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The Journal of Neuroscience, December 1, 2002, 22(23):10142-10152
Systematic Identification of Splice Variants in Human P/Q-Type
Channel  12.1 Subunits: Implications for Current Density
and Ca2+-Dependent Inactivation
Tuck Wah
Soong1, 2, 3,
Carla D.
DeMaria3,
Rebecca
S.
Alvania3, 4,
Larry S.
Zweifel4,
Mui Cheng
Liang1,
Scott
Mittman5, 6,
William S.
Agnew5, and
David T.
Yue3, 4
1 National Neuroscience Institute, Singapore 308433, 2 Department of Physiology, National University of
Singapore, Singapore 119260, and Departments of
3 Biomedical Engineering, 4 Neuroscience,
5 Physiology, and 6 Anesthesiology, Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
P/Q-type (Cav2.1) calcium channels support a host of
Ca2+-driven neuronal functions in the mammalian
brain. Alternative splicing of the main  1A
(12.1) subunit of these channels may thereby represent a
rich strategy for tuning the functional profile of diverse
neurobiological processes. Here, we applied a recently developed
"transcript-scanning" method for systematic determination of splice
variant transcripts of the human 12.1 gene. This screen identified seven loci of variation, which together have never been
fully defined in humans. Genomic sequence analysis clarified the
splicing mechanisms underlying the observed variation.
Electrophysiological characterization and a novel analytical paradigm,
termed strength-current analysis, revealed that one focus of
variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary
effect on Ca2+-dependent channel inactivation. These
findings significantly expand the anticipated scope of functional
diversity produced by splice variation of P/Q-type channels.
Key words:
alternative splicing; P/Q-type calcium channel; 1A subunit; 12.1 subunit; transcript
scanning; human brain; Ca2+-dependent inactivation; calmodulin
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INTRODUCTION |
P/Q-type calcium channels,
potentially the most abundant voltage-gated calcium channels in the
mammalian brain (Mori et al., 1991 ), constitute prominent pathways of
Ca2+ entry at both presynaptic and
somatodendritic loci throughout the CNS (Catterall, 1998). As such,
these channels not only represent a predominant trigger for
neurotransmitter release in the CNS but also support a host of
essential neuronal responses (Wheeler and Tsien, 1999). Moreover,
spontaneous mutations within these channels underlie a growing list of
inherited forms of ataxia, epilepsy, and migraine (Ophoff et al., 1996;
Zhuchenko et al., 1997). Fitting with the diverse roles of these
channels, their function can be fine-tuned by numerous mechanisms
rather than being limited to a single profile. On the moment-to-moment
time scale, gating can be modulated by channel phosphorylation (Zamponi et al., 1997) and G-protein interactions (Colecraft et al., 2001). More
enduring functional adjustments ensue from variations in the
hetero-oligomeric composition of channels, comprising a principal pore-forming 1A (Cav2.1)
subunit, complexed with auxiliary  , 2 , and
possibly  subunits (Dunlap et al., 1995). P/Q-type channels may
assemble from several different or 2 gene
products, yielding channels with distinctive functional properties
(Patil et al., 1998). Another potential strategy for generating
longer-term functional diversity is alternative splicing of transcripts
for the principal 12.1 subunit. Because the
human 12.1 gene comprises >47 exons (Ophoff
et al., 1996), alternate splicing at just a few exon boundaries could
yield an enormous number of variants, especially when considering the
combinatorial possibilities.
Previous studies of 12.1 have established
instances of splice variation and related functional effects. For
example, in human brain, splicing results in optional translation of
amino acids encoded by exon 47, which includes a poly-glutamine tract
whose abnormal expansion triggers an inherited form of cerebellar
ataxia (SCA6; Zhuchenko et al., 1997). However, a limitation of studies to date arises from the method used to discover splice variants of
12.1, which involves explicit sequencing of
various partial-length cDNA clones of the 12.1
subunit. Because isolation of each clone from a conventional cDNA
library is labor-intensive, extensive screening for splice variation
becomes impractical. Accordingly, it is possible that only a fraction
of splice variants has been revealed by traditional methods.
Here, we applied a recently developed, transcript-scanning approach
(Mittman et al., 1999a,b) to the systematic identification of splice
variation of the human 12.1 gene. This
PCR-based approach rapidly amasses cDNA replicas of all transcript
variants in a specified gene region and thus promises practical
detection of most, if not all splice variants of the transcripts of
this gene. This screen identified seven loci of variation, some of
which are novel and the complete set of which has never been fully
defined in humans. Electrophysiological experiments and a novel
paradigm of analysis revealed that combinatorial inclusion and
exclusion of exons 43 and 44 exerted a primary effect on current
amplitude and a corollary effect on
Ca2+-dependent channel regulation.
Parts of this paper have been published previously in abstract form
(Soong et al., 2000).
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MATERIALS AND METHODS |
Transcript scanning by PCR and sequence analysis. The
overall transcript-scanning approach has been described previously, as
applied to T-type Ca2+ channels (Mittman
et al., 1999a,b). Here, to scan cDNA replicas of human
1A (12.1)
transcripts, we designed PCR oligonucleotide primers to span 1-4 exons
of this gene (Figs. 1A,B), using Oligo 6.1 software
(Molecular Biology Insights, Cascade, CO). The sequence of
oligonucleotides used for all scanning reactions (Fig. 1C) is detailed in Table S-1 (available as on-line supplementary
information at www.jneurosci.org). PCR amplification from whole-brain
(7400-1; Clontech, Palo Alto, CA), cerebellar (7401-1), thalamus
(7188-1), substantia nigra (7193-1), hippocampus (7169-1), cerebral
cortex (7110-1), amygdala (7190-1), and hypothalamus (7429-1) cDNA
libraries produced products that were electrophoresed on an agarose
gel, purified, and cloned into the pCR2.1-TOPO vector by
high-efficiency topoisomerase-based ligation (TOPO TA cloning kit;
Invitrogen, Carlsbad, CA). The ligation products were efficiently
transformed into TOP bacterial cells (TOPO TA cloning kit). Splice
variation sometimes produced gel bands with different mobility, and
these were purified separately before TA subcloning. Eight to 28 individual bacterial colonies from each TA subcloning reaction were
screened by PCR with exon scanning primers (supplemental on-line
information, Table S-1, at www.jneurosci.org), followed by automated
sequencing and display using Prism DNA Sequencing software (Applied
Biosystems, Foster City, CA). Sequences were compared against the
National Center for Biotechnology Information (NCBI) public databases
by using the BLAST program. DNA sequence alignments to detect splicing of exons or alternate use of junctional sites were performed using the
Lasergene Software (DNAStar, Madison, WI). In some instances, an
initial analysis based on size differentiation during gel
electrophoresis was performed.
