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The Journal of Neuroscience, March 1, 2002, 22(5):1573-1582
Alternative Splicing of the  4 Subunit Has
 1 Subunit Subtype-Specific Effects on
Ca2+ Channel Gating
Thomas D.
Helton 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 |
Ca2+ channel  subunits are important
molecular determinants of the kinetics and voltage dependence of
Ca2+ channel gating. Through direct interactions
with channel-forming  1 subunits, subunits enhance
expression levels, accelerate activation, and have variable effects on
inactivation. Four distinct subunit genes each encode five
homologous sequence domains (D1-5), three of which (D1, D3, and D5)
undergo alternative splicing. We have isolated from human spinal cord a
novel alternatively spliced 4 subunit containing a short
form of domain D1 (4a) that is highly homologous
to N termini of Xenopus and rat 3
subunits. The purpose of this study was to compare the gating
properties of various 1 subunit complexes containing
4a with those of complexes containing a 4
subunit with a longer form of domain D1, 4b. Expression
in Xenopus oocytes revealed that, relative to
1A and 1B complexes containing
4b, the voltage dependence of activation and
inactivation of complexes containing 4a were shifted to
more depolarized potentials. Moreover, 1A and
1B complexes containing 4a inactivated at
a faster rate. Interestingly, 4 subunit alternative splicing did not influence the gating properties of 1C
and 1E subunits. Experiments with 4
deletion mutants revealed that both the N and C termini of the
4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are 1 subunit specific. Our data are best explained by a
model in which distinct modes of activation and inactivation result
from -subunit splice variant-specific interactions with an
1 subunit gating structure.
Key words:
4 subunit; alternative splicing; N terminus; calcium
channel; gating; voltage clamp; spinal cord
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INTRODUCTION |
Neuronal high voltage-activated
Ca2+ channels (L, N, P/Q, and R) consist
of at least four subunits, 1,
2/ , and (Liu et al., 1996 ), with
a fifth subunit,  , being recently described (Letts et al., 1998).
Different Ca2+ channel phenotypes arise
primarily from the expression of five unique 1
subunit genes
(1A-1E). These genes
encode large pore-forming proteins (>2200 amino acids) that are
differentially distributed throughout the nervous system (Westenbroek
et al., 1990, 1998). Synaptic N-, P/Q-, and R-type channels, formed by
1B, 1A, and 1E subunits, respectively, play a principal
role in regulating neurotransmitter release (Turner et al., 1992;
Takahashi and Momiyama, 1993; Wheeler et al., 1994; Wu et al.,
1999).
Ca2+ channel subunits (subtypes 1-4)
are highly homologous intracellular proteins with primary sequences
ranging from 480 to 630 amino acids (for review, see Birnbaumer et al.,
1998). The sequence can be divided into five domains on the basis of the regions of amino acid identity between subtypes. All subunits contain a highly conserved interaction domain (BID) in domain 4, which has been shown to interact with high affinity to an interaction domain (AID) on the I-II linker of
1 subunits (Pragnell et al., 1994; De Waard
and Campbell, 1995). Structure prediction methods using the Prodom
and Pfam protein databases have established a domain structure
(A-E domains) for the 1b subunit (Hanlon et al., 1999) that primarily overlaps with sequence domains 1-5. The A
domain [100 amino acids (aa)] shows some homology to PDZ domains, the B domain (61 aa) to SH3 domains, and the D domain (210 aa) to guanylate-kinase, although it lacks a functional
ATP-binding P-loop motif. Domains C and E were without precedent in the
Prodom and Pfam protein databases; however, Domain C is rich in serine residues, suggesting that it serves a linker function between domains B
and D. Thus, in many respects, Ca2+
channel subunits resemble members of the membrane-associated guanylate kinase (MAGUK) protein family, which are known to
cluster ion channels, receptors, adhesion molecules, and cytosolic
signaling proteins at synapses and cellular junctions (Fanning and
Anderson, 1999).
Previous studies have shown that the kinetics and voltage sensitivity
of 1 subunit gating are affected profoundly by
subunits (Lacerda et al., 1991; Singer et al., 1991), and the
extent to which these parameters are altered varies significantly with
subunit subtype (Ellinor et al., 1993; Olcese et al., 1994). For example, although 1 and
3 subunits shift the voltage dependence of
1E subunit inactivation to more hyperpolarized
potentials, 2 subunits have a marked
depolarizing effect (for review, see Birnbaumer et al., 1998).
Moreover, the responsiveness of 1 subunits to
subunit modulation can be modified by alternative splicing of both
(Olcese et al., 1994; Qin et al., 1996) and
1 subunits (Krovetz et al., 2000; Pan and
Lipscombe, 2000). In this study, we demonstrate for the first time that
alternative splicing of the N terminus of the
4 subunit alters
Ca2+ channel gating and that this effect
is specific to 1A and
1B subunits.
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MATERIALS AND METHODS |
Human spinal cord library screening. Calcium channel
4 subunits were isolated from an oligo-dT and
random-primed human spinal cord  gt11 5'-Stretch Plus cDNA library
(Clontech, Palo Alto, CA) using 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 and female Caucasians, ages
16-75 years, who died of sudden death syndrome. The insert size range of the library was 0.8-7.0 kb (average size 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 (all endonucleases used were from Roche Molecular Biochemicals). All
cDNA isolates were ligated into pBluescriptII (Stratagene, La Jolla,
CA) for PCR-based cycle sequencing (FS chemistry; PE Biosystems,
Foster City, CA) with universal and custom internal primers (Genosys,
The Woodlands, TX). Sequences were 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 (DNA Star, Madison, WI).
