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The Journal of Neuroscience, October 15, 2000, 20(20):7564-7570
C-Terminal Alternative Splicing Changes the Gating Properties of
a Human Spinal Cord Calcium Channel  1A Subunit
Howard S.
Krovetz,
Thomas D.
Helton,
Anne L.
Crews, and
William A.
Horne
Department of Anatomy, Physiological Sciences, and Radiology, North
Carolina State University College of Veterinary Medicine, Raleigh,
North Carolina 27606
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ABSTRACT |
The calcium channel  1A subunit gene codes for
proteins with diverse structure and function. This diversity may be
important for fine tuning neurotransmitter release at central and
peripheral synapses. The 1A C terminus, which serves a
critical role in processing information from intracellular signaling
molecules, is capable of undergoing extensive alternative splicing. The
purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of 1A subunits. Specifically, the biophysical
properties of two alternatively spliced 1A subunits were
compared. One variant was identical to an isoform identified previously
in human brain, and the other was a novel isoform isolated from human
spinal cord. The variants differed by two amino acids (NP) in the
extracellular linker between transmembrane segments IVS3 and IVS4 and
in two C-terminal regions encoded by exons 37 and 44. Expression in
Xenopus oocytes demonstrated that the two variants were
similar with respect to current-voltage relationships and the voltage
dependence of steady-state activation and inactivation. However, the
rates of activation, inactivation, deactivation, and recovery from
inactivation were all significantly slower for the spinal cord variant.
A chimeric strategy demonstrated that the inclusion of the sequence
encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can
influence the way in which 1A subunits respond to a
depolarizing stimulus and add to the developing picture of the C
terminus as a critical domain in the regulation of
Ca2+ channel function.
Key words:
spinal cord; cerebellum; calcium channel; 1A subunit; voltage clamp; alternative splicing; C terminus
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INTRODUCTION |
Multiple types of
high-voltage-activated Ca2+ channels (L,
N, P, Q, and R) coordinate a variety of
Ca2+-dependent processes, including gene
expression, signal propagation, and neurotransmitter release (Tsien et
al., 1991 ; Zhang et al., 1993; Dunlap et al., 1995). These channels can
be distinguished by their biophysical and pharmacological properties.
L- and R-type channels, found on cell bodies and proximal dendrites,
regulate gene transcription and signal propagation (Westenbroek et al., 1990; Murphy et al., 1991; Yokoyama et al., 1995), whereas synaptic N-,
P-, and Q-type channels regulate neurotransmitter release (Turner et
al., 1992; Takahashi and Momiyama, 1993; Wheeler et al., 1994). The
Ca2+ channel complex consists of four
subunits, 1, 2/ ,
 , and  . With the exception of P- and Q-type channels, the
different neuronal Ca2+ channel phenotypes
arise primarily from the expression of five unique
1 subunit genes (Tsien et al., 1991; Jun et
al., 1999). These genes (A-E) are differentially distributed
throughout brain and spinal cord (Murphy et al., 1991; Takahashi and
Momiyama, 1993; Westenbroek et al., 1998) and encode large proteins
consisting of four homologous domains (I-IV) containing six
transmembrane segments each (S1-S6) (Tsien et al., 1991; Zhang et al.,
1993).
Alternative splicing of the 1A gene results in
the expression of multiple Ca2+ channel
phenotypes (Sutton et al., 1998; Bourinet et al., 1999; Hans et al.,
1999; Jun et al., 1999). Splicing of two amino acids (NP) in the
1A IVS3-IVS4 linker affects rates of
activation and inactivation, the voltage dependence of
inactivation, and the affinity of 1A for
 -Aga IVA (Sutton et al., 1998; Bourinet et al., 1999; Hans et al.,
1999; Lin et al., 1999). The biophysical and pharmacological properties
of 1A Ca2+
channels are also influenced by intracellular signaling molecules. Coexpression of 1A with different subunit
subtypes alters inactivation rates and -Aga IVA affinity (Moreno et
al., 1997). Generally, 1A subunit function is
inhibited by interactions with G-proteins or syntaxin-1A (Zhang et al.,
1996; Qin et al., 1997; Sutton et al., 1999) and enhanced by protein
kinase C (Zamponi et al., 1997; Bourinet et al., 1999).
Calcium-activated calmodulin has dual effects on
1A subunit function (Lee et al., 1999).