Intronic sequence analysis. Human genomic DNA (Clontech) was
used to PCR amplify introns 9, 31, and 46 to determine or confirm the
intron and exon organization across some sites for alternative splicing. The primers used for these introns are as follows: for intron
9, int9 forward (F), 5'-TCG CCG AGG ATG AAA CTG AC-3'; and int9 reverse
(R), 5'-CAG CCT CTT CGG GGT TGA GC-3'); for intron 31, int31F, 5'-CGA
TAT CCT CGT GAC TGA-GT-3'; and int31R, 5'-AGC AGA CAG ACA TAA GGC
AG-3'); and for intron 46, int46F, 5'-GGG CCG CTA CAC CGA TGT GG-3';
and int46R, 5'-GTT GAG GGG GCT GGG CTT CC-3'). For other introns, data
mining of the NCBI/European Molecular Biology Library (EMBL) database
proved sufficient to specify key sites controlling splice variation. In
particular, partial intronic sequences at most exonal boundaries were
deposited (Ophoff et al., 1996) under accession numbers Z80114-Z80155. Partial intronic sequence surrounding exon 37b is available under accession number AF144098. Finally, large stretches of the genomic
sequence spanning the human 12.1 gene are
available under accession numbers AC098781 (exon 4), AC022436 (exons
5-10), AC026805 (exons 11-22), AC005305 (exons 22-31), and AC011446 (exons 31*-47); all contained within the consensus reference sequence NT-031915.
Generation and characterization of full-length 1A
(12.1) clones by PCR. A full-length, single-gene
12.1 cDNA library was isolated by heminested
PCR amplification of whole-brain and cerebellar cDNA libraries
(Clontech; Regan et al., 2000). Two sets of primers were used: (1) F1,
5'-CCG GCA GCC TCA GCA TCA GC-3'; and R1, 5'-GGA TCA CAG GGG AAT AGG
AC-3'; and (2) F2, 5'-GCG TAA CCC GGA GCC CTT TG-3'; and R2, 5'-CGG ATC
ACA GGG GAA TAG GAC-3'. The first round of PCR, using oligonucleotides
F1 and R1, followed a touch-down protocol, which was initiated by
denaturation at 94°C for 2 min. This was followed by one cycle of PCR
comprising denaturation at 94.5°C for 25 sec, annealing at 65°C for
30 sec, and extension at 68°C for 8 min. In the next five cycles, the
annealing temperature was initially 63°C and progressively decreased
by 1°C on each cycle. The terminal 28 cycles maintained the annealing
temperature at 58°C. The buffer contained 1× pCRx and 2 mM Mg2+ (Elongase; Invitrogen,
Gaithersburg, MD). The second round of PCR (performed with flanking
oligonucleotides) was performed similarly, except that the initial
annealing temperature was 67°C, and the touch-down protocol was
programmed to decrease annealing temperature by 2°C over the next
five cycles until reaching 55°C. The terminal 15 cycles of PCR
maintained the annealing temperature at 55°C. The ~7 kb PCR product
was subcloned into the pCRXL vector (Invitrogen), transformed into TOP
bacterial cells, and picked into 96-well microtiter plates for PCR
screening and analyses. Individual clones were verified to be
full-length by performing restriction enzyme digestions with
BamHI and XbaI, and observing a resultant band of
~7 kb.
The particular type of splice variant encoded by a given full-length
1A (12.1) clone was
determined by performing custom PCRs spanning the exonal region of
interest on individual clones (Fig. 1C). Size or direct sequence
analysis of scanning reaction products or both uniquely identified the
type of splice variant. Compilation of like results for many such
clones yielded the aggregate summary in Table 1.
Construction of 1A (12.1) cDNA
clones with variable splicing at exons 43 and 44. The parental
human 1A (12.1) clone
(Sutton et al., 1999) was a gift from Dr. Terry Snutch (University of British Columbia), and its splice variant content at the seven loci
(Fig. 2) is the following:  10A (+G);
16+/17+; 17 (-VEA), 31* - (-NP);
37a (EFa);
43+/44 ;
47A. To generate 1A
(12.1) clones containing the other exon 43/44
splice combinations, a human brain cerebellar cDNA library (7401-1;
Clontech) was amplified using a forward primer (5'-GAA AGC GGC CTC AAG
GAG AG-3') and a reverse primer containing an XbaI site
(5'-tgc gtc tag aTC GCC CGG GCT TAG CAC CAA-3'). The PCR
products were cloned into pCR2.1-TOPO vectors (TOPO TA cloning kit),
and TOP bacterial transformants were placed into 96-well plates for PCR
screening to determine the presence or absence of exons 43 and 44. Fifteen clones were then selected for DNA sequencing, and appropriate
clones were substituted by BstEII and XbaI
restriction enzyme sites into the original full-length 1A (12.1) clone to
generate the various constructs subjected to electrophysiological
study. In the context of electrophysiological results, these four
constructs are referred to as
43+/44+,
43/44+,
43+/44, and
43/44.
Transient expression of Cav2.1 splice variants in
HEK293 cells. cDNA for one of four human
1A subunits
(43+/44+,
43/44+,
43+/44, and
43/44)
was transiently cotransfected with auxiliary
2a and 2b subunits in HEK293 cells, according to previously described methods (Peterson et
al., 1999). 2a minimized voltage inactivation
(Patil et al., 1998), thereby enhancing resolution of
Ca2+-dependent regulation. Two to 3 d
later, whole-cell recordings were obtained at room temperature.
Whole-cell electrophysiology and analysis. The bath solution
contained (in mM): 140 TEA-MeSO3, 10 HEPES, pH 7.3, and 5 CaCl2 or
BaCl2, 300 mOsm, adjusted with glucose. The
internal pipette solution contained (in mM): 135 Cs-MeSO3, 5 CsCl2, 0.5 EGTA, 1 MgCl2, 4 MgATP, and 10 HEPES, pH 7.3, 290 mOsm, adjusted with glucose. Reported voltages are uncorrected for a
-11 mV junction potential, and true voltage may be obtained by
subtracting 11 mV from reported values. Whole-cell currents, obtained
under voltage clamp with an Axopatch 200A amplifier (Axon Instruments),
were filtered at 2 kHz and sampled at 10 kHz. Series resistance was typically 1-2 M after >70% compensation. Leaks and capacitive transients were subtracted by a P/8 protocol. Test pulse
depolarizations were delivered every 60 sec (facilitation protocol) or
100 sec (inactivation protocol).