The library-screening process was initiated with a human brain
4 cDNA probe obtained from the National Center
for Biotechnical Information dbEST database (1.5 kb human fetal brain
4 fragment; GenBank number R15035). Of nine
first-round 4 cDNAs isolated, the 1.6 kb
4-7 clone was the largest, extending from
nucleotide 216 to beyond an in-frame stop codon (the human brain
4 cDNA, GenBank number U95020, was used as a
reference for all 4 nucleotide and amino acid
positions). The 4-7 clone contained 134 nucleotides of 5' untranslated sequence. A second round of screening,
using a probe consisting of the N-terminal portion of
4-7 from an internal BamHI site
(550) to the 5' untranslated region, yielded seven additional
4 cDNAs, 4-15 to
4-22. Clone 4-17
possessed an in-frame start codon and novel exon 1 sequence but lacked
the last 33 nucleotides of the human brain 4
C-terminal coding sequence. Therefore, to create a full-length
4 cDNA, the N terminus of the
4-17 clone from the BamHI site at
nucleotide position 550 to the BamHI site in the pBluescript
II was ligated into a BamHI-prepared 4-7 clone. Sequence analysis was used to
confirm that the 4-17/7 ligation occurred in
the proper orientation. This full-length 4
cDNA was referred to as 4a (GenBank number
AY054985). We used RT-PCR to isolate the previously published human
brain 4 N terminus (U95020). A 694 bp fragment
was obtained using a commercially available RT-PCR kit (Stratagene),
custom oligonucleotide primers (4 25F:
5'-CTCCGCCCACCGCACACG; 4 719R:
5'-CTAACACCACCGGACGCAT), and human spinal cord
poly(A+) RNA (Clontech). Complete
sequence analysis determined that the 694 bp fragment was identical to
the U95020 N terminus, that it contained a start codon, and that it
extended beyond the BamHI restriction site at position 550. Therefore, to make a second full-length 4
subunit, this fragment was cloned into a BamHI-prepared pBluescriptII SK+ vector containing 4-7.
Sequence analysis was used to confirm correct reading frame and proper
N-terminal orientation. This full-length 4
cDNA was referred to as 4b.
Construction of 4 N,
4aC,
4bC, and
4N/C
deletion mutants. A 4 cDNA lacking
exon 1 (4N) was obtained by using PCR to
replace exon 1 of 4a with an idealized Kozak
sequence (Kozak, 1991) and start codon. Custom oligonucleotide primers
4NF (5'-GCCACCATGG-GTTCAGCGGATTCC), containing the Kozak sequence and start codon and beginning at nucleotide 215, and 4 719R were used in a PCR
reaction with the 4-17 clone as template to
generate the fragment, 4NT( ). This fragment
was then cloned into the BamHI-prepared
4-7 cDNA and sequenced to confirm correct
reading frame and proper N-terminal orientation. The
4aC, 4bC, and
4N/C cDNAs were obtained by using PCR to
remove the C-terminal nucleotide sequence 3' to nucleotide 1286 (corresponding to amino acid position 404). Custom oligonucleotide
primers 4 849F (5'-GCTGACATTTCTCTTGCTAA
upstream of a unique BglII site) and
4CR (5'-TCAGGTTGTGTG-GGTGGCAC, which ended at 4 nucleotide 1286 and included an
in-frame stop codon) were used in a PCR reaction with the
4-17 clone as template to generate the
truncated fragment, 4C(). This fragment was
then cloned into the pT-Advantage vector (Clontech) and sequenced to determine correct orientation. The 4C()
fragment was then cut with BglII and XhoI (from
pT-Advantage poly-linker) and cloned into BglII- and
XhoI-prepared 4a,
4b, and 4N cDNAs.
The resulting cDNAs were then sequenced with internal primers flanking
the C-terminal deletion to confirm sequence orientation and fidelity.
The BI-2 (1A) and
2a/-1 clones used in this study were
provided by T. Tanabe (Tokyo Medical and Dental University, Tokyo, Japan). The rat 1B and rabbit
1C clones were kindly provided by D. Lipscombe
(Brown University, Providence, RI) and E. Perez-Reyes (University of
Virginia, Charlottesville, VA), respectively.
Electrophysiology and data analysis. Complementary RNAs
(cRNAs) were synthesized in vitro using Ambion's mMessage
mMachine RNA transcription kit [T3 or T7 depending on clone
orientation in pBluescript II S/K+ or
pBSTA (1B)]. Standard Xenopus
laevis oocyte expression methods were used to characterize
subunit splice variants. Briefly, full-length
1, 2/, and cRNAs were injected in equimolar ratios (5.6 ng 1A
or 1B, 2.4 ng
2/, and 1.6 ng in 46 nl; 17 ng 1C or 1E, 7 ng
2/, and 5 ng in 50 nl) into
defolliculated oocytes (stage V-VI). (The 2-1
subunit was used in this study.) Calcium channel currents were recorded
2-8 d after oocyte injection by standard two-electrode voltage clamp
using a Warner amplifier (OC-725B) at 20-22°C, and data were
collected using pCLAMP6 software (Axon Instruments, Foster City, CA).
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 in a chloride-free bath containing 5 mM
Ba(OH)2, 5 mM HEPES, 85 mM TEA-OH, and 2 mM KOH, pH
adjusted to 7.4 with methansulfonic acid (1A
and 1B), or 40 mM
Ba(OH)2, 5 mM HEPES, 85 mM TEA-OH, and 2 mM KOH, pH
adjusted to 7.4 with methansulfonic acid (1C
and 1E). Currents used to generate the data in
this study ranged from 0.5 to 2.9 µA. For activation and inactivation
experiments, the average current sizes for 1A
and 1B complexes containing either
4a or 4b were 1.2 and
1.6 µA, respectively. Leak currents were between 20 and 100 nA. Only recordings with minimal tail currents were used for
each data set (see representative traces in Fig. 5). Data were analyzed
using pCLAMP6 software (Axon Instruments) and Excel 7.0 (Microsoft
Corp., Redmond WA). The leak and capacitive currents were subtracted
on-line using a standard P/4 protocol. Boltzmann fits to the
activation and inactivation data were performed using Sigma Plot
version 5.0 (SSPS Inc., Chicago IL) with the equations
%IBa = 1/[1 + exp((Vtest V1/2)/k)] and %IBa = 1/ [1 + exp((Vpre V1/2)/k)], respectively, where
Vtest = I-V test
potential, Vpre = prepulse potential,
V1/2 = midpoint of activation or
inactivation, and k = slope factor. An estimate of
gating charge, z, was calculated by dividing 25 (approximate value for RT/F at room temperature, where
R = gas constant, T = temperature, and
F = Faraday constant) by the slope factor. Statistical analysis was performed with a Student's two-sample equal
variance t test with a two-tailed distribution (Microsoft Excel 97 SR-2). Data are presented as mean ± SEM.