Determination of genomic exon-intron boundaries (Ophoff et al., 1996)
and isolation of several 1A C-terminal splice
variants (Zhuchenko et al., 1997) suggest that there are four exons
between IVS3-IVS4 and the C-terminal stop codon that undergo
alternative splicing. This implies that 16 combinations of these
four exons are possible. Eight combinations have been isolated
previously from human brain (Ophoff et al., 1996; Zhuchenko et al.,
1997; Hans et al., 1999). In this study, a ninth is isolated from human spinal cord. Biophysical characterization of this
1A variant reveals that its rates of
activation, inactivation, deactivation, and recovery from inactivation
are all significantly slower than that of an
1A variant from cerebellum. The rate of
inactivation was especially affected by sequence encoded by a single
exon. These findings suggest that variation in
1A C-terminal structure provides an added
mechanism for functional diversity among neuronal Ca2+ channel subtypes.
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MATERIALS AND METHODS |
Human spinal cord library screening. Calcium channel
subunit cDNAs were isolated from an oligo-dT and random-primed human spinal cord  gt11 5' stretch cDNA library (Clontech, Palo Alto, CA)
by the use of a nonradioactive digoxigenin-labeling and colorimetric detection system (Roche Molecular Biochemicals, Indianapolis, IN). The
library was constructed from mRNA isolated from whole spinal cords
pooled from 26 male or female Caucasians, ages 16-75, who died of
sudden death syndrome. The insert size range of the library was
0.8-7.0 kb (average size of 1.7 kb). Plaque-purified phage DNAs were
isolated using a Lambda Prep Kit (Qiagen, Santa Clara, CA) and digested
with the restriction endonuclease EcoRI (Roche Molecular
Biochemicals, supplier of all endonucleases used). Southern blot
analysis was used to assess insert size. cDNA isolates were ligated
into pBluescriptII (Stratagene, La Jolla, CA) for sequencing. The
inserts were subjected to exonuclease III/SI nuclease digestion
(Erase-A-Base; Promega, Madison, WI) before sequencing. PCR-based cycle sequencing (FS chemistry; PE Biosystems, Foster City,
CA) with universal primers and custom internal primers (Genosys, The
Woodlands, TX) was used for each clone. Sequence was obtained using an
ABI Prism 310 Genetic DNA analyzer, and data were analyzed using ABI
Prism DNA Sequencing Software (version 2.12; PE Biosystems). Sequence
comparisons, alignments, and restriction maps were performed using
Lasergene Software (DNAStar, Madison, WI). 5' RACE (Roche Molecular
Biochemicals) was used with human spinal cord
poly(A+) RNA (Clontech) to create a 5'
probe for the 1A screening. This RNA came from
a pool of 92 male or female Caucasians, ages 16-75, who died from
sudden death syndrome.
The library-screening process was initiated with a cDNA probe
(1-EST) obtained from the NCBI
dbEST database (1.8 kb human fetal brain
1A fragment; GenBank number H14053). Clone
1-9, extending from nucleotide 3416 to the
3'-untranslated region, was the longest of 24 3' cDNAs isolated in the
first rounds of screening. Five 1 cDNAs were
isolated in a second round, in which a 1 kb 5' EcoRI
fragment of clone 1-9 was used as the probe. The longest clone isolated in this round, extending from nucleotides 2399 to 4551, was subsequently labeled and used as a probe to identify
1-38 (which contained an
EcoRI/EcoRI fragment from 1800 to 4551). To
isolate the 5' portion of the 1A sequence, a
probe containing nucleotides 946 to 1800 was created by reverse
transcription-PCR with human spinal cord RNA. Library screening with
this probe yielded 14 cDNAs, one of which, clone
1-80, extended from the 5'-untranslated region
to a region beyond the EcoRI site at nucleotide 1800.
Clone construction and sequencing. Full-length spinal cord
1A cDNAs were assembled in several steps.
Initially, PCR was performed using PFU Polymerase (Stratagene)
and custom primers (Genosys) to truncate the 5'-untranslated region of
1-80 and to insert an idealized Kozak (Kozak,
1991) sequence into the shortened 5' end of the construct. The
resulting 1.1 kb fragment was then ligated into the pT-Adv vector
(Clontech) and sequenced to ensure that there were no polymerase
errors. This PCR product was cut with the restriction endonuclease
NotI, and the resulting fragment was ligated into
EcoRV/NotI-prepared pBluescriptII. The
untruncated 1-80 was cut with NotI,
and the resulting 900 bp fragment was then ligated into the shortened
5' clone by the use of NotI. The resulting clone (referred
to as 5'short) spans from the shortened 5'-untranslated sequence to the
EcoRI site at nucleotide1800.