Ca2+-dependent facilitation was determined
from the normalized charge difference, Q, obtained by
integrating the difference between normalized traces ± prepulse
(see Fig. 4, middle). Test pulse current traces were
normalized to unity at the end of 50 msec depolarizing pulses to 0 or 5 mV. The fraction of channels facilitated by prepulse
(Ffacilitated) is directly
proportional to Q divided by the time constant ( ) of
facilitation, yielding relative facilitation (RF = Q/). This follows by assuming that all channels are
initially in the normal mode at test pulse onset, and subsequent shifts
to the facilitated mode occur monoexponentially with time constant .
Then, RF = Ffacilitated × [Po,facilitated  Po,normal]/Po,facilitated,
where Po,facilitated and
Po,normal are steady-state open
probabilities in facilitated and normal modes, respectively. was
explicitly determined from Ca2+ traces in
each cell before calculation of RF.
Ba2+ RF was calculated by using
values determined from Ca2+ traces in
the same cell. All average data are presented as mean ± SEM after
analysis by custom-written software in MATLAB 6.1 (MathWorks, Natick,
MA). Smooth-curve fits to data are by eye.
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RESULTS |
The entire 1A
(12.1) cDNA, encoding the principal
pore-forming subunit of P/Q-type calcium channels (Fig.
1A), was systematically screened for alternative splicing of its constituent exons (Fig. 1B). We exploited a transcript-scanning strategy
(Mittman et al., 1999a,b), the crux of which was to perform multiple
PCRs that amplified overlapping segments spanning the entire
12.1 cDNA (Fig. 1C), as represented
in human brain cDNA libraries. Each "scanning reaction" serves to
rapidly pool and amplify numerous copies of all splice variations
within a specified region of the 12.1 subunit,
thus greatly improving the practicality and sensitivity of discovering
splice variants compared with traditional cloning and identification of
partial-length channel cDNAs from a brain library. Furthermore, because
of the overlapping and all-encompassing coverage of the scanning
reactions, most of which spanned at least two exon boundaries, analysis
of the resulting set of PCR products should in principle reveal most,
if not all, splice variations. To identify splice variations
represented within a given scanning reaction, the PCR product was
cloned in bacteria, and individual bacterial colonies were subject to
PCR with exon scanning primers (supplemental on-line information, Table
S-1, at www.jneurosci.org). The identity of the splice variant
composition contained in individual clones was then determined by
heterogeneity of product size, specificity of primers used, and direct
sequencing. Figure 1D illustrates the size analysis
for PCR 5, spanning exons 16 and 17. The larger species (650 bp)
results from amplification of transcripts containing both exons 16 and
17 (+16/17), and the smaller species (454 bp) results from variant
transcripts lacking both exons 16 and 17 (-16/17).
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Figure 1.
Schematic representation of P/Q-type calcium
channel 1A (12.1) subunit and PCR
scanning reactions used to detect splice variation therein.
A, Diagram of 12.1 channel backbone
structure, showing four homologous domains
(I-IV), each with six transmembrane-spanning
regions (1-6). The C-terminal tail contains
structures postulated to be important for Ca2+
regulation of the channel. EF, EF-hand;
IQ, IQ-like CaM interaction domain; CBD,
CaM interaction domain. B, Locations of exon transcripts
corresponding to the backbone diagram in A.
C, Locations of scanning reactions to detect splice
variation (for details, see on-line Table S-1, at
www.jneurosci.org). D, Size differentiation of
splice variants (±16/17), revealed by scanning reaction 5.
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The transcript-scanning strategy revealed a total of seven loci for
alternative splicing of the 12.1 gene, as
summarized in Figure 2: (1) at the
beginning of exon 10 in the I-II interdomain loop (10/10A/10B),
there can be insertion of a valine and glycine, insertion of a glycine
alone, or no insertion of either amino acid; (2) in the IIS6 region,
exons 16 and 17 can be present or absent (±16/17); (3) near the end of
exon 17 in the II-III interdomain loop (17/17A), there can be
optional omission of the tripeptide VEA; (4) near the beginning of exon
32 in the IVS3-IVS4 loop (±31*), there can be insertion of the
dipeptide NP; (5) at the proximal end of the C-terminal tail, there is
mutually exclusive use of exon 37a or
37b
(37a/37b), resulting in
channels with one of two versions of an EF-hand motif (Kretsinger,
1976), which often represents a canonical
Ca2+ binding site; (6) downstream of the
CBD domain, exons 43 and 44 can be present or absent in all four
combinations (43±/44±); and (7) at the distal end of the C terminus,
there can be insertion of a pentanucleotide GGCAG 5' of the beginning
of exon 47, thus allowing in-frame translation of exon 47 to produce a
long version of the C terminus (47). Otherwise, omission of the GGCAG
in variant transcripts causes a frameshift, leading to stop codon
termination of channel subunits near the beginning of exon 47 (47).
Although isolated subsets of these splice variants have been reported
for rat, mouse, and human (see Discussion), specific variations at loci
2 and 6 are novel, and the full set has not been delineated in
human.
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Figure 2.
Seven loci of 12.1 splice variation
detected by transcript scanning. The postulated schematic diagram
(top) shows a more detailed secondary structure of
12.1, along with loci of splice variation
(1-7), labeled according to transcript variant
names. CI, Ca2+ inactivation region,
containing structures believed important for CDI such as the EF-hand
and IQ domain. Detailed changes in amino acid composition resulting
from splice variation at each of seven loci are shown below. At
locus 3, deletion of exons 16 and 17 would remove half
the P-loop and the entire IIS6 segment, ostensibly producing a
nonfunctional channel.
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To infer the mechanisms underlying the observed splice variation, we
determined pertinent intronic sequences from the genomic DNA sequences
corresponding to 1A
(12.1). With regard to 10/10A/10B variation in the I-II interdomain loop (Fig.
