The Journal of Neuroscience, July 23, 2003, 23(16):6537-6545
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Critical Residues of the Caenorhabditis elegans unc-2 Voltage-Gated Calcium Channel That Affect Behavioral and Physiological Properties
Eleanor A. Mathews,1
Esperanza García,1
Celia M. Santi,1
Gregory P. Mullen,2
Colin Thacker,1
Donald G. Moerman,2 and
Terrance P. Snutch1
1Biotechnology Laboratory and
2Department of Zoology, University of British
Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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Abstract
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The Caenorhabditis elegans unc-2 gene encodes a voltage-gated
calcium channel 
1 subunit structurally related to mammalian
dihydropyridine-insensitive high-threshold channels. In the present paper we
describe the characterization of seven alleles of unc-2. Using an
unc-2 promoter-tagged green fluorescent protein construct, we show
that unc-2 is primarily expressed in motor neurons, several subsets
of sensory neurons, and the HSN and VC neurons that control egg laying.
Examination of behavioral phenotypes, including defecation, thrashing, and
sensitivities to aldicarb and nicotine suggests that UNC-2 acts
presynaptically to mediate both cholinergic and GABAergic neurotransmission.
Sequence analysis of the unc-2 alleles shows that e55, ra605,
ra606, ra609, and ra610 all are predicted to prematurely
terminate and greatly reduce or eliminate unc-2 function. In
contrast, the ra612 and ra614 alleles are missense mutations
resulting in the substitution of highly conserved residues in the C terminus
and the domain IVS4-IVS5 linker, respectively. Heterologous expression of a
rat brain P/Q-type channel containing the ra612 mutation shows that
the glycine to arginine substitution affects a variety of channel
characteristics, including the voltage dependence of activation, steady-state
inactivation, as well as channel kinetics. Overall, our findings suggest that
UNC-2 plays a pivotal role in mediating a number of physiological processes in
the nematode and also defines a number of critical residues important for
calcium channel function in vivo.
Key words: calcium channel; mutation; behavior; electrophysiology; presynaptic; C. elegans
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Introduction
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Voltage-gated calcium (Ca) channels play a central role in a number of
biological processes, including neurotransmitter release,
excitation-contraction coupling, regulation of gene expression, and neuronal
migration. In addition, compounds that directly affect either Ca channels or
proteins that modulate their activity are used to treat a number of
cardiovascular and neurological pathologies
(Miller, 2001
). Understanding
the nature of Ca channel functional diversity will provide insight into both
normal calcium signaling mechanisms, as well as contributing to our
understanding of the roles that these molecules play in various pathological
conditions.
High-threshold Ca channels are multisubunit complexes consisting of four to
five subunits designated 1, 2
,
, and 
(for review, see
Catterall, 2000). The
1 subunit forms the voltage-sensor and channel proper,
whereas the remaining proteins interact with the 1 subunit
to modulate channel activity. In mammals, five pharmacologically distinct
types of Ca channels have been described (designated L-, N-, P/Q-, T-, and
R-types) and that are encoded by 10 distinct 1 subunit genes
(Stea et al., 1995b;
Catterall, 2000). Genetic and
molecular studies have revealed the presence of three 1
subunit genes in the nematode Caenorhabditis elegans: unc-2
(Schafer and Kenyon, 1995),
egl-19 (Lee et al.,
1997), and cca-1 (for review, see
Bargmann 1998) and which appear
to be ancestral to the mammalian N/P/Q-, L-, and T-type channels,
respectively. Genes encoding other Ca channel subunits have also been
identified in the C. elegans genome: unc-36 and T24F1.6
encode 2 subunit homologues and
ccb-1 (T28F2.5) and W10C8.1 both encode putative subunits
(Bargmann, 1998).
Schafer and Kenyon (1995)
first noted that the slow, uncoordinated movement and constitutive egg-laying
defects displayed by unc-2 mutants resembled the affects of adding
exogenous serotonin to wild-type worms. In addition, unlike wild-type animals,
unc-2 worms were unable to adapt to either serotonin or dopamine
(Schafer and Kenyon, 1995).