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RESULTS |
Cloning of a Ca2+ channel 4
subunit with an N terminus similar to that of 3
subunits
Two 4 subunit N-terminal splice variants,
4a and 4b (Fig.
1), are the focus of this study. Both
were isolated from a human spinal cord cDNA library using routine
screening techniques. The amino acid sequence of the
4b variant is identical to a previously published sequence (GenBank number U95020), whereas this is the first
reporting of the 4a sequence. The difference
in the two variants lies solely in the nucleotide sequence of exon 1, the translated region of which is referred to as domain D1 (Birnbaumer et al., 1998). The remaining sequence of both
4a and 4b is composed of 1410 nucleotides that encode the 470 amino acids of domains 2-5
(data not shown). As shown in Figure 1, exon 1 of
4a encodes a 15 amino acid sequence that is
highly homologous to the N-terminal sequences of several previously
identified Ca2+ channel
3 subunits. This indicates that
4a exon 1 must have been present in the genome
before the time that an ancestral gene duplicated to form distinct
3 and 4 genes.
Interestingly, amino acids 5-11 (LYLHGIE) are identical to those found
in the Xenopus subunit, x32,
but quite divergent from the same region of the human
3 subunit. This could imply that a particular
function of this sequence has been purposely conserved throughout
evolution. Also of note in the human 4a
sequence are two D to N conversions at positions 4 and 12 (asterisks)
that eliminate two negative charges that appear to be highly conserved
among 3 subunits. Figure 1 also demonstrates
that D1 of 4a is not at all homologous to D1
of 4b. It can be seen, however, that D1 of
1b and 4b are more
closely related than D1 of 4a and
4b. Domain 1 of 4b contains 49 amino acids, 2 of which are negatively charged, and 8 of
which are positively charged. Six of these positive charges are
clustered in the center of the sequence close to consensus sites (TTR
and TRR) for phosphorylation by protein kinase C. No further
Prosite-listed consensus sites were found in the D1 sequences of either
4a or 4b.
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Figure 1.
Sequence comparisons of human spinal cord
Ca2+ channel 4a and 4b
subunits and other subunit subtypes. Top, The amino
acid sequence of domain 1 and a short segment of domain 2 of the human
4a subunit (h4a) is
shown aligned with comparable domains of two Xenopus
3 subunits (x32 and
x28) (Tareilus et al., 1997) and a
human 3 subunit
(h3). Amino acids identical to the
h4a sequence are boxed. Asterisks denote
D to N amino acid conversions in the human 4a sequence.
Bottom, The amino acid sequence of domain 1 and a short
segment of domain 2 of the human 4b subunit
(h4b) is shown aligned with
comparable domains of the human 1b subunit. Identical
amino acids are boxed. Dashed lines indicate gaps in the
sequence. The bar denotes consensus sites for
phosphorylation by protein kinase C.
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Alternative splicing of the 4 subunit N terminus
affects Ca2+ channel expression
Critical to the interpretation of our expression data is the fact
that some populations of Xenopus oocytes have been shown to
express low levels of an endogenous 3-like
subunit that is capable of binding to and altering the gating
properties of injected 1 subunits (Tareilus et
al., 1997). To test for this possibility in our oocytes, we conducted
experiments in which we measured the time required for
1A/2,
1A/2 + 4a, and
1A/2 + 4b complexes to reach levels of expression
that we thought suitable for electrophysiological recording (1 µA of
peak current). Figure 2 shows that
channel complexes containing 4b expressed at a
much faster rate than those containing 4a,
reaching adequate levels within 1-2 d. Complexes containing
4a took 3-4 d to reach similar levels,
whereas complexes that did not contain a subunit required 7-8
d to express 1 µA of current. Similarly,
1B/2 + 4b
complexes reached adequate levels in 1-2 d, whereas
1B/2 + 4a complexes took 3-4 d to reach similar
levels. 1B complexes expressed without 4 subunits did not reach suitable current size
until day 7-8 (data not shown). 1C/2 and
1E/2 expressed
with either 4a or 4b
reached adequate current size in 6-8 d, whereas complexes without
4 subunits showed no appreciable current even
after 8 d. Expression rates and levels for
1C/2 and
1E/2 + 4a and 4b were
essentially identical (data not shown). As shown in Figure 2, a sixfold
increase in the amount of subunit cRNA injected into oocytes
relative to that of 1A did not affect
expression rates or levels, suggesting that subunit binding sites
on 1A are saturated even when the two subunits
are coinjected at a 1:1 ratio. This is consistent with the findings of
Qin et al. (1996). We concluded from these experiments that the
endogenous Xenopus 3-like subunit
would not significantly influence the examination of exogenous currents
measured in the 2-6 d time period.
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Figure 2.