In the next step, a 3' cDNA extending from nucleotide 3826 to the
3'-untranslated region of the clone, 1-10, was
cut with NotI to eliminate the EcoRI site located
in the polylinker of pBluescriptII. The construct was then cut with
SacI, blunted using the Klenow fragment (Roche Molecular
Biochemicals), and cut with EcoRI to yield a 3.5 kb fragment
spanning from the EcoRI site at 4551 to the 3'-untranslated
region. This fragment was ligated into
EcoRI/SmaI-prepared pBluescriptII, removed with
EcoRI/BamHI, and ligated into the
EcoRI/BamHI-cut vector containing 5'short. Thus,
the resulting construct (5'short + 3') extended from the shortened
5'-untranslated region to the EcoRI site at 1800 and continued from the EcoRI site at 4551 to the 3'-untranslated
region. In the final step, an EcoRI fragment of
1-38 extending from 1801 to 4550 was cloned
into EcoRI-cut 5'short + 3'. The fully constructed clone
1A-C1 was then sequenced to ensure the
fidelity of the construction process. Exchanging the C-terminal domain
of 1A-C1 with that of
1-EST and 1-9 created
1A-C2 and 1A-C16,
respectively. Thus, both 1A-C2 and
1A-C16 are constructed of the same cDNAs up to
nucleotide 4550 but then contain segments of different cDNAs beyond
this point.
The rabbit Ch1a and
2a/ clones used in this study were provided
by T. Tanabe (Tokyo Medical and Dental University, Tokyo, Japan). The
Ch1b, Ch3, and
Ch4 subunits were cloned from the same human
spinal cord library described above and are nearly identical to
previously reported sequences (Ch1b, GenBank
number M923303; Ch3, GenBank number U07139;
and Ch4, GenBank number U95020).
Electrophysiology and data analysis. Standard Xenopus
laevis oocyte expression methods were used to characterize the
1A splice variants. Briefly, full-length
1A subunit cDNA was in vitro
transcribed (Ambion, Austin, TX). The resulting cRNA was injected into
defolliculated X. laevis oocytes (stage V-VI) along with
equimolar ratios of rabbit 2a/ and
Ch cRNAs (0.40 µg/ml 1A; 0.16 µg/ml
2a/; 0.08 µg/ml Ch in a total of 46 nl). Ca2+ channel currents were recorded
by standard two-microelectrode voltage-clamp techniques using a Warner
amplifier (OC-725B) at room temperature (20-22°C), and data were
collected using pCLAMP6 software (Axon Instruments, Foster City, CA).
The bath solution contained the following: 40 mM
Ba(OH)2, 40 mM TEA-OH, 2 mM KOH, and 5 mM HEPES,
with pH adjusted to 7.4 with methanesulphonic acid. Microelectrodes
were filled with 3 M KCl, and the resistances of
the current and voltage electrodes were 0.3-1.5 M . Data were filtered at 2 kHz and sampled at 10 kHz. Currents were recorded 6-18 d
after injection. The holding potential of all experiments was  100 mV
unless otherwise noted. Control oocytes (1A
alone or uninjected oocytes) did not yield currents >50 nA. Because of
lower expression levels when Ch subunits other than the rabbit Ch1a were used, currents as low as 150 nA were
included in the initial portion of the study. Only currents >0.5 µA
were analyzed for the remainder of this work. To diminish the
contamination caused by the Ca2+-activated
Cl channels, currents that exhibited
slow deactivation ( > 10 msec) were excluded from analysis.
The leak current and capacitive current transients were subtracted
on-line by a standard P/4 protocol. Data were analyzed using pCLAMP6
and Excel 7.0 (Microsoft, Redmond, WA). For statistical analysis, a
one-way ANOVA test was used followed by a Fisher's PLSD test using
Statview software (SAS Institute, Cary, NC).
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RESULTS |
Two 1A C-terminal splice variants,
1A-C2 and 1A-C16, are
the focus of this study (Fig.
1A). These variants
were assembled from a parent spinal cord construct,
1A-C1, and thus are identical up to homology
domain IV. Sequencing of the 1A-C1 cDNA
through domain IV revealed only three differences when compared with an 1A cDNA isolated from human cerebellum
(GenBank number AF004883). A deletion ( G419) identified previously
in rat brain was found in the region encoding the intracellular linker
between homology domains I and II (Bourinet et al., 1999). A mutation
resulting in a charge change (M537R) was found in the region encoding
transmembrane segment IIS2. A nine nucleotide deletion resulting in the
loss of amino acids 726-728 (VEA) was found in the region encoding the
intracellular linker between homology domains II and III. This deletion
has been identified in another 1A variant from human brain (GenBank number U79666). The spinal cord
1A-C1 cDNA did not contain a six nucleotide
insertion that codes for the amino acid sequence NP in the
extracellular linker between IVS3 and IVS4.