Subsequently, it was shown that mutations in the unc-36 gene
exhibited similar mutant phenotypes to that of unc-2, suggesting that
these two genes act together in a Ca-dependent pathway to modulate responses
to serotonin (Schafer and Kenyon,
1995; Schafer et al.,
1996). Genetic mosaic analysis and in situ hybridization
experiments have shown that UNC-2/UNC-36 both act in the HSN and/or VC neurons
known to control egg laying in C. elegans
(Schafer and Kenyon, 1995;
Schafer et al., 1996).
Mutations in unc-2 have also been shown to affect migration of
specific neuronal cells. For example, the sensory neuron AVM and the
interneuron SDQR undergo aberrant postembryonic migrations in unc-2
mutants (Tam et al., 2000).
unc-2 also plays an integral role in a Ca-dependent signal
transduction pathway that controls the asymmetric expression of the
str-2 odorant receptor gene in the AWC sister sensory neurons
(Troemel et al., 1999). In
both processes, mutations in unc-2 and unc-36 show similar
phenotypes, implying that these two gene products act in the same functional
complex. Mutations in genes encoding factors that are known to regulate Ca
channel 1-subunits, such as CaM kinase II (unc-43)
(Reiner et al., 1999;
Rongo and Kaplan, 1999),
syntaxin (unc-64) (Saifee et al.,
1998), and G-proteins (goa-1:
Lochrie et al., 1991;
gpb-1: Brundage et al.,
1996; Zwaal et al.,
1996), have also been identified. Interestingly, genetic analysis
of the Ca signaling pathways involving UNC-2 that affect cell migration and
str-2 odorant receptor gene expression have demonstrated that one of
the downstream effectors is unc-43/CaM kinase II
(Troemel et al., 1999;
Tam et al., 2000). Taken
together, these findings suggest that both potential modulators of Ca channel
1-subunits and the 1-subunits themselves
function in a variety of complex physiological processes. Consequently, the
coordinated analysis of these genes in C. elegans using genetic,
behavioral, and electrophysiological methodologies should add to our
understanding of Ca channel physiological functions in vivo.
Here we describe a comprehensive characterization of the unc-2
gene and the isolation of six new alleles of unc-2 as well as the
canonical allele, e55. We also present evidence that unc-2
acts as a presynaptic Ca channel to mediate both acetylcholine and GABA
release in the nematode. Molecular analyses of the mutant unc-2
alleles identifies highly conserved residues that contribute to Ca channel
function in vivo. Introduction of the ra612 allele into the
mammalian P/Q-type channel shows that disruption of a conserved site in the C
terminus dramatically affects channel voltage-dependent and kinetic
properties.
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Materials and Methods
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Nematode strains and growth conditions. Nematodes were grown on
nematode growth media (NGM) plates streaked with Escherichia coli
(OP50 strain), as described (Brenner,
1974). Strains in this work include the wild-type strain N2;
unc-2(e55), CB55; unc-2(ra605) TS79, unc-2(ra606)
TS80; unc-2(ra609) TS82; unc-2(ra610), TS83;
unc-2(ra612), TS85; unc-2(ra614), TS118; unc-2(e55)
dpy-3(e27), DM2601; unc-25(e265), CB265;
unc-36(e251), CB251; lin-15(n765); and
vaIs9, TS67. The identification of the ra605 and
ra610 mutations were previously reported in Tam et al.
(2000).
Isolation of new unc-2 alleles. A non-complementation
screen was used to isolate new alleles of unc-2. Briefly, N2 males
were mutagenized with 50 mM ethyl methane sulfonate and crossed to
dpy-3(e27) unc-2(e55) hermaphrodites. Twenty mating plates, each with
six males and three hermaphrodites, were set up, and their progeny were
screened for the presence of Unc non-Dpy progeny. Eleven independent Unc
non-Dpy animals were identified and picked to new plates. From each plate,
single Unc animals were transferred to new plates, and their progeny were
scored for the presence of Dpy Uncs. Animals that failed to segregate Dpy Unc
progeny were presumed to be homozygous for the new unc-2 mutation.