Expression rates of 1A
Ca2+ channel complexes with different subunit
compositions. Peak currents elicited by depolarization to +10 mV
(1A/2), +5 mV
(1A/2 + 4a), or 0 mV
(1A/2 + 4b) from a holding potential of 80 mV are
plotted against days after injection. Barium (5 mM) was the
charge carrier. Oocytes were maintained in ND96 culture media at
18°C. Comparisons between experiments in which the 4a
or 4b subunits were injected at 1:1 (1×) or 6:1 (6×)
ratios relative to the 1A are shown. Each data point
represents a minimum of six recordings. The SEM for each point is shown
unless the values were smaller than the symbol.
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Alternatively spliced 4 subunits have
1-subunit subtype-specific effects on voltage-dependent
activation and inactivation
To determine whether 4 N-terminal
splicing affected Ca2+ channel gating
properties, we expressed either 4a or
4b with rabbit 2
and with rabbit 1A (BI-2) (Mori et al., 1991),
rat 1B (21 1B)
(Pan and Lipscombe, 2000), rabbit 1C (Mikami
et al., 1989), or marine ray 1E (doe-1) (Horne
et al., 1993) in Xenopus oocytes. (The
2-1 subunit is included in all experiments
in this study.) Figure
3A,B,E,F
shows comparisons of normalized current-voltage (I-V) curves for the four different
1 subunits expressed with either
4a or 4b. Figure 3,
A and B, illustrate that the peaks of the
current-voltage curves for 1A and
1B complexes containing 4b were shifted to more hyperpolarized
potentials relative to complexes containing
4a. In contrast, Figure 3, E and
F, shows that the I-V curves for
1C and 1E complexes
containing either 4a or
4b were essentially superimposed. The
difference in 1 subunit responsiveness was not
caused by differences in charge carrier concentrations used in the
experiments (5 mM
Ba2+ for 1A and
1B; 40 mM
Ba2+ for 1C and
1E), because we observed identical
hyperpolarizing shifts for both 1A and
1B with 4b, even in
40 mM Ba2+ (data not
shown). We concluded from these first experiments that alternative
splicing of the 4 subunit N terminus affects
activation of Ca2+ channel complexes
containing 1A and 1B
subunits but not those containing 1C or
1E. To estimate the
V1/2 of activation for the different
1A and 1B
combinations, we averaged Boltzmann fits to the
I-V data generated over the range of 40 to +10
mV for 1A complexes and 40 to +20 mV for
1B complexes containing either
4a or 4b (Fig.
3C,D). The results show that the
V1/2 of activation for both
1A and 1B complexes
containing 4b were shifted to the left
relative to complexes containing 4a by ~5 mV
and ~7 mV, respectively (Table 1). The
results also show that the slopes of the 4b
fits were somewhat steeper than for 4a.
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Figure 3.
4a and 4b subunits
have 1 subunit subtype-specific effects on the voltage
dependence of activation. A, B,
E, F, Normalized, averaged peak
current-voltage (I-V) plots for
1A (A), 1B
(B) 1C (E), and
1E (F) coexpressed with
2 and either 4a or 4b.
A, B, The 1A (BI-2) and
1B (21) subunits used in these and subsequent
experiments are those described by Mori et al. (1991) and Pan and
Lipscombe (2000), respectively. Currents were activated by 300 msec
depolarizations to various test potentials (40 to +40 mV in 5 mV
increments) from a holding potential of 80 mV. Barium (5 mM) was the charge carrier for both 1A and
1B. C, D, Voltage
dependence of activation up to +10 mV for 1A
(C) and +20 mV for 1B
(D) as determined from averaged
I-V data in A and
B. Data points represent the means of the normalized
data at a given membrane potential. The SEM for each point is shown
unless the values were smaller than the symbol. Smooth
curves represent a single Boltzmann fit to the averaged data.
Values for V1/2 and k for
1A and 1B plus
2/ and either 4a or
4b are listed in Table 1. E,
F, The 1C (cardiac) and 1E
(doe-1) subunits used in these and subsequent experiments are
those described by Mikami et al. (1989) and Horne et al. (1993),
respectively. Currents were activated by 300 msec depolarizations to
various test potentials (40 to +40 mV in 5 mV increments) from a
holding potential of 80 mV (1C + 4a, n = 12;
1C + 4b,
n = 13; 1E + 4a, n = 9;
1E + 4b,
n = 9). Barium (40 mM) was the charge
carrier.
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Table 1.
Effects of 4 subunit alternative splicing
and N- and C-terminal deletions on voltage-dependent activation and
inactivation of 1A and 1B
Ca2+ channel subunits
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We next examined whether alternative splicing of the
4 subunit affected isochronal inactivation. We
used a 20 sec conditioning prepulse over a wide range of potentials
followed by a 300 msec test pulse to near-peak potentials to generate
the data. Figure 4A-D shows
that, as was the case for activation, alternative splicing of the
4 subunit N terminus affects inactivation of
Ca2+ channel complexes containing
1A and 1B subunits
but not those containing 1C or
1E. The figure illustrates that the voltage dependence of inactivation of both 1A (Fig.
4A) and 1B (Fig. 4B) complexes containing 4b
was shifted to more hyperpolarized potentials relative to complexes
containing 4a. In contrast, inactivation
curves for 1C (Fig. 4C) and
1E (Fig. 4D) complexes containing
4a or 4b were
essentially identical. The Boltzmann-derived V1/2 for inactivation of both
1A and 1B complexes
containing 4b were shifted to the left
relative to complexes containing 4a by
~10-11 mV (Table 1). Interestingly, the hyperpolarizing shift in
V1/2 for 1A
complexes (Fig. 4A) occurred as the result of a
parallel shift in the voltage dependence of inactivation, whereas for
1B complexes (Fig. 4B), the
shift in V1/2 occurred primarily as
the result of a change in slope. Slope factors for 1B complexes containing
4a and 4b complexes
were ~14 and 7 mV, respectively (Table 1).
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Figure 4.
4a and 4b subunits
have 1 subunit subtype-specific effects on the voltage
dependence of inactivation. A-D,
Normalized, averaged isochronal inactivation curves for
1A (A), 1B
(B), 1C (C),
and 1E (D) coexpressed with
2 and either 4a or 4b.