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Figure 1.
Alternative splicing of 1A domain
IV and C-terminal exons. A, Alternatively spliced exon
patterns of 1A-C2 (middle bar) and
1A-C16 (bottom bar) cDNAs relative to a
consensus protein that is a composite of known exons (top
bar). This figure is drawn on the basis of our data and that of
others (Ophoff et al., 1996; Zhuchenko et al., 1997; Hans et al.,
1999). Note that the NP sequence is located in the extracellular loop
between transmembrane domains IVS3 and IVS4. B,
The amino acid sequences of exons 37a, 37b, and 44. Regions of
variation are highlighted in black. Charged amino acids
are denoted by an asterisk.
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The 1-EST and 1-9
C-terminal cDNAs used to construct 1A-C2 and
1A-C16, respectively, differed from the C terminus of
1A-C1 and other 1A
clones in sequences that could be traced to specific exon-intron
boundaries (Ophoff et al., 1996). The 1-EST
cDNA did not extend to domain IV, and therefore
1A-C2, like 1A-C1, lacks NP (Fig. 1A). The 1-9
cDNA contained the NP exon, and this region is therefore present in
1A-C16. The 1-EST
cDNA contained one version of exon 37 (37a), whereas
1-9 contained a second (37b). There are nine
amino acid changes in this region including one charge change (Fig.
1B, L1851K). The 1-EST cDNA
contained 12 amino acids encoded by exon 44, whereas
1-9 did not. Interestingly, five of these
amino acids are positively charged (Fig. 1B). A 3'
GGCAG insert in exon 46 that would extend the open reading frame to
exon 47, although present in 1A-C1 (data not
shown), was not found in either 1-EST or
1-9 (Fig. 1A). Thus, neither 1A-C2 nor 1A-C16
contains the ~240 amino acid domain encoded by exon 47. The end
result is that 1A-C2 is identical to the human
cerebellar clone described above, and 1A-C16
represents a novel spinal cord clone containing a unique arrangement of
NP and C-terminal exons.
To determine the functional consequences of C-terminal splicing, both
1A-C2 and 1A-C16 were
coexpressed with rabbit 2a/ and a rabbit
Ch1a or a human Ch1b,
Ch3, or Ch4 subunit
in Xenopus oocytes. Representative current traces
demonstrate that 1A-C2 inactivates more
rapidly than does 1A-C16 when associated with
Ch1a, Ch1b, or
Ch3 subunits (Fig.
2A-C; see below). When coexpressed with the Ch4 subunit, the
1A-C2 complex appears to inactivate at
approximately the same rate as 1A-C16 (Fig. 2D) although the low expression levels of this
complex make interpretation difficult. As seen in Figure
2A, the deactivation rate of the 1A-C2 variant was slightly faster than that of
1A-C16 [deactivation (1A-C2) = 5.6 ± 0.3 msec at 80 mV
(n = 7); deactivation
(1A-C16) = 6.6 ± 0.3 msec at 80 mV
(n = 6)]. When expressed with each of the Ch
subunits, the current-voltage relationships of
1A-C2 and 1A-C16 were
essentially indistinguishable (Fig. 2). With all Ch subunits, both
1A variants had peak inward currents at +15 to
+20 mV. Additionally, the voltages at which 1% of the maximal current
was obtained were nearly identical
(Ch1a/1A-C2 = 19.2 ± 4.4 mV, whereas
Ch1a/1A-C16 = 19.5 ± 5.0 mV; this measurement was not obtained with the other
Ch subunits because of poor expression levels). Coexpression of the
rabbit Ch1a subunit yielded currents two to
five times higher than that with any of the human Ch subunits (Fig.
2). The goal of this study was to determine the effect of C-terminal
1A splicing on the function of the channel,
independent of Ch binding to the region. Therefore, the remainder of
the study was performed with the Ch1a subunit
because it does not appear to bind to this region of the
1A subunit and has the most robust expression
levels (Walker et al., 1998).
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Figure 2.
Biophysical properties of the 1A-C2
and 1A-C16 subunits coexpressed with rabbit
2a/ and Ch1a,
Ch1b, Ch3, or
Ch4 subunits. A-D, Representative
current traces of 1A-C2
(top) and 1A-C16
(middle) between 40 and +40 mV with the
Ch1a (A), Ch1b
(B), Ch3
(C), or Ch4
(D) subunit. Normalized current-voltage
relationships (bottom) of 1A-C2
(open diamonds) and 1A-C16
(filled diamonds) with the Ch1a
(n = 14 and 20, respectively),
Ch1b (n = 21 and 14, respectively),
Ch3 (n = 10 and 10, respectively),
or Ch4 (n = 26 and 22, respectively)
subunit.