3A), we obtained a 2.8 kb
amplicon from PCR amplification of human genomic DNA across intron 9. Sequencing of the exon-intron boundaries suggests a possible mechanism
for the three possible outcomes of splicing, involving alternative
nonconsensus splice acceptors. The 10 (+VG) and 10A (+G) scenarios
appear to exploit an unconventional GT/TG acceptor-donor site pair,
whereas the no insertion (-) 10B case uses a more common GT/AG
pair. Although much less common, the use of a TG acceptor has been
found to represent ~0.04% of 22,489 mammalian expressed sequence
tag-verified splice pairs (Burset et al., 2000). Our sequencing result
differs from that provided by Ophoff et al. (1996) (accession number
Z80123) regarding the junction between intron 9 and exon 10, where
their database information indicates the sequence ccattgtagGAG instead
of the ccattgttgGAG that we have determined. However, our sequence is identical to deposited genomic sequence found in GenBank AC022436. Concerning the dual cassette, ±16/17 splicing (Fig. 2, locus
2), the published sequence for introns 15 and 17 (accession
numbers Z80127-Z80129 and AC026805) points to customary alternative use of a rather canonical GT/AG donor-acceptor site pair (Fig. 3B). With respect to 17/17A (Fig. 2, locus 3),
the published intronic sequence (accession numbers Z80128-Z80129 and
AC026805) suggests that there is alternative use of junctional donor
sites after exon 17 (Fig. 3C). Turning to ±31* splicing
(Fig. 2, locus 4), EMBL partial sequence for the
margins of intron 31 (accession numbers Z80142-Z80143) failed to
reveal alternative use of acceptor-donor sites near exon-intron
boundaries as a plausible basis for this splice variation (Fig.
3D). However, data mining the interior region of intron 31 (accession number AC011446) revealed a potential six-nucleotide exon
(exon 31*) that encodes NP, and the putative exon 31* is flanked by
canonical GT/AG acceptor-donor sites (Fig. 3D).
Furthermore, a pyrimidine-rich tract resides just upstream of the
acceptor AG site, further supporting this region as a legitimate exon
(Sharp and Burdge, 1997; Zhang, 1998). Thus, cassette inclusion of a
putative exon 31* appeared to be the mechanism of optional NP
insertion. Nonetheless, database annotations failed to acknowledge this
region as a potential exon, and there was no independent confirmation
of the putative exon 31* sequence. Accordingly, we used exonic primers
from exons 31 and 32 to amplify an ~10 kb fragment encompassing
intron 31 in its entirety. Nested PCR of this large, intronic DNA
fragment enabled the cloning of a 0.9 kb fragment, the sequencing of
which confirmed the essential features of the presumed exon 31*
sequence. Hence, we conclude that ±31* splice variation involves
optional exclusion and inclusion of a novel exon 31* encoding NP. To
understand mutually exclusive cassette splicing of exons 37a and 37b
(accession numbers Z80146-Z80148, AF144098, and AC011446) (Fig. 2,
locus 5), as well as combinatorial splicing at exons 43 and 44 (accession numbers Z80150-Z80153 and AC011446) (Fig. 2, locus
6), data mining of exon-intron border sequences revealed alternative use of canonical GT/AG acceptor-donor site pairs (Fig. 3E,F). Finally, with regard to locus 7 (Fig. 2), we
used PCR to explicitly confirm published intron-exon boundaries among
exon 46, intron 46, and exon 47 (accession numbers Z80154-Z80155 and
AC011446). The sequence (Fig. 3G) suggests that alternate use of a canonical acceptor site just 5' of exon 47 can generate the
insertion of a pentanucleotide of GGCAG, which would result in a
frameshift whereby exon 47 would be read in-frame after exon 46. Otherwise, GT/AG splicing precisely at the exon boundaries would yield
an in-frame stop codon immediately after exon 46, yielding an
1A (12.1) subunit
with a shorter C-terminal tail. In summary, these results indicate that
alternative splicing produces a rich ensemble of channel
customizations, exploiting both traditional and rather unexpected
mechanisms that include a six-nucleotide exon and noncanonical splice
acceptor-donor site pairs.
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Figure 3.
Postulated mechanisms underlying splice variation
of 12.1. A-G, Top
row, Nucleotide sequence of relevant exon-intron boundaries.
Bottom row, resultant transcript and encoded amino acids
of each variant. Mechanisms for splicing were (A,
G, alternate acceptors (A, G), cassette
(B, D-F), and alternate donor
(C).
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Beyond establishing the spectrum of possible splice variations, it was
important to gauge how frequently the various splice configurations
occur. Moreover, because our exon scanning was performed on a cDNA
library whose constituent clones need not span complete channel
transcripts, it was also worth establishing whether the observed
variants were present in full-length 1A (12.1) cDNAs. To address these issues, we
reexamined key transcript-scanning reactions, this time performed on a
different library as a template, a full-length, single-gene
1A (12.1) library,
obtained by long PCR amplification (Regan et al., 2000) of the original
human cerebellar library. Results of the analysis are summarized in
Table 1, inspection of which reveals
clear preferences for certain splice variants. For example, the -16/17
variant is undetectable or extremely rare, whereas 17A (-VEA) and
+31* (+NP) variants heavily predominate. Other loci demonstrate more
even representation of splice options, such as the distribution between
37a (EFa) and
37b (EFb) variants. Although two of the variants,
43/44+ and
43/44
(Table 1), are just detectable, it is quite possible that such "rare" splice variants could be more prevalent within specific types of neurons of the cerebellum. Furthermore, it would be quite interesting if these variants were more prevalent in other portions of
the brain, fitting with a general theme in which regional distribution profiles of splice variants are customized by development or external and internal cues. In fact, this latter possibility was strongly supported by transcript scans across exons 43 and 44 in human brain
region-specific cDNA libraries (Table 2).
Here, the fraction of
43/44+
transcripts reached 10% in amygdala, whereas the fraction of 43/44
transcripts exceeded 20% in amygdala, thalamus, and substantia nigra.
Overall, the analysis establishes that, except for the 16/17
variant, the ensemble of observed splice variants (Fig. 2) contains legitimate customizations of full-length channel transcripts, and that
variations in the distribution within the ensemble of possibilities
represents a potentially rich dimension for functional regulation.