Curves were generated from peak currents elicited by a 300 msec test
depolarization to +5 mV (1A + 4a),
0 mV (1A + 4b), +10 mV
(1B + 4a), +5 mV
(1B + 4b), or +20 mV
(1C and 1E with 4a and
4b) after a 20 sec conditioning prepulse to
voltages ranging from 80 to +30 mV (A,
C, D) or 100 to +10 mV
(B). Barium (5 mM for
1A and 1B; 40 mM for
1C and 1E) was the charge carrier.
Data points represent the means of the normalized data at a given
membrane potential. The SEM for each point is shown unless the values
were smaller than the symbol. Smooth curves represent a
single Boltzmann fit to the averaged data. Values for
V1/2 and k for inactivation
of 1A and 1B plus 2 and
either 4a or 4b are listed in Table
1.
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Because 1C and 1E
subunits were not affected by alternative splicing of
4 subunits, we next directed our experiments
toward characterizing the 1A and
1B responses in more detail. Figure 5 shows representative current traces of
1A (Fig. 5A) and
1B (Fig. 5B) complexes containing
either 4a (top) or
4b (bottom) expressed in
Xenopus oocytes. Traces shown were generated by step depolarization to 10, 0, 10, 20, and 30 mV. The arrows
indicate that the potentials at which peak currents were reached varied with each complex. Regardless of the 1 subunit
subtype, however, complexes containing 4a
inactivated faster than those containing 4b,
with a difference in rates being more apparent for complexes containing
1B. Figure 5, C and D,
shows the averaged currents remaining after 300 msec
(R300) step depolarizations to each
potential for 1A and
1B, respectively. The results indicate that
the rate of inactivation for all four complexes is voltage dependent
and that the differences in rates between complexes containing
4a versus 4b become
apparent primarily with depolarizations beyond 0 mV.
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Figure 5.
1A and 1B complexes
containing 4a inactivate faster than those containing
4b. A, B, Representative
current traces of 1A (A) and
1B (B) plus 2
and either 4a (top) or 4b
(bottom). Currents were elicited by step depolarizations
to a range of test potentials (10 to +30 mV in 10 mV increments) from
a holding potential of 80 mV. Barium (5 mM) was used as
the charge carrier. Traces were fit with a single exponential from 25 msec beyond the peak inward current to the end of the depolarization.
Averages of  inactivation at the peak current potential
were 1A + 4a, 226.6 ± 12.5 msec (n = 12); 1A + 4b, 307.2 ± 19.2 msec
(n = 10); 1B + 4a, 160.1 ± 20.0 msec
(n = 10); 1A + 4b, 213.9 ± 15.6 msec
(n = 10). C, D,
Current remaining at the end of a 300 msec test pulse
(R300), elicited as in the protocol
above, for 1A (C) and
1B (D) plus 2
and either 4a or 4b. The SEM for each bar
is shown. Asterisks denote statistical significance
(p < 0.05) as determined by a Student's
two-sample equal variance t test.
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1 subunit-specific responses to 4
subunit N- and C- terminal deletions
The results to this point indicated that the N terminus of the
4 subunit plays an important role in setting
the kinetics and voltage-dependence of
Ca2+ channel gating, with some differences
in responsiveness noted between 1A and
1B subunits. We next sought to determine
whether the 4 N terminus could be acting in
concert with the 4 C terminus to exert its
effects on gating. Because previous studies had shown that the
4 C terminus binds directly to the
1A subunit (Walker et al., 1998, 1999), it was
of particular interest to determine whether the gating properties of
1A would change in comparison to
1B if the 4 C
terminus were deleted. To address this issue, we made four
4 subunit deletion constructs that along with
4a and 4b provided us
with all the possible +/ combinations of 4
N- and C termini (Fig.
6A). We found that all
four constructs augmented Ca2+ channel
expression to a level that was comparable to or exceeded (i.e.,
4NC) the expression levels we observed
with 4b. The effects of these constructs on
activation and inactivation of 1A and
1B subunits are shown in Figure 6,
B and C, and Figure 7, A and B,
respectively. (Our initial results with 4a and
4b are included as dashed lines for
reference in Figs. 6 and 7). Interestingly, it was readily apparent
from both the activation and inactivation results shown in Figures 6
and 7 that despite testing six different 4
subunit constructs, our data could be grouped into two activation
modes, A1 and A2
(1A and 1B), and two
(1B) or three (1A)
inactivation modes, I1-I3,
on the basis of the curve position alone. As can be seen from the data,
the distinction between activation and inactivation modes was most clearly delineated in experiments involving 1B
(Figs. 6C, 7B). Table 1 shows that the
distinction between modes is quite evident when comparing
Boltzmann-derived values for V1/2 and
slope factor, and along with Figures 6 and 7 reveals that the
4 subunit constructs responsible for setting
each mode differ between 1A and
1B subunits.
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Figure 6.
Effects of 4 subunit N- and
C-terminal deletions on the voltage dependence of activation of
1A and 1B Ca2+
channels. A, Schematic diagrams of the wild-type and
artificial 4 subunits used in this series of
experiments. The 15 amino acid 4a and 49 amino acid
4b N termini (alternatively spliced forms of domain 1)
are denoted by filled and open bars,
respectively. Domains 2-4 are represented by a single
cross-hatched bar. The C terminus (domain 5) is denoted
by a diagonally striped bar. B, C,
Voltage dependence of activation up to +10 mV for 1A
(B) and +20 mV for 1B
(C) as determined from averaged
I-V data. Data points represent the
means of the normalized data at a given membrane potential. The SEM for
each point is shown unless the values were smaller than the symbol.