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The voltage dependency of activation, as determined from tail current
measurements, was similar between 1A-C2 and
1A-C16 [Fig.
3A, V1/2
(1A-C2) = 9.5 ± 1.4 mV;
V1/2 (1A-C16) = 7.3 ± 1.1 mV], as was the voltage dependency of steady-state
inactivation (Fig. 3B, V1/2
(1A-C2) = 37.9 ± 1.4 mV;
V1/2 (1A-C16) = 34.3 ± 1.5 mV]. The time constants of activation and
inactivation were described with a single exponential (Fig.
3C,D). The time constants of activation of the
1A-C16 variant, however, were much slower than
that of 1A-C2 and varied with membrane
potential (Fig. 3E). The values of activation of
1A-C16 ranged from 6.15 ± 0.40 msec at 0 mV to 1.24 ± 0.06 msec at +30 mV, whereas those of
1A-C2 ranged from 2.07 ± 0.51 msec at 0 mV to 0.75 ± 0.12 msec at +30 mV. The time constants of
inactivation of the 1A-C16 variant were also
slower than that of 1A-C2 (Fig.
3F). The values of inactivation of 1A-C2 ranged from 347 ± 69 msec at 10 mV to 175 ± 11 msec at +50 mV, whereas those of
1A-C16 ranged from 984 ± 325 msec at
10 mV to 338 ± 46 msec at +50 mV. Analysis of the correlation
between peak current and the of inactivation demonstrates that slow
activation of the ClCa current did not contaminate the apparent rates of inactivation (r = 0.304).
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Figure 3.
Voltage dependencies and rates of inactivation and
activation of 1A-C2 and 1A-C16 subunit
complexes. A, Voltage dependence of activation of
1A-C2 and 1A-C16 as measured from tail
current measurements. Inset, Tail currents that were
measured after the capacitive current (dashed vertical
line) at 0 mV after a 20 msec depolarization to the test
potential (40 to +75 mV in 5 mV increments). Tail currents were
normalized to the largest tail current in each series of test pulses.
These data were fit with a Boltzmann equation: % I = 1/[1 + exp((Vtest V1/2)/k)]. B, Isochronal
inactivation of 1A-C2 and 1A-C16. A 20 sec conditioning pulse ranged from 100 to +40 mV in 10 mV increments.
The conditioning pulse was followed by a test pulse to +20 mV for 300 msec. Data were fit with a Boltzmann equation: % I = 1/[1 + exp((Vtest V1/2)/k)]. C,
Single-exponential fits of activation of 1A-C2 and
1A-C16 at +20 mV. Fits are shown as solid black
lines. D, Single-exponential fits of
inactivation of 1A-C2 and 1A-C16 at +20
mV. Fits are shown as solid black lines.
E, Average activation of
1A-C2 and 1A-C16 between the voltages 0 and +30 mV. Traces were fit with a single exponential
from the onset of the inward current to the time of peak current. For
this and all following figures, the asterisks denote
statistical significance (p < 0.01) by the
use of an ANOVA test. F, Average
inactivation between 10 and +50 mV for
1A-C2 and 1A-C16. Each
point represents a minimum of six recordings. The SEM
for each point is shown unless the values were smaller
than the symbol. Traces were fit with a
single exponential from the peak inward current to the end of the
depolarization.
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Because 1A subunits are expressed primarily at
synapses and high-frequency trains of action potentials govern
neurotransmitter release, recovery from inactivation was characterized
as a means of predicting how 1A-C2 versus
1A-C16 might respond to repetitive stimulation. Interestingly, at 40 and 60 mV, the rates of recovery from inactivation of 1A-C2 and
1A-C16 are not significantly different (Fig.
4A,D). However, at more
negative potentials, the 1A-C2 subunit
recovers from inactivation more rapidly than does the
1A-C16 subunit (Fig. 4B-D).
A comparison of the fits of these recovery rates with a
single-exponential function reveals that the differences in recovery
rates become more apparent at increasingly negative potentials (Fig.
4D), showing that the rate of recovery from
inactivation of the 1A-C2 subunit is more
voltage dependent than that of 1A-C16.
Although this difference is greater at potentials not likely to be seen
physiologically (100 to 120 mV), the recovery rate is still
significantly different between 70 and 90 mV: at 70 mV,
recovery 1A-C2 = 398 ± 19 msec, and
recovery 1A-C16 = 469 ± 23 msec; at
80 mV, recovery 1A-C2 = 234 ± 11 msec, and recovery
1A-C16 = 321 ± 19 msec; and at 90 mV,
recovery 1A-C2 = 178 ± 6 msec, and recovery 1A-C16 = 251 ± 15 msec. The voltage dependence of the recovery from
inactivation is shifted by ~10 mV in the physiological range (Fig.