Splice variation of 1A (12.1)
channels could have important functional correlates, as already shown
by previous studies of partial sets of variants (Bourinet et al., 1999;
Hans et al., 1999; Krovetz et al., 2000; Tsunemi et al., 2002). The
more exhaustive suite of variants delineated here by systematic exon
scanning raises expectations that splice-related functional diversity
may well be considerably larger than currently appreciated. As a first step in defining the potentially broader spectrum of functional sequelae, we focused on novel splice variations in the
1A C-terminal tail, because this region
contains critical structural determinants for
Ca2+ feedback regulation of corresponding
channels. In particular, Ca2+/CaM binding
to an IQ-like binding motif in exon 40 (DeMaria et al., 2001), or
possibly to a CBD binding motif in exon 42 (Lee et al., 1999, 2000),
initiates both channel facilitation and inactivation by
Ca2+ (DeMaria et al., 2001). The proximity
of these structures to exons 43 and 44 (Figs. 1, 2), which are frequent
sites of alternative splicing, suggested that combinatorial splice
variation at this locus might tune Ca2+
regulation, especially given the significant representation of each
combinatorial possibility (Table 1).
Figure 4 compares the
Ca2+ facilitation properties of
1A (12.1) channels
with all the possible variations at exons 43 and 44, with background
splice variant structure delineated in Materials and Methods. Figure
4A shows the behavior for the
43+/44+
construct, using our previously reported methods of characterization (DeMaria et al., 2001), Facilitation was readily resolved in exemplar test pulse currents (+0 mV) with Ca2+ as
the charge carrier (middle traces). Without a prepulse (to +20 mV), the test pulse waveform showed an initial rapid component of
activation, followed by a slower phase of
Ca2+ current increase (gray
trace, arrow). This overall waveform morphology provides a direct
indication of facilitation, because the biphasic kinetics arises from
fast activation in a normal mode of gating, followed by more gradual
Ca2+-driven conversion to a facilitated
gating mode with greater open probability (Takahashi et al., 2001). For
test pulse waveforms preceded by a prepulse (middle, black
trace), channels were initially "prefacilitated" by
Ca2+ entry during the prepulse. Hence,
these currents activated rapidly to the fully facilitated level, as
would be expected for fast activation of channels that reside
predominantly within the facilitated gating mode at the test pulse
onset. With Ba2+ as the charge carrier,
exemplar test pulse currents (top traces) activated rapidly,
with no appreciable slow phase, regardless of the presence or absence
of a prepulse, consistent with "trapping" of channels in the normal
mode of gating. To quantify facilitation, we integrated the difference
between normalized test pulse currents in the absence and presence of a
prepulse (see Materials and Methods), and this integral was used to
determine the RF induced by a voltage prepulse. With
Ca2+, relative facilitation demonstrated a
bell-shaped dependence on prepulse voltage, providing a hallmark of a
Ca2+-driven process. By contrast, the
corresponding relationship with Ba2+ was
far smaller, reflecting weak background G-protein modulation (DeMaria
et al., 2001). The difference between Ba2+
and Ca2+ relationships for a +20 mV
prepulse, g, then provides a convenient quantifier of pure
Ca2+-dependent facilitation. With respect
to the effects of splice variation at exons 43 and 44, Figure
4B-D revealed no detectable change in the profile of
Ca2+ facilitation.
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Figure 4.
Splicing at exons 43 and 44 does not affect CDF
properties. A-D, Top,
Prepulse voltage protocol used to reveal facilitation, with fixed test
pulse depolarization to 0-5 mV and 30 msec prepulse depolarization.
Middle, Exemplar Ba2+ and
Ca2+ current traces corresponding to
specific voltage pulses diagrammed at top. The
gray trace corresponds to the trial without a prepulse.
The arrow in A marks slow activation
phase characteristic of CDF. Bottom, Population averages
(from n cells) of strength of CDF
(RF) plotted as a function of prepulse potential.
Ca2+ data are plotted as filled
symbols; Ba2+ data are plotted as
open symbols. g is a metric for the
strength of pure Ca2+-dependent facilitation, and
mean values and SEM are shown.
|
|
By contrast, splice variation appeared to have substantial effects on
Ca2+-dependent inactivation (CDI) of
channels, as well as on the overall amplitude of the current (Fig.
5). Figure 5A shows the method of characterization for the
43+/44+
construct. When viewed on a longer time base than used to resolve facilitation, specimen test pulse currents evoked by step
depolarization to +10 mV could be seen to exhibit marked CDI, because
currents decayed clearly faster with Ca2+
versus Ba2+ as the charge carrier
(top). The Ba2+ trace has been
scaled downward approximately two times to enhance visual comparison of
kinetics here and throughout. We quantified inactivation by determining
the fraction of the peak current remaining after depolarizing for 800 msec (r800), plotted as a function of
test pulse voltage (middle). Population means for these
relationships confirmed a clearly deeper decay of
r800 with
Ca2+ versus
Ba2+, and the unmistakable U shape of the
relationship with Ca2+ provided a classic
hallmark for a Ca2+-driven process. The
comparatively modest decline of the Ba2+
relationship is believed to reflect a separate voltage-dependent inactivation process (Jones, 1999), so the distance between
Ca2+ and Ba2+
relationships at +10 mV, f, furnished a convenient index of
pure CDI. Overall current size was assessed by averages of peak
Ca2+ current amplitude plotted as a
function of the test pulse voltage. Inspection of the corresponding
analysis for the other splice variants (Fig. 5B-D) revealed
two effects. First, CDI was clearly more pronounced in the
43/44
construct, with f values of ~0.4. Other constructs showed
less CDI, with f values hovering between 0.2 and 0.3. Second, the
43/44
construct produced twofold larger currents than the
43+/44
construct (Fig. 5, compare A, D, bottom), whereas the other
two constructs manifested intermediate current amplitudes (Fig.
5B,C, bottom). No clear differences in the voltage
dependence of activation or inactivation were observed among the
variants (Fig. 5 legend). One plausible mechanism for the effects on
current size is that exons 43 and 44 may regulate the number of
expressed channels, possibly by altered turnover rates secondary to
complexation of this region of the channel with adaptor molecules
(Maximov et al., 1999). Overall, splice variant effects on CDI and
levels of current hold enormous potential biological impact for
synaptic plasticity (von Gersdorff and Borst, 2002) and
neurodegenerative disease (Pietrobon, 2002).
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Figure 5.
Splicing at exons 43 and 44 affects Ca2+ current amplitude and CDI.