Smooth curves represent a single Boltzmann fit to the
averaged data. Broken curves represent activation data
shown in Figure 3, C and D, and are
included in this figure for reference. Values for
V1/2 and k for
1A and 1B plus
2/ and each of the six 4
constructs are grouped according to curve similarities in Table
1.
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Figure 7.
Effects of 4 subunit N- and
C-terminal deletions on the voltage dependence of inactivation of
1A and 1B Ca2+
channels. A, B, Normalized, averaged
steady-state inactivation curves for 1A
(A) and 1B
(B) coexpressed with 2 and one
of the six 4 constructs shown in Figure
6A. Curves were generated from peak currents
elicited by a 300 msec test depolarization to 5 mV (1A + 4NC), 0 mV (1A + 4b, 4aC), +5 mV
(1A + 4a, 4N,
and 4bC; 1B + 4b and
4N), +10 mV (1B + 4a, 4NC), or +15 mV
(1B + 4aC and 4bC)
after a 20 sec conditioning prepulse to voltages ranging from 80 to
+10 mV (A) or 100 to +10 mV
(B). Barium (5 mM) was the charge
carrier for both 1A and 1B. Data points
represent the means of the normalized data at a given membrane
potential. The SEM for each point is shown unless the values were
smaller than the symbol. Smooth curves represent a
single Boltzmann fit to the averaged data. Values for
V1/2 and k for
inactivation of 1A and 1B plus
2 and each of the six 4 constructs are
grouped according to curve similarities in Table 1.
|
|
The details of the deletion results are best understood by examining in
sequence the data that we obtained with individual subunit
constructs. Our first experiments were directed toward determining what
effect deletion of both the 4 N and C
termini (4NC) would have on
1A and 1B gating
properties. Unexpectedly, both 1A and
1B complexes containing the
4NC subunit had activation properties
very similar to complexes containing full-length
4b (Fig.
6B,C, mode
A1). This indicated that
1 subunits could not distinguish
4 subunits without an N or C terminus from
4 subunits with the longer form of N terminus
and the C terminus present. Relative to 1
complexes containing 4a, however,
4NC caused a 6-7 mV hyperpolarizing
shift and a slight increase in slope of activation of both
1A and 1B (Table 1).
Figure 7, A and B, shows that, although the
inactivation curve for 1A complexes containing
4NC fell between those for complexes
containing 4a and 4b,
the inactivation properties of 1B complexes
containing 4NC and
4b were also indistinguishable. For both
1A and 1B, it can be
seen that relative to complexes containing 4a,
4NC caused a qualitatively similar
hyperpolarizing shift in the voltage dependence of inactivation and
decrease in slope (shift from mode I2 to mode
I1). As shown in Figure 7B, this
effect was most dramatic for 1B complexes,
where relative to 4a,
4NC caused a ~10 mV hyperpolarizing
shift in inactivation and a nearly 50% decrease in slope (Table 1).
We next characterized the effects of the construct
4N (4NC plus
the 4 C terminus) on the gating properties of
1A and 1B subunits.
Interestingly, as shown in Figure 6, A and B, the 4N construct had different effects on
activation of 1A as compared with
1B. Although addition of the C terminus had a
depolarizing effect on 1A activation relative
to 4b and 4NC,
there was no change in the activation properties of
1B. Moreover, as can be seen in Figure
6B and Table 1, the activation properties of 1A complexes containing
4N were essentially identical to those containing 4a (mode A2).
Similarly, as shown in Figure 7, A and B,
4N, like 4a, had a
noticeable depolarizing effect on 1A inactivation (mode I2) relative to complexes
containing 4NC but caused no change in
the inactivation properties of 1B. These results indicated that, at least in the absence of the N terminus, the
4 C terminus has 1A
subunit-specific effects on the voltage dependence of both activation
and inactivation.
To define further the role of the 4 N termini
in gating, we next characterized the effects of two constructs,
4aC and 4bC, that lacked the 4 C terminus but contained the
N termini of 4a and
4b, respectively
(4NC plus 4a or
4b N terminus). Interestingly, the pattern of
results that we obtained with these constructs in many respects was
just the opposite of what we saw with 4N. Although 4N had 1A
subunit-specific effects on gating, 4aC and
4bC had, for the most part,
1B subunit-specific effects. Figure
6A shows that relative to
4NC, 4bC,
like 4a, caused a depolarizing shift in
activation of 1A subunits, but
4aC was without effect. Figure
7A shows that relative to
4NC, neither 4aC nor 4bC had
effects on inactivation of 1A subunits. In contrast, Figures 6B and 7B show that
relative to 4NC both 4aC and 4bC
caused a depolarizing shift in activation and inactivation of
1B. Moreover, the gating properties of
1B complexes containing
4aC or 4bC were
essentially identical to those containing 4a
(modes A2 and I2). The
results of these experiments indicate that, at least in the absence of
the C terminus, the 4b but not the
4a N terminus has effects on
1A activation, whereas neither affects
1A inactivation. In contrast, both the 4a and 4b N termini
have effects on 1B activation and inactivation.
With the results from the deletion experiments, it was informative to
reexamine the data from our initial experiments (Figs. 6 and 7,
dashed lines) with the idea that full-length
4a and 4b subunits
were constructed by adding back the 4a and
4b N termini to
4N. As the data reveals, this also had
1 subunit-specific effects on both activation
and inactivation. With respect to activation, Figure
6A shows that, relative to
4N, adding back the
4a N terminus had no effect on
1A activation, suggesting that the short form
of N terminus could not overcome the
1A-specific 4 C-terminal effect noted with 4N previously.
Adding back the 4b N terminus, however, did
supercede the C-terminal effect and caused a hyperpolarizing shift in
1A activation relative to
4N (back to mode A1).