4D).
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Figure 4.
Voltage dependency of recovery from inactivation
of the 1A-C2 and 1A-C16 subunit
complexes. A-C, Average recoveries of
1A-C2 and 1A-C16 at 60 mV
(A), 80 mV (B), and 100
mV (C). A two-pulse protocol was used with an
initial test pulse of 600 msec to +30 mV followed by a conditioning
pulse (ranging from 40 to 120 mV) of intervals ranging from 20 to
1100 msec. This was followed by a second 200 msec test pulse to +30 mV,
I2. The percentage recovery was measured as
% Recovery = (I2 Iend of pulse
I1)/(I1 Iend of pulse I1). Each point
shown is the average of six to nine different recordings. The SEM for
each point is shown unless the values were smaller than
the symbol. The solid lines are the
single-exponential fits of the % Recovery. D, Voltage
dependency of the rate of recovery from inactivation of
1A-C2 and 1A-C16. recovery
is the average value derived from single-exponential fits of individual
experiments. The asterisks (p < 0.01) and cross (p < 0.05) denote statistical significance by the use of an ANOVA
test.
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Because the most obvious structural difference between
1A-C2 and 1A-C16 is
the presence of the highly charged 12 amino acid segment encoded by
exon 44 (Fig. 1A), a chimera was constructed to
characterize the influence of this segment on channel gating. The
chimera 1A-C14 is identical to
1A-C16 except that it contains the 12 amino
acids encoded by exon 44. As expected, the voltage dependencies of
activation and steady-state inactivation of
1A-C14 were similar to those of
1A-C16 [V1/2activation
(1A-C14) = 6.7 ± 1.3 mV;
V1/2inactivation (1A-C14) = 33.0 ± 1.1 mV]. The differences between the rates of activation (Fig.
5C) and deactivation [at 80
mV, deactivation
(1A-C14) = 6.3 ± 0.4 msec] and the
rates of recovery from inactivation (at 100 mV,
recovery = 216 ± 19 msec; at 80 mV,
recovery = 316 ± 31 msec; at 60 mV,
recovery = 520 ± 63 msec) were not
statistically significant. The most striking difference between the two
variants was the enhancement of the rate of inactivation of
1A-C14 relative to that of 1A-C16 (Fig.
5A,B,D). Moreover, these rates were similar to those of 1A-C2, ranging from 198 ± 14 msec at 0 mV to 133 ± 16 msec at +50 mV.
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Figure 5.
Exon 44 enhances the rate of inactivation.
A, Current traces of
1A-C14, a clone that contains the 12 amino acids
encoded by exon 44, between 50 and +40 mV. B, Current
traces of 1A-C16, a clone that
does not contain exon 44, between 50 and +40 mV. C,
Average activation of 1A-C14 and
1A-C16 between 0 and +30 mV. D, Average
inactivation between 0 and +50 mV for
1A-C14 and 1A-C16. Each
point represents a minimum of five recordings. The SEM
for each point is shown unless the values were smaller
than the symbol. Traces were fit with a
single exponential from the peak inward current to the end of the
depolarization.
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To determine whether the effects of the amino acid segment encoded by
exon 44 on the rate of inactivation were background dependent, this
exon was removed from 1A-C2 to create
1A-C4. Thus, 1A-C4 is
identical to 1A-C2 except that it lacks the 12 amino acids encoded by exon 44. The effect of the removal of exon 44 was indeed to slow the rate of inactivation (Fig.
6A,B,D) without significantly affecting the voltage dependency of activation
[V1/2 (1A-C4) = 7.7 ± 1.0 mV] or inactivation [V1/2
(1A-C4) = 31.4 ± 1.2 mV].
Interestingly, this construct had a slower rate of activation (Fig.
6A-C).
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Figure 6.
In a different C-terminal combination, the exon 44 slows the rate of activation but still enhances the rate of
inactivation. A, Current traces of
1A-C2, a clone that contains the 12 amino acids
encoded by exon 44, between 50 and +40 mV. B, Current
traces of 1A-C4, a clone that does
not contain exon 44, between 50 and +40 mV. C, Average
activation of 1A-C2 and
1A-C4 between 0 and +30 mV. D, Average
inactivation between 0 and +50 mV for
1A-C2 and 1A-C4. Each
point represents a minimum of five recordings. The SEM
for each point is shown unless the values were smaller
than the symbol. Traces were fit with a
single exponential from the peak inward current to the end of the
depolarization.