A-D, Top, One second
depolarizing voltage pulse used to reveal CDI, along with exemplar
Ba2+ and Ca2+ current
traces. The gray trace was obtained with
Ca2+ as the charge carrier; the black
trace was obtained with Ba2+. Scale bar
corresponds to the Ca2+ trace;
Ba2+ traces were scaled downward to facilitate
comparison of decay kinetics. Middle, Population
averages (from n cells) for inactivation properties, as
gauged by r800, the fraction of peak
current remaining after an 800 msec depolarization plotted as a
function of test pulse potential. Ca2+ data are
plotted as filled symbols; Ba2+ data
are plotted as open symbols. f is a
metric for the strength of pure Ca2+-dependent
inactivation, and mean values and SEM are shown. Bottom,
Peak current versus test pulse potential with Ca2+
as the charge carrier. Data were averaged from same cells as the
r800 plot above. A versus
D, Ca2+ current amplitude
(bottom) and CDI are clearly greater in the
43/44 versus
43+/44+ variant, with other
splice forms showing intermediate behavior (B,
C). No significant difference in the voltage dependence
of activation or inactivation was observed. Half-activation voltages,
as determined from current-voltage relationships
(bottom) with Ca2+ as the charge
carrier, were (in mV): 6.0 ± 1.6 (43+/44+; n = 8), 1.8 ± 0.7 (43/44+;
n = 4), 1.7 ± 1.3 (43+/44; n = 5), and 5.6 ± 1.3 (43/44; n = 11). Half-inactivation voltages, as determined from 1 sec prepulse
inactivation protocols with Ca2+ as the charge
carrier, were (in mV): 2.4 ± 2.3 (43+/44+; n = 8), 6.5 ± 1.6 (43/44+;
n = 4), 0.1 ± 1.3 (43+/44; n = 5), and 2.1 ± 1.1 (43/44; n = 11).
|
|
Before concluding that splice variation at exons 43 and 44 entails two
independent functional outcomes, we questioned whether the effects
might be coupled through a single mechanism. We had postulated
previously that 1A
(Cav2.1) CDI is driven by a "global" Ca2+ concentration signal that integrates
Ca2+ influx through multiple channels
(DeMaria et al., 2001). By contrast, we also postulated that
Ca2+-dependent facilitation (CDF) responds
primarily to a "local" Ca2+ signal
that predominantly reflects the activity of individual channels. If
these postulates hold true, then simply augmenting the number of
expressed channels could increase the global
Ca2+ signal and thereby enhance CDI, even
without any change in the intrinsic properties of individual channels.
Thus, the only direct effect of splice variation may be to increase the
number of expressed channels, and the enhanced CDI may result as a
corollary outcome of elevated channel number. Alternatively, splice
variation could change the inherent propensity for each channel to
undergo CDI, given the same global Ca2+
signal. Finally, a combination of the above two scenarios would also
accord with the functional results presented thus far.
To distinguish among these, we pursued two sets of experiments that
consolidated and refined our global
Ca2+-CDI hypothesis in a manner
permitting discrimination among the three possible scenarios. In the
first, we examined the effects of a high intracellular concentration
(10 mM) of the rapid Ca2+
chelator BAPTA on CDF and CDI. Under these conditions, processes driven
by global Ca2+ signals should be strongly
attenuated, whereas those responding with extreme selectivity to local
Ca2+ influx might be mostly spared
(Deisseroth et al., 1996). Figure 6,
A and B, displays striking results in this regard
for the
43+/44+
construct. Exemplar traces (Fig. 6A) suggest complete
sparing of CDF in the face of virtual elimination of CDI. Population
data (Fig. 6B) entirely confirmed these trends,
clearly demonstrating that CDF responds very selectively to local
Ca2+ influx through individual channels,
and that CDI requires a more global Ca2+
signal. This experiment did not, however, exclude the possibility that,
although the locus of Ca2+ sensing for CDI
lies somewhat more distant from the channel pore than does the sensor
for CDF (accounting for BAPTA sensitivity), a single channel could
still manage to inactivate itself. Moreover, the first experiments did
not gauge how sensitively different splice variants might undergo CDI
in response to a given global Ca2+ signal,
and splice variation could have produced CDI effects via changes in
such sensitivity.
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Figure 6.
CDF responds selectively to local
Ca2+ influx through individual channels, whereas CDI
responds selectively to global Ca2+ influx through
many channels. A, Exemplar traces
illustrating complete sparing of CDF and total elimination of CDI by
intracellular 10 mM BAPTA. Format is identical to that of
Figures 4 and 5. B, Population data, corresponding to
exemplar traces in A, confirm selective
elimination of CDI by BAPTA. Format is identical to that of Figures 4
and 5. C, Theoretical predictions of strength-current
analysis for Ca2+ regulatory processes with
different selectivities for local versus global Ca2+
influx. The y-axis plots the strength of
Ca2+ regulation; the x-axis plots
peak Ca2+ current; curves are
schematic. Left, Scenario for a Ca2+
regulatory process selective for local Ca2+ influx
through individual channels. Right, Case for a
Ca2+ regulatory process triggered by both local
Ca2+ influx through individual channels (positive
y-axis intercept) and global Ca2+
influx through many channels (increasing regulatory strength with
growing I). D, Strength-current
analysis for CDF and CDI properties of various splice variants. Each
symbol corresponds to one cell. Different
symbols correspond to splice variants as follows:
filled circle,
43+/44+; filled
triangle, 43/44+;
plus sign,
43+/44; and open
square, 43/44.
Left, CDF follows prediction of a process completely
selective for local Ca2+ influx through individual
channels, fitting with insensitivity to BAPTA in A and
B. Right, CDI follows prediction of a
process entirely selective for global Ca2+ influx
through many channels (with 0-valued y-intercept),
fitting with elimination of CDI by BAPTA in A and
B. To resolve the y-intercept of the
right CDI I plot, some
43/44 (open
square) data here were obtained from cells only 12-18 hr after
transfection rather than the usual 48-72 hr used for the bulk of the
data, including those in Figure 5.
|
|
To address the remaining ambiguities, a novel experimental paradigm was
devised. We reasoned that the strength of channel regulation would
respond differently to progressive increases in the global
Ca2+ signal, depending on the selectivity
and sensitivity of the modulatory process for global versus local
sources of Ca2+ influx (Fig.
6C). As a practical measure of global
Ca2+ levels, we chose to measure peak
Ca2+ current amplitudes
(I). If a process were completely selective for
Ca2+ influx through individual channels,
without regard for influx through adjacent or neighboring channels,
then regulatory strength should be independent of overall
Ca2+ current amplitude (Fig. 6C,
left). Alternatively, if a regulatory process were sensitive to
both local and global Ca2+ influxes, then
regulatory strength should increase with increasing Ca2+ current amplitude (Fig. 6C,
right), and the y-axis intercept of the relationship
should indicate the regulatory strength resulting from
Ca2+ influx through an individual channel.
