is relatively rapid (Mintz et al.,
1992) and thus not inconsistent with P-type currents. Furthermore, in the cerebellum and hippocampus, where significant levels of both NP1A and +NP1A mRNAs are found (Fig.
7), both P- and Q-type Ca channel currents have been described (Llinas
et al., 1989; Wheeler et al., 1994; Randall and Tsien, 1995). The
dominance of NP 1A mRNA in the thalamus (Fig. 7), on
the other hand, does not correlate well with the significant Q-type
current component reported in this region of the brain (Kammermeier and
Jones, 1997).
As yet we have no evidence that splice variants of the N-type Ca
channel 1B subunit differ in their pharmacological
properties. -conotoxin GVIA, the widely used high-affinity N-type Ca
channel blocker, inhibits both variants equally well (Z. Lin and D. Lipscombe, unpublished observations), consistent with studies of native
N-type Ca channels in peripheral and central neurons (Wang et al.,
1998). This result is not surprising, however, considering that the
IVS3-S4 extracellular linker is not the main site of -conotoxin
GVIA binding (Ellinor et al., 1994). Drugs or toxins that bind to the S3-S4 linkers of the Ca channel 1B subunit that
discriminate between S3-S4 splice variants may be identified in the
future. The S3-S4 linkers of several voltage-gated channel
1 subunits, including 1A, are
known to be important targets of toxin binding (Rogers et al., 1996;
Swartz and MacKinnon, 1997; Cestele et al., 1998; Sutton et al., 1998;
Hans et al., 1999). In the case of the voltage-gated Na channel subunit, a single amino acid (Gly) within the IIS3-S4 linker plays a
major role in -scorpion toxin binding (Cestele et al., 1998).
Predicting the differential effect of the different Ca channel
1B splice variants on action potential-induced calcium
entry
Our modeling provides a useful first step toward understanding how
differences in the gating kinetics and voltage dependence of activation
between the 1B splice variants might affect action potential-induced Ca entry. Under the specific condition of the model
used here, the CNS-dominant Ca channel variant ET1B
supports a larger and slightly faster rising intracellular calcium
signal in response to action potential-induced depolarization compared with +ET1B (Fig. 8). However, the significance of these
differences with respect to excitation-secretion coupling efficiency
at synapses that use the N-type Ca channel is difficult to predict.
Neurosecretion is thought to be triggered by intracellular calcium
levels in the range of 500 nM to a few micromoles
(Augustine and Neher, 1992; Heidelberger et al., 1994),
and differences in the rate of rise of the calcium transient may be
more important than differences in the absolute levels of calcium. Our
model, however, does not take into account how the presence of Ca
buffers might shape the Ca signal close to the Ca channel in the
microdomain where excitation-secretion coupling occurs (Naraghi and
Neher, 1997). Furthermore, many other factors such as (but not limited
to) Ca channel density, the shape of the presynaptic action potential,
and neurotransmitter and second messenger-mediated Ca and K channel
modulation will affect presynaptic calcium levels. For example, the
effectiveness of rn1B-d-type currents to couple
depolarization to Ca entry in a neuron is enhanced relative to
rn1B-b with relatively brief depolarizations, but
declines as the stimulus duration is increased. Furthermore, if the
N-type Ca channel current activates fully during the rising phase of
the action potential, then differences in the rate or voltage
dependence of Ca channel opening will have relatively insignificant
effects on Ca entry. In contrast, a slowing of Ca channel gating
kinetics, such as is observed during G-protein-mediated modulation,
would serve to amplify the different effectiveness of the two splice
variants to couple depolarization to Ca entry. The primary purpose of
the model presented here is to emphasize that although they are
small, the functional differences between the 1B
splice variants may, under certain conditions, significantly impact
action potential-induced Ca entry.
Regulated alternative splicing is an important mechanism used
throughout the nervous system for generating functionally distinct products from a single gene in different regions of the nervous system
and at different stages during development (Grabowski, 1998). The
continued characterization of alternatively spliced loci of Ca channel
1 subunits in neurons will likely lead to a greater
understanding of the scope of Ca channel diversity and the
physiological importance of the expression of splice variants in the
mammalian nervous system.
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FOOTNOTES |
Received Jan. 13, 1999; revised April 20, 1999; accepted April 22, 1999.
This work was supported by National Institutes of Health (NIH) grants
NS 29967 and NS 01927 (D.L.) and NIH Training Grant MH19118
(Z.L.). We thank Drs. Hans and colleagues for providing a copy
of their manuscript before publication.
Correspondence should be addressed to Dr. Diane Lipscombe, Department
of Neuroscience, Brown University, Box 1953, Providence, RI 02912.
Dr. Lin's current address: Cold Spring Harbor Laboratory, Beckman
Building, 1 Bungtown Road, Cold Spring Harbor, NY 11724.
| |
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ABSTRACT
INTRODUCTION