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DISCUSSION |
The gating properties of neuronal
Ca2+ channels are determined by multiple
structural domains within the 1 subunit (Tsien
et al., 1991; Catterall, 1995). Our results indicate that C-terminus alternative splicing can influence some of these properties. The C
terminus of the 1A subunit is encoded by 12 exons, numbered 36-47 (Ophoff et al., 1996), of which 4 exons, 37, 44, 46, and 47, undergo alternative splicing (Zhuchenko et al., 1997).
Depending on the pattern of splicing, the C terminus may vary from 436 to 517 amino acids. Binding sites for several intracellular signaling molecules, which affect channel gating, have been identified within this sequence. An EF-hand Ca2+-binding
motif can be aligned to sequence encoded by exons 36 and 37 (de Leon et
al., 1995). Calmodulin has been shown to bind to a region of the C
terminus encoded by exon 40 (Peterson et al., 1999) and to a
calmodulin-binding domain (CBD) identified in the region encoded by
exon 42 (Lee et al., 1999). Calcium-dependent binding of calmodulin to
the CBD motif speeds inactivation and recovery from inactivation and
produces a long-lasting facilitation of
Ca2+ current (Lee et al., 1999). Calcium
channel 4 subunit binding to a region encoded
by exons 43-47 has been shown to enhance the rate of inactivation
(Walker et al., 1998). In addition, the C terminus appears to be
essential for modulation by
G  -proteins (Zhang et
al., 1996), particularly those regions encoded by exons 45 and 46 (Qin
et al., 1997). G-proteins modulate the kinetics of channel activation
and inactivation, the voltage dependence of activation, and
recovery from inactivation (Bean, 1989; Patil et al., 1998; Zamponi
and Snutch, 1998). Polyglutamine tract expansions in exon 47 (Zhuchenko et al., 1997) shift the voltage dependence of activation to
more negative potentials, dramatically increase current density
(Piedras-Rentería et al., 1999), and alter activation and inactivation kinetics (Resituito et al., 1999). These latter effects appear to be dependent on coexpression of the
4 subunit.
This study focuses on two naturally occurring splice variants of the
1A subunit gene that differ in nucleotide
sequences corresponding to the NP exon of the IVS3-IVS4 extracellular
linker and C-terminal exons 37 and 44. One variant,
1A-C2, has been identified previously in human
cerebellum but has not been expressed and characterized (Zhuchenko et
al., 1997) (GenBank number U79663). It lacks the NP exon but contains
exons 37a and 44. The other variant, 1A-C16,
which has not been identified previously, contains the NP exon and exon
37b but lacks exon 44. Neither 1A-C2 nor 1A-C16 contains exon 47, which is present in
the well characterized 1A subunits BI-1
and BI-2 isolated from rabbit brain (Mori et al., 1991; Sather et al.,
1993). Because of the central role of the 1A C
terminus in defining channel-gating characteristics, we hypothesized
that the difference in splicing pattern between the two variants would
be reflected by changes in their biophysical properties.
Expression in Xenopus oocytes demonstrated that
1A-C2 and 1A-C16 are
similar with respect to current-voltage relationships and the voltage
dependency of activation and inactivation. This is different from
previously reported results. The presence of the NP exon shifted the
voltage dependence of activation and inactivation of a rat brain
1A subunit (Bourinet et al., 1999). In a study conducted with a human 1A subunit, the NP exon
only shifted the voltage dependence of inactivation (Hans et al.,
1999). Taken together, these data suggest that regions in addition to
NP are important for determining the voltage dependence of activation and inactivation. However, other differences, such as the isoform of
the Ch subunit coexpressed with the 1A
subunit, differing expression systems, and the lack of complete
inactivation at the end of the conditioning pulse could shift the
voltage dependence of inactivation (Hans et al., 1999).
Further characterization reveals differences in the respective rates of
activation, inactivation, deactivation, and recovery from inactivation
of channels containing the 1A-C2 or
1A-C16 subunits. Complexes containing the
1A-C16 subunit activate more slowly than do
those that contain 1A-C2. This may be
explained, in part, by the presence of the NP amino acids in the
extracellular IVS3-IVS4 linker, which have been shown to decrease the
rate of activation of 1A and
1B channel complexes by ~1.5-fold at +10 mV
(Hans et al., 1999; Lin et al., 1999). The
1A-C2 complex has an apparent rate of
activation that is similar to that of other 1A
subunits that lack NP (1.6 ± 0.2 vs 1.2 ± 0.5 msec) (Hans et al., 1999). The activation rate of 1A-C16
is considerably slower than that of other subunits that contain NP
(4.8 ± 0.5 vs 2.2 ± 1.1 msec) (Hans et al., 1999),
suggesting that other regions of the C terminus also affect activation.