In contrast, Figure 6B shows that adding back the
4a N terminus to
4N caused a depolarizing shift in the activation of 1B (back to mode
A2), but adding back the
4b N terminus had no effect. This goes along
with the 4N data showing that with
1B there is no 4
C-terminal effect to overcome, and that the 4a
N terminus alone causes an 1B-specific
depolarizing shift in activation. With respect to inactivation, Figure
7B shows that, as was the case for activation, adding back
the 4a N terminus to
4N had little effect on
1A inactivation, but adding back the
4b N terminus caused a significant
hyperpolarizing and, in this case, parallel shift in the curve for
1A inactivation (mode I3). It is worth noting that this shift is
different from and goes beyond the curve for
4NC, and that this effect on
1A gating is unique to
4b. Figure 7B shows that, as
expected from the 4aC results with
1B, without a C-terminal effect to overcome, adding back the 4a N terminus to
4N caused a depolarizing shift in
1B inactivation (back to mode
I2). Not expected, however, was the result that
adding back the 4b N terminus had no effect, recalling that the 4b N terminus alone causes
a depolarizing shift in 1B inactivation. This
suggests that the presence of the 4 C
terminus, although not having effects on its own, interferes in some
way with the ability of the 4b N terminus to
influence 1B channel gating.
| |
DISCUSSION |
Our results provide the first evidence that alternative splicing
of the 4 subunit alters
Ca2+ channel gating and that this effect
is specific to 1A and
1B subunits. The physiological relevance of
our findings lies in the fact that 1A,
1B (Westenbroek et al., 1998), and
4 subunits (Wittemann et al., 2000) colocalize
in nerve terminals and that 1A and
4 (Liu et al., 1996) and
1B and 4 subunits
(Scott et al., 1996) are directly associated. In many respects, our
experiments were similar to those of Olcese et al. (1994) and Qin et
al. (1996), which characterized the effects of various subunit
splice variants, chimeras, and deletion mutants on human
1E subunit gating. Their studies yielded five
results pertinent to our findings. (1) Relative to
1E alone, all subunit constructs tested
caused a nearly identical hyperpolarizing shift in the
V1/2 of activation and decrease in
slope factor [see also Jones et al. (1998)]. (2) Deletion of the N
terminus of the 1b,
2a, and 3 subunits
had no effect on the fast component of activation. (3) Alternative
splicing of the N terminus, C terminus, and internal domain 3 of
1 and 2 subunits had
opposing effects on the V1/2 of
steady-state inactivation but did not affect slope. (4) Deletion of the
N terminus of the 1b and
3 subunits caused a depolarizing shift in the
V1/2 of inactivation without affecting
slope, whereas deletion of the N terminus of the
2a subunit caused a hyperpolarizing shift. (5)
C-terminal alternative splicing did not affect gating properties. The
principal conclusion of these experiments was that, independent of
effects on activation, the N terminus of the subunit plays a
dominant role in governing the voltage sensitivity of
1E subunit inactivation. This suggested to
Olcese et al. (1994) that there were two separate
1 and subunit interaction sites regulating activation and inactivation.
Our results point similarly to the N terminus of the
4 subunit as a key determinant of
1A and 1B gating
properties but show some dissimilarity to the five
1E results listed above. (1) Unlike
1E with
1-3 subunits,
alternatively spliced 4 subunits had
differential effects on activation of both 1A
and 1B. Relative to the short
4a N terminus, the longer
4b form caused a hyperpolarizing shift in
activation of both 1A and
1B subunits (but not
1C or 1E). (2)
Relative to 4b, deletion of the N terminus of
the 4 subunit caused a depolarizing shift in
activation of 1A but not
1B. (3) Alternative splicing of the
4 subunit affected both the
V1/2 and slope of inactivation of
1B, whereas only shifting the
V1/2 of 1A
inactivation without a change in slope. Alternative splicing of the
4 subunit did not affect inactivation of
1C or 1E. (4)
Relative to 4b, deletion of the N terminus of
the 4 subunit caused a depolarizing shift in
inactivation of 1A but not
1B. (5) C-terminal deletion experiments
revealed that the 4 N and C termini work in
concert to set gating parameters of 1A and
1B subunits. Taken together, these results
indicate that alternatively spliced subunits can affect both
activation and inactivation of Ca2+
channels, and the responsiveness of Ca2+
channels to subunit splicing varies with 1
subunit subtype.
To explain our results, we devised a structural model for potential
1-4 subunit domain
interactions based on a subunit modular structure (domains A-E)
(Hanlon et al., 1999) and actual molecular weights of the potential
1A and subunit domains involved (Fig.
8). Although highly speculative, the
model integrates related structure-function results from a number of
different laboratories that point to the subunit D domain
interaction with the 1 subunit I-II linker as
a key determinant of Ca2+ channel gating
properties (Herlitze et al., 1997; Bourinet et al., 1999; Stotz et al.,
2000; Berrou et al., 2001). Moreover, it incorporates results showing
that regulation of activation and inactivation are separable functions
of subunits (Olcese et al., 1994). Of particular relevance to our
model are studies showing that the 1 subunit D
domain was all that was required to reproduce the inactivation rate of
L-type channels coexpressed with full-length 1
(Cens et al., 1999) and that a single point mutation (R378E) in the subunit binding site of the 1 I-II linker
(AID domain) had a depolarizing effect on the voltage dependence of
both activation and inactivation of an 1E
subunit (Berrou et al., 2001).
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Figure 8.