Strains were outcrossed at least three times before phenotypic analysis.
Phenotypic analyses of unc-2 mutants. Resistance of
wild-type and mutant worms to aldicarb was performed as described
(Miller et al., 1996).
Briefly, 20 animals were placed on plates containing 0.5 mM
aldicarb and assayed for paralysis at 10 min intervals for 3 hr. Animals,
except for five individuals, were removed from the plates and then scored
periodically over 1, 2, and 3 weeks for the production of viable progeny. To
test for sensitivity to nicotine, plates were flooded with 1% nicotine
solution.
To examine defects in movement, young adult hermaphrodites were transferred
to a microtiter well containing 60 µl of M9 buffer. After a 2 min recovery
period, thrashes were counted for 2 min
(Miller et al., 1996). A
thrash was defined as a change in direction of bending at the mid-body. Ten
animals from each strain were examined. Defects in defecation were determined
by examining young adult hermaphrodites with a dissecting microscope for the
presence or absence of an expulsion event after each posterior body muscle
contraction (pBoc) of the defecation cycle
(Thomas, 1990;
Miller et al., 1996). Ten
animals from each strain were observed for 10 consecutive cycles.
cDNA cloning. Overlapping cDNAs encoding the unc-2 gene
were isolated from several sources. These included RT-PCR of total RNA
prepared from a mixed population of worms and both PCR amplification and
screening of a cDNA library (
ACT-RB2; kindly provided by Robert
Barstead, Oklahoma Medical Research Foundation, OK). The entire predicted
unc-2 open reading frame (ORF) is encoded by five overlapping cDNA
clones, namely punc2.1, which included the putative initiator methionine
codon; cDNA 82-43; cDNA10; cDNA1, and yk131b1 which contained the predicted
unc-2 poly(A) site (AATAAA) and the 3' untranslated region
(3' UTR) (Fig. 1
A). The cDNA clone yk131b1 was generously provided by
Yuji Kohara (National Institute of Genetics, Japan). The composite sequence of
the overlapping cDNA clones was submitted to GenBank (accession number
AY264781
[GenBank]
). Existing GenBank entries for unc-2 differ considerably
from the sequence reported in this work. An explanation for this discrepancy
is that previous GenBank submissions have been based on annotated predictions
using the program GeneFinder rather than on actual physical comparison of cDNA
and genomic sequences. Differences between the cDNA sequence described in this
work and that from Tam et al.
(2000) is attributable to the
discovery of additional 5' sequences and a nucleotide sequencing change
in the 3' end of the gene that altered the reading frame of the
predicted gene product (see Results).
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Figure 1. unc-2 gene structure and phylogenetic comparison. A,
Organization of the unc-2 gene structure. Shown is the intron-exon
structure of the unc-2 gene (Gene), including the genomic region used
to generate the unc-2::GFP fusion construct (top). Also shown are the
regions of the predicted gene product (Protein) that are encoded by the
corresponding exons and the overlapping cDNAs or RT-PCT products used to
determine the gene structure (cDNAs). B, Phylogenetic comparison of
UNC-2 with other C. elegans and mammalian Ca channel
1 subunits. The predicted amino acid sequences of
representatives of each class of Ca channel 1 subunit and
the novel NCA ion channel family 1 subunits were compared
pairwise, and the percentage similarities were plotted. GenBank accession
numbers for the Ca channels: rat 1A/
Cav2.1, M64373
[GenBank]
; rat 1B/Cav2.2,
M92905
[GenBank]
; rat 1C/Cav1.2, M67515
[GenBank]
; rat
1E/Cav2.3, L15453
[GenBank]
; human
1F/Cav1.4, AJ224874
[GenBank]
; rabbit
1S/Cav1.1, M23919
[GenBank]
; rat
1D/Cav1.3, (E. Mathews and T. Snutch,
unpublished results); UNC-2, AY264781
[GenBank]
(this work); EGL-19, AF023602
[GenBank]
; CCA-1,
(AY313898
[GenBank]
); rat brain NCA, AAC68885
[GenBank]