Finally, if a process were selectively responsive to global
Ca2+ influx, whereas the influx via an
isolated channel alone were unable to inactivate itself, then the
regulatory strength relationship would be as shown in Figure
6C (right), except that the y-axis intercept value would be 0.
Figure 6D shows the results for such a
"strength-current" experiment, in which the strength of CDF (or
CDI) and peak Ca2+ current amplitudes for
individual cells contribute separate data points. Plots of the metric
g (CDF strength) versus I (Fig. 6D, left) reveal a completely flat relationship, indicating entirely selective reliance on local Ca2+, a result
fitting with deductions from the BAPTA experiments above (Fig.
6B, left). The ability of a single flat relationship to fit data from all splice variants, plotted as different symbols, suggests that the CDF sensitivity to local
Ca2+ influx is the same among splice
variants, as might be expected from the similarity of aggregate
averages for CDF analysis (Fig. 4A-D, bottom). By
contrast, plots of the descriptor f (CDI strength) versus
I strikingly demonstrate a positive correlation (Fig.
6D, right), with a 0-valued y-axis
intercept. This indicates that CDI is selectively reliant on global
Ca2+ influx, and that an isolated channel
would be incapable of inactivating itself. Because data from all splice
variants, plotted as different symbols, appear to define a single
relationship, CDI sensitivity to global
Ca2+ influx is probably identical among
splice variants, at least within the resolution of this experimental paradigm.
In summary, in-depth biophysical analysis (Fig. 6) indicates that the
direct effect of splice variation at exons 43 and 44 is to regulate
overall current amplitude, possibly by variations in channel number.
Variations in CDI, especially prominent with the
43/44
construct (Fig. 6D), result as corollary outcomes of
fluctuations in current amplitude playing through an intrinsic
sensitivity of CDI for global Ca2+ levels.
| |
DISCUSSION |
Transcript scanning compared with previous screens for
splice variation
Transcript scanning (Mittman et al., 1999a,b) was applied to
screen systematically for splice variants of the human
1A (12.1) gene. Locus
1 (Fig. 2) is novel for human, although there are rat analogs (Bourinet
et al., 1999). Valine insertion slowed voltage inactivation but
enhanced G-protein inhibition and PKC upregulation (Bourinet et al.,
1999).
Locus 2 (±16/17) is new, although there are human analogs in
1B (12.2, N-type) and
1E (12.3, R-type)
(Mittman and Agnew, 2000). Absence of the -16/17 variant in
full-length 12.1 transcripts (Table 1)
suggests that this variant may only be present in P/Q-type hemichannels
(Scott et al., 1998). Mutant 12.1 hemichannels
may cause episodic ataxia 2 (Ophoff et al., 1996), and there are
hemichannel analogs for 1C
(11.2, L-type; Wielowieyski et al., 2001) and 12.2 (Mittman and Agnew, 2000), the latter of
which may dominant negatively suppress full-length subunits (Raghib et
al., 2001).
Optional VEA insertion (locus 3), within the "synprint" region for
channel-SNARE-complex interaction (Rettig et al., 1996; Zhong
et al., 1999), has not been explicitly linked to splice variation, but
suggestions of such have been raised (Hans et al., 1999).
Locus 4 splicing has been found in rat brain (Bourinet et al., 1999)
and human spinal cord (Krovetz et al., 2000), although the hypothesized
mechanism used obscured splice acceptor-donor pairs (Bourinet et al.,
1999). Identification of candidate exon 31* provides a simple
explanation involving conventional GT/AG splice pairs. NP insertion
decreases  -Aga IVA sensitivity (Bourinet et al., 1999; Hans
et al., 1999) and shifts the voltage dependence of activation (Bourinet
et al., 1999) and inactivation (Hans et al., 1999; Toru et al., 2000).
In 12.2, an analogous variant, encoding ET
insertion in the corresponding IVS3-S4 loop, also affects activation
and inactivation, and a similar cassette-exon mechanism was advanced
(Lin et al., 1999).
Mutually exclusive splicing at locus 5 was detected in
12.1 clones from rat brain (Bourinet et al.,
1999), human brain (Zhuchenko et al., 1997), and human spinal cord
(Krovetz et al., 2000). Such splicing yields two versions of an
EF-hand-like structure, typically supporting
Ca2+ binding (Kretsinger, 1976). The
analogous EF-hand motif in 11.2 is
essential for its CDI (DeLeon et al., 1995; Zuhlke and Reuter, 1998;
Peterson et al., 2000), but the motif probably transduces CDI rather
than binds Ca2+ (Peterson et al., 2000).
Instead, the trigger for Ca2+ regulation
of either 11.2 or
12.1 is Ca2+/CaM
interaction with a distinct IQ-like region (Peterson et al., 1999; Qin
et al., 1999; Zuhlke et al., 1999; DeMaria et al., 2001). Hence, there
is reason to wonder whether EF-hand splicing could alter
Ca2+ regulation of P/Q-type channels
(Bourinet et al., 1999; Chaudhuri et al., 2001).
Splicing of both exons 43 and 44 (locus 6) is novel; previous reports
describe optional inclusion of exon 44 alone (Zhuchenko et al., 1997;
Krovetz et al., 2000). Omitting exon 44 may diminish voltage-dependent
inactivation (Krovetz et al., 2000), although we did not detect this
effect (Fig. 5). The differing results may arise from our use of the
auxiliary 2a subunit to diminish voltage-dependent inactivation and enhance quantification of
Ca2+-dependent regulation (Bourinet et
al., 1999; DeMaria et al., 2001). A previous study (Krovetz et al.,
2000) used 1a, 1b, 3, and 4 subunits;
all would enhance voltage-dependent inactivation (Patil et al.,
1998).
Locus 7 (47/47) is well documented in human brain (Zhuchenko et al.,
1997; Hans et al., 1999), human spinal cord (Krovetz et al., 2000), and
mouse (Toru et al., 2000). In the 47 variant (Mori et al., 1991;
Ophoff et al., 1996), a stop codon terminates translation before exon
47. Pentanucleotide insertion in the 47 form causes a frameshift
permitting translation of exon 47. Expansion of a poly-glutamine tract
encoded in exon 47 triggers SCA6 (Zhuchenko et al., 1997), and we
speculate that developmental enhancement of the 47 variant could help
explain the delayed adult onset of this disease.