Potential 1A and subunit domain
interactions as viewed from inside the cell looking out through the
pore. Top, 1A alone. Transmembrane
domains I-IV are represented as gray circles
and intracellular domains as white circles. Middle,
1A + 4b. The 4b subunit
A-E domains are shown as black circles
superimposed on 1A. Bottom,
1A + 4a. The radius of each
circle was calculated from the spherical volume
(V = 4/3  r3) of
each subunit domain, where V = [(0.73
cm3/gm × 1024
Å3/cm3 × molecular
weight)/6.02 × 1023] and the average
molecular weight of an amino acid is 120 Da. For 1A
(BI-2): N terminus, 98 aa; transmembrane domains I-IV, 229-268 aa;
I-II linker, 127 aa; II-III linker, 537 aa; III-IV linker, 54 aa; C
terminus, 604 aa. For 4b [nomenclature as in Hanlon et
al. (1999)]: A domain, 92 aa; B domain, 61 aa; C domain, 37 aa; D
domain, 210 aa; E domain, 144 aa. For 4a: A domain, 44 aa. Interactions of the 4 D domain with the
1A I-II linker (Pragnell et al., 1994) and
4 E domain with 1A N and C termini have
been well documented (Walker et al., 1998, 1999). Dashed
arrow in the bottom diagram
indicates the potential for a conformational change when the
4a N terminus is substituted for the 4b N
terminus.
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|
Previous studies have shown that the D domain of the
4 subunit binds with high affinity to the
1A I-II linker (Pragnell et al., 1994) and
that the E domain of the 4 subunit binds to both the N and C terminus of the 1A subunit
(Walker et al., 1998, 1999). As shown in Figure 8, this indicates that
the D and E domains likely establish the N- to C-terminal orientation
of the 4 subunit relative to the
1A subunit. Although little is known about subunit N-terminus interactions with the 1
subunit, a modular structure for the subunit A, B, and C domains
suggests that the interactions could occur over a wide range. As
suggested in Figure 8, a change in the size and sequence of the subunit A domain could have an effect on the way the subunit D
domain interacts with the 1 I-II linker. Such
a change might be responsible for the different gating properties
observed between Ca2+ channel complexes
containing 4a versus
4b.
The salient feature of the model is that a core subunit structure
encoded by exons 2-12 (4NC), through
interactions with the 1 subunit I-II linker,
sets separate default parameters for 1
activation and inactivation (Table 2,
mode A1I1). This mode likely represents a specific 1 I-II linker
conformation that, through its connection to the
1 IS6 transmembrane domain, influences the
mobility of the gating charges within 1 IS4
(Zhang et al., 1994). [The effects of 4
N-terminal alternative splicing on apparent gating charge (z
value) are shown in Table 1. Note the significant difference in calculated z values during inactivation of
1B complexes containing
4a versus 4b.] As
shown in Table 2, in mode
A1I1 the two presumed
1 and subunit interaction domains are in
alignment (1, filled symbols; , open
symbols). Changes from default parameters occur when either the subunit N or C terminus, or both, interacts with, or is acted on by,
other regions of the 1 subunit such that the
I-II linker changes mode conformations. For example, in mode
A2I2 (row 2), the two
presumed 1 and subunit interaction domains
would be out of alignment. Displacing the subunit D domain in
either the C- or N-terminal direction would enable mode 2 conformation,
whereas a balance of these two forces favors mode 1.
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Table 2.
Gating mode model describing the effects of
4 subunit constructs on 1A and
1B subunit activation and inactivation
|
|
Describing our data in terms of the model (Table 2), the default
activation and inactivation parameters of 1A
and 1B complexes containing
4NC are denoted as mode
A1I1 (row 1). Steep
activation and shallow inactivation typify this mode. Row 2 shows that
adding back the C terminus to 4NC
(4N) shifts 1A
complexes to mode A2I2,
whereas 1B complexes remain in mode
A1I1. This could be explained by the
1A-4 C-terminal
binding event described by Walker et al. (1998, 1999) causing the
4 D domain to be displaced in the C-terminal
direction. Relative to mode
A1I1, activation in mode
A2I2 is shallower and
inactivation is steeper. Rows 3 and 4 show that by adding back the N
terminus, 1B complexes containing either the
4aC or 4bC
construct shift to mode
A2I2. This shift might be
explained by 4
N-terminal-1B interactions causing the 4 D domain to be displaced in the N-terminal
direction. Alternatively, steric changes resulting from the presence of
the N terminus may shift the 4 D domain in the
C-terminal direction (row 4, 4bC(1B)). Whatever
the cause, it is likely to be different for 1B
complexes containing 4aC versus
4bC. Adding back the C terminus to
4aC has no effect on
1B mode
A2I2 (row 5), whereas
addition of the C terminus to 4bC causes a
shift to mode A1I1 (row 6).
Row 4 also shows that 4bC causes an
1A subunit mode change that is limited to
activation (A2I1). This was
the one instance in our experiments in which regulation of activation
and inactivation were separable functions. The addition of the C
terminus to 4aC shifts
1A to mode
A2I2 (row 5), which again
is likely the result of a 4 C-terminal binding
event. The addition of the C terminus to
4bC creates a distinct
1A mode,
A1I3, characterized by
steep activation and inactivation.
In conclusion, our results add to the developing picture of the
intracellular domains surrounding the Ca2+
channel pore being composed of modular "hot spots" for channel regulation by -subunits, protein kinases, G-proteins, syntaxin, and
calmodulin (for review, see Walker and DeWaard, 1998; Levitan, 1999).
Our future experiments will be directed toward understanding how
interactions between these diverse regulatory components might contribute to the dynamic molecular events giving rise to synaptic plasticity.
| |
FOOTNOTES |
Received June 29, 2001; revised Dec. 5, 2001; accepted Dec. 7, 2001.
This work was supported by National Institutes of Health Grant R29-NS
32094, North Carolina Biotechnology Center Academic Research Grant 9905 ARG 0044, and a College of Veterinary Medicine State Research Support
Grant. We thank Dr. Robert Rosenberg for advice on data analysis and
manuscript preparation.
Correspondence should be addressed to Dr. William A. Horne, Department
of Anatomy, Physiological Sciences, and Radiology, North Carolina State
University College of Veterinary Medicine, 4700 Hillsborough Street,
Raleigh, NC 27606. E-mail: bill_horne{at}ncsu.edu.
| |
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ABSTRACT
INTRODUCTION