.1; and C. elegans NCA-1 and NCA-2
1 subunits (cosmids C11D2.6 and C27F2.5; and K. Hamming and
T. P. Snutch, unpublished results).
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Identification of unc-2 lesions. To identify the
nucleotide changes in the unc-2 mutants, two approaches were used.
The first involved the use of an RNase protection assay performed according to
the procedure outlined in the Mismatch Detect II kit (Ambion). In the second
approach genomic DNA encompassing the unc-2 gene from the
corresponding homozygous mutant animals was amplified, and the DNA sequence
was determined directly.
unc-2::green fluorescent protein fusion. Long-range PCR was used
to amplify the 5' region of the unc-2 gene and the predicted
first three exons (Fig. 1
A). Two fragments were amplified from genomic DNA. One
included 
4 kb of 5' sequence upstream from the first exon, along
with a 6.8 kb region encompassing exons one and two. The second long-range PCR
product was 4.1 kb in length, overlapping with the first product by 694 bp and
ending within exon three. The second product was cloned into the vector pGEM-T
easy. A green fluorescent protein (GFP) cassette isolated from the expression
vector pPD95.70 (kindly provided by A. Fire) was then cloned in-frame with
exon 3, generating the construct punc24.70. The 10.8 kb long-range PCR product
was injected into the gonads of adult lin-15(n765ts) hermaphrodites
along with punc24.70 and pJM23, which contains a wild-type copy of the
lin-15 gene. Transformants were rescued for the lin-15
multivulval phenotype, and stable lines were established. The array from one
of these lines was integrated, and GFP expression was examined in this strain,
called TS67. Cell identification was based on the position and characteristic
morphology of GFP-positive cell nuclei using fluorescence and Nomarski
differential interference microscopy.
Construction of rat brain 1A
mutant. unc-2 mutations corresponding to e55 and
ra612 were introduced into the rat brain
1A/Cav2.1 cDNA by site-directed mutagenesis.
Details of the procedures used can be obtained from the authors on request.
All mutations were made in the rat 1A/Cav2.1
clone pc3RBA1 (Starr et al.,
1991). To generate the corresponding e55 mutation the
1A/Cav2.1 cDNA was truncated by the introduction
of a stop codon after the R477 codon. The construct p1A55
encodes only the N terminus of the rat 1A/Cav2.1
subunit ending in the domain I-II linker. Plasmid pm1A-612
contains the corresponding ra612 mutation in which the G1817 codon
(GGC) was changed to arginine (CGT).
Electrophysiology and data analysis. Human embryonic kidney
(HEK)tsa201 cells were transiently transfected by the Ca phosphate
precipitation method with an equimolar ratio of cDNAs encoding either the
wild-type rat brain Ca channel 1A/CaV2.1 or one
of the plasmids carrying the engineered mutation plus the rat brain 1b
and 2 ancillary subunits. Coexpression of CD8 antigen was used
to visually identify cells for electrophysiological experiments through the
binding of anti-CD8 antibody-coated microspheres (Dynal, Great Neck, NY).
Whole-cell inward currents were recorded 24 -48 hr after transfection with an
Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA).