Our results indicate that the region encoded by exon 44 appears to be
acting in a modular manner to enhance the rate of inactivation of
1A complexes twofold to threefold. It is
unclear whether this results from a direct conformational change or
whether the presence or absence of this charged sequence affects
modulation by intracellular signaling molecules. Further experiments
are required to resolve this issue. Interestingly, the regions encoded
by exons 43-47 have been shown to bind 4 and
2a subunits but not 1
or 3 subunits (Walker et al., 1998). Our
results with the 1 subunit suggest that
changes in inactivation imparted by exon 44 are independent of
C-terminal subunit binding. The deactivation rate of the 1A-C16 complex is slightly slower than that of
1A-C2. Because the settling time of the oocyte
clamp is ~2 msec, these rates are likely to be faster than we report.
Regardless, the 1A-C16 complexes are
consistently slower over a wide voltage range (60 to 100 mV; data
not shown). Thus, as is true for the 1C
subunit of L-type Ca2+ channels (Soldatov
et al., 1997, 1998), C-terminal alternative splicing does affect the
biophysical properties of channel complexes composed of
1A subunits.
The slower activation, inactivation, and deactivation of
1A-C16 splice variants relative to
1A-C2 could translate to differences in
neurotransmitter release at subtype-specific synapses. Although differences in channel number and density, second messenger effects, and Ca2+ buffering make it difficult to
predict what the combined effects of C-terminal splicing on
Ca2+ entry will be (Park and Dunlap, 1998;
Lin et al., 1999), our results do allow for some generalizations to be
made. The slower rate of activation of channels containing the
1A-C16 subunit should lead to a decrease in
the initial rate of calcium influx. However, the slower inactivation
rate of the 1A-C16 subunit would enhance later
calcium entry, providing the channel does not recover from inactivation
by passing through the open state (Slesinger and Lansman, 1991). The
slower deactivation rate of 1A-C16 channel complexes would increase Ca2+ flux through
these channels at a time when the driving force is relatively large.
Therefore, it is likely that in response to a single action potential,
the initial rate of Ca2+ entry would be
slower through complexes containing the 1A-C16 subunit, but these channels would allow for a greater total
Ca2+ influx.
The rate of recovery from inactivation at negative potentials plays an
important role in determining Ca2+ influx
and neurotransmitter release in response to a train of action
potentials. The voltage dependence of the rate of recovery of channel
complexes containing the 1A-C16 subunit
differs from those containing 1A-C2. At less
negative potentials, the two splice variants behave similarly. After
500 msec at 60 mV, ~30% of channels composed of either variant are
fully recovered. However, at more negative potentials, complexes
containing the 1A-C16 subunit recover more
slowly. After 500 msec at 80 mV, ~73% of 1A-C2 complexes are recovered, compared with
~62% of 1A-C16 complexes. Thus,
Ca2+ entry after a train of action
potentials would be less in nerve terminals expressing the
1A-C16 subunit relative to those expressing the 1A-C2 subunit. These differences may be
important from the standpoint of
Ca2+-mediated synaptic plasticity.
Our results add to a developing picture of neuronal calcium channels
that suggests that 1 subunit genes express
channels with a wide array of structures and functions. These channels may have evolved to meet the specific needs of highly specialized synapses. Our results, combined with those of several laboratories (Bourinet et al., 1999; Hans et al., 1999), point to the fact that it
is no longer sufficient to refer to 1A
channels as simply P- and Q-type. Minor splicing events in critical
domains of the 1A subunit alter both
pharmacological and physiological properties in ways in which we are
only beginning to understand. Determination of where specific
1A subunit splice variants are expressed, how they respond to trains of action potentials, and how they interact with
various signaling molecules should greatly enhance our understanding of
the physiology of the synapse.
| |
FOOTNOTES |
Received Jan. 13, 2000; revised Aug. 1, 2000; accepted Aug. 4, 2000.
This work was supported by National Institutes of Health Grant R29-NS
32094, North Carolina State University Faculty Research and
Professional Development Grant 09223-350791, and North Carolina Biotechnology Center Academic Research Initiation Grant 9905 ARG 0044. We would like to thank Drs. Robert Rosenberg and Mark Chapman for
advice in the preparation of this manuscript.
Correspondence should be addressed to Dr. William A. Horne, College of
Veterinary Medicine, North Carolina State University, 4700 Hillsborough
Street, Raleigh, NC 27606. E-mail: Bill_Horne{at}ncsu.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