Finally, still other variants probably exist. Transcript scanning
poorly resolves alternate versions of bracketing exons (1 and 47),
because little is known about the respective upstream or downstream
sequences against which scanning reaction oligonucleotides can be
designed. Thus, the upstream oligonucleotide for scanning reaction 1 (Fig. 1C) was designed against the 5' untranslated region of
the known version of exon 1. Likewise, the downstream oligonucleotide
for reaction 15 (Fig. 1C) was designed against the 3'
untranslated region of the known exon 47. These reactions would detect
use of alternate splice acceptor-donor sites with the known exons
(e.g., 47/47) but not of potential alternate versions of exons 1 and
47 (Tsunemi et al., 2002). Also, rare cell-specific RNA editing
restricted to certain cells (Tsunemi et al., 2002) could elude our
screen of cell population libraries.
Strength-current analysis
According to strength-current analysis, CDF fits with a process
completely selective for Ca2+ influx
through individual channels (Fig. 6C,D, left), and CDI fits
with a process completely selective for global
Ca2+ (Fig. 6C,D, right).
Moreover, splice variation of exons 43 and 44 did not cause deviation
from a baseline relationship, arguing against changes in intrinsic
Ca2+ sensitivity.
To refine this interpretation and broaden understanding, we explore two
alternative scenarios. Consider first a hypothetical outcome in which
splice variation increases the intrinsic sensitivity of channel CDI to
global Ca2+ (Fig.
7A).
Ca2+ current (I)
produces the same global Ca2+ signal, but
channels inactivate more strongly in response to the same global
Ca2+, causing an upward-shifted relation.
Also, the y-axis intercept would probably shift upward,
because increased CDI sensitivity for global
Ca2+ would likely pertain to
Ca2+ influx through individual channels.
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Figure 7.
In-depth consideration of strength-current
analysis. A, Theoretical curves
illustrating the anticipated shift (arrow) that would
result from increased intrinsic Ca2+ sensitivity of
a regulatory process for global Ca2+. The curvature
of relationships reflects saturation of Ca2+
responsiveness, the metric quantifying regulatory strength, or both.
B, Underlying transformations connecting peak
Ca2+ current amplitude
(I) and regulatory strength. Increased
intrinsic Ca2+ sensitivity would affect the second
transformation, whereas cell-geometric factors such as channel
clustering would affect the first. Changes in either transformation
could impact the overall strength-current relationship.
C, Diagram illustrating how increased channel clustering
could enhance the transformation between I and the
global Ca2+ signal relevant to channel
regulation.
|
|
Considering the subtransformations linking CDI and I (Fig.
7B) reveals another interpretation of the upward shift (Fig.
7A). Channel Ca2+ influx must
first be transformed into a relevant global
Ca2+ signal, and the latter signal drives
Ca2+ regulation (e.g., CDI). The first
transformation reflects cell channel geometry, such as channel
clustering (Fig. 7C); the second pertains to intrinsic
Ca2+ sensitivity of channels for global
Ca2+. Hence, enhanced channel clustering
(Fig. 7C) could amplify the transformation from I
to global Ca2+, thereby causing the net
effect in Figure 7A. In addition, if channels are only
expressed as tightly packed clusters but not as individual channels,
the y-intercept would reflect CDI resulting from
Ca2+ influx through an individual channel
cluster rather than through one channel. Also, increasing cluster size
would possibly elevate the y-intercept.
Fortunately, our CDI data (Fig. 6D, right) indicate
no shift in the relationship with splice variation. This outcome has
only one likely interpretation: that splice variation changed neither Ca2+ sensitivity of channels for CDI nor
channel clustering. The differences in CDI among splice variants solely
reflect variations in the amplitude of the current.
Mechanism of current enhancement by exon 43/44 splicing
The remaining uncertainty concerns the basis for alterations of
current amplitude with exon 43/44 splicing. Because I = N Po i, where N is
channel number per cell, Po is the
open probability, and i is the unitary current, changes in
N, Po, and i
could underlie the variation in I with exonal splicing.
Which mechanism(s) underlies the data?
Fortunately, the invariance of CDF with splice variation (Fig.
6D, left) favors a single mechanism, as follows.
Given the complete insensitivity of CDF to buffering by 10 mM BAPTA (Fig. 6B, left) and
the flat relationship between the strength of CDF and I
(Fig. 6D, right), CDF must be selectively triggered
by a local Ca2+ domain driven by
Ca2+ influx through an individual channel
(Deisseroth et al., 1996; Peterson et al., 2000). In this "local
domain regimen," the relevant domain
Ca2+ concentration would be virtually
synchronized in time with single-channel openings and directly
proportional to i (Sherman et al., 1990), such that
[Ca2+]domain  i when the channel is open or 0 when closed. The strength of
CDF (g) should be a function of the
"effective rate constant" governing the transition from
nonfacilitated to facilitated channels at +10 mV (Peterson et al.,
2000). In the local domain regimen, this rate constant would be a
function of
[Ca2+]domain and
Po, where
Po is the peak open probability of
unfacilitated channels at +10 mV. Specifically, the strength of CDF
(g) should be closely proportional to
Po × [Ca2+]domain2,
where the square term reflects triggering of CDF by two
Ca2+ ions binding to the C-terminal lobe
of CaM (Peterson et al., 1999; DeMaria et al., 2001). Considering that
[Ca2+]domain i, we have that g Po
i2. The invariance of
g with splice variation (Fig. 6D, left)
thus implies the invariance of both Po
and i (absent unlikely cancellation of changes in
Po and
i2). By exclusion, variation in
channel number (N) alone is the most likely mechanism
underlying splice variant effects on I. One explanation for
changes in N could be that the channel turnover rate is
affected by channel complexation at or near exons 43 and 44 (Maximov et
al., 1999) with as-yet-unknown adaptor molecules. Customization of
channel interaction with adaptor molecules would be a most intriguing
dimension for splice variant modulation.
| |
FOOTNOTES |
Received June 19, 2002; revised Sept. 12, 2002; accepted Sept. 13, 2002.
The work was supported by the National Medical Research Council,
Singapore (T.W.S.), a National Institutes of Health National Research
Service Award fellowship (C.D.D.), National Institute of Mental Health
Grant RO1-MH65531 (D.T.Y.), and a Johns Hopkins Singapore travel grant
(T.W.S. and D.T.Y.). We thank T. P. Snutch for the gift of the
human 12.1 cDNA clone.
Correspondence should be addressed to David T. Yue, Calcium Signals
Laboratory, Departments of Biomedical Engineering and Neuroscience,
Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: dyue{at}bme.jhu.edu.
| |
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ABSTRACT
INTRODUCTION