Recordings were filtered at 2 kHz and acquired using pClamp software, version
6.03 (Axon Instruments). The extracellular recording solution contained either
5 mM BaCl2 or5mM CaCl2
and1mM MgCl2,10mM HEPES, 40 mM
TEACl, 10 mM glucose, and 87.5 mM CsCl, pH 7.4. Pipettes
of typical resistances ranging from 2 to 4 M
were filled with internal
solution containing (in mM): 105 CsCl, 25 TEACl, 1
CaCl2, 11 EGTA, and 10 HEPES, pH 7.2. The voltage dependence of
activation was analyzed by step depolarizations from a holding potential of
-120 mV to various test potentials ranging from -60 to +30 mV. Normalized
current amplitudes were plotted as a function of membrane potential, and
I-V curves were fitted according to equation: I =
{Gmax
(Vm-Vrev)}/{1 +
exp[(Vm-V0.5)/ka]},
where G is membrane conductance, Vrev is the
reversal potential, V0.5 is the midpoint, and
ka the slope of the voltage dependence. To measure
steady-state inactivation profiles, conditioning prepulses (15 sec) from -120
to 40 mV in 10 mV steps were applied, and the membrane was then stepped to the
peak of the I-V curve. Currents were normalized to the maximal value
obtained at the test pulse and plotted as a function of the prepulse
potential. Data were fitted with Boltzmann equations
(I/Imax = {1 + exp[(V -
V0.5)/ki]} - 1). All experiments were
performed at room temperature. Recordings were analyzed using Clampfit 6.03
(Axon Instruments); figures and nonlinear regressions were done using the
Origin software (version 6.0; Microcal, Northampton, MA). Data are presented
as mean ± SEM. Significant differences were determined using Student's
t test with the significance value set at p < 0.01.
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Results
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Isolation and characterization of the unc-2 gene
Previous analysis of the unc-2 gene suggested that it encoded the
1 subunit of a non-L-type high voltage-activated (HVA) Ca
channel (Schafer and Kenyon,
1995). To characterize the complete unc-2 gene structure
and predicted coding region, a combination of cDNA cloning and RT-PCR were
used. Comparison of the cDNA and nematode genomic sequences show that the
unc-2 gene consists of at least 28 exons and 27 introns encompassing
a region of 25 kb (Fig.
1A). The exons range in size from 47 to 879 bp. Exon one
contains the putative ATG initiation methionine, and exon 28 (determined
through cDNA sequence analysis) contains an in frame termination codon (TAG)
and a potential polyadenylation sequence (AATAAA) located 688 bp downstream.
The majority of introns within unc-2 are between 40 and 60 bp,
although introns one and two are 5.4 and 4.9 kb, respectively. With the
exception of intron eight, all introns conform to the GU-AG splice site
consensus sequence. Intron eight starts with a GC dinucleotide, which has been
previously noted, albeit rarely, at 5' splice sites in C.
elegans (for review, see Blumenthal
and Steward, 1997).
Compared with the previously reported primary sequence for unc-2
(Schafer and Kenyon, 1995;
Tam et al., 2000), we found an
additional 161 nucleotides in the 5' coding and non-coding regions and
that resulted in 42 additional amino acids, including the putative initiator
methionine. In the 3' coding region, we also found that an additional
nucleotide insertion caused a frame shift and resulted in replacement of the C
terminal 53 residues by 335 new amino acids (GenBank accession number
AY264781
[GenBank]
). Overall, the longest open reading frame of unc-2 encodes a
2027 amino acid polypeptide with a predicted molecular weight of 231 kDa
(Figs. 1A,
2). UNC-2 has features common
to all voltage-gated Ca channels, including four homologous domains (I-IV)
each containing six hydrophobic membrane-spanning segments (S1-S6), which
share significant sequence similarity with the analogous regions of other Ca
channels. In addition, UNC-2 possesses other features that are well conserved
among vertebrate high voltage-activated Ca channels, including a
G (De Waard et al.,
1997; Zamponi et al.,
1997) and Ca channel subunit binding motifs
(Pragnell et al., 1994) in the
domain I linker, EF-hand (Babitch,
1990) and IQ motifs (Lee et
al., 1999; DeMaria et al.,
2001) in the C terminus distal to domain IV, and four conserved
glutamate residues in the P-loops that confer Ca2+
selectivity (Heinemann et al.,
1992; Yang et al.,
1993).
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Figure 2. Schematic representation of the location of molecular lesions within
UNC-2. A, The major structural domains including the four domains
(I-IV), the EF-hand domain in the C terminal region, and the locations of
identified mutations are indicated. B, Alignment of the UNC-2
sequence within the boxed area indicated in A with the corresponding
region of the mammalian and C. elegans EGL-19 L-type Ca channel
1 subunits. Identical residues (*), conserved
residues (:). The C terminal region shown encompasses the conserved EF-hand
domain (single overlined). Note that the unc-2(ra612)
mutation changes a conserved glycine residue (bold), which resides between the
EF-hand and an IQ-like motif further downstream.
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Comparatively, UNC-2 is more closely related to the mammalian
DHP-insensitive 1 subunit classes (rat brain
1B = 73% similarity; 1A = 67%;
1E = 66%) than to the L-type channels (55% similarity)
(Fig. 1b). A defining
feature of L-type channels is their sensitivities to DHPs and phenyalkylamines
and the specific amino acids of the L-type channels required to interact with
these agents have been described (Moreno,
1999). Of the four specific residues in domain IV identified to
confer DHP and phenyalkylamine binding in the L-type channels
(Tang et al., 1993;
Grabner et al., 1996;
Schuster et al., 1996), UNC-2
does not match exactly at any position but instead possesses residues
identical to those found only in the non-DHP-sensitive high voltage-activated
Ca channel subfamily.
Isolation and characterization of novel unc-2 mutations
Using a non-complementation screen, six new EMS-induced alleles of
unc-2 were isolated from 20,000 F1 progeny examined. All alleles
showed locomotor defects typical for unc-2 mutants, as described
previously (Schafer and Kenyon,
1995; Schafer et al.,
1996). However, differences in the mobility of mutant animals were
noted. For example, the canonical unc-2 allele, e55, showed
a severe movement deficit, as did ra605, ra606, ra609, and
ra610. In contrast, the ra612 and ra614 alleles
showed moderate to mild defects in movement. To understand the underlying
differences between the mutants, the molecular lesions of several
unc-2 alleles were determined (see Materials and Methods). The
results show that five mutations, e55, ra605, ra606, ra609, and
ra610 are all single base substitutions predicted to result in
premature stop codons and, hence, truncated proteins
(Fig. 2A). The
e55 allele is a change of glutamine 511 to a stop in the domain I-II
linker, the ra605 allele a change of glutamine 1288 to stop in domain
IV S4, ra606 a change of glutamine 1039 to a stop in the domain
III-IV loop, ra609 a change of tryptophan 179 to a stop in the IS3
transmembrane domain, and ra610 a change of arginine 1379 to stop in
domain IV S5-S6. All of these mutations result in a severe uncoordinated
phenotype, and together with their identification as premature stop codons in
highly conserved structural regions of the channel are therefore likely to
represent the unc-2 null phenotype (see below).
Two alleles, ra612 and ra614, were found to be missense
mutations. The ra612 mutation altered glycine 1477 to an arginine in
the C terminus in a region flanked by a conserved EF-hand and a downstream-IQ
domain (Fig. 2B). The
ra614 allele was found to possess an A to G mutation that alters a
conserved tyrosine 1255 to a cysteine in the domain IVS4-IVS5 linker.
Interestingly, the residues altered in both mutants are conserved in all
vertebrate high voltage-activated Ca channels cloned to date including all
splice variants of 1A/Cav2.1 and
1B/Cav2.2
(Stea et al., 1995b).
Phenotypic analysis of unc-2 alleles
In an attempt to further correlate the various unc-2 molecular
lesions with locomotor activity, several phenotypic analyses were performed.
Initially, e55, ra605, ra609, and ra612 were examined for
their sensitivities to aldicarb and nicotine. unc-2 mutants have
previously been shown to be resistant to the effects of the AChE inhibitor
aldicarb, implicating the UNC-2 protein in cholinergic transmission
(Miller et al., 1996). Within
1 hr of being placed on NGM plates containing 0.5 mM aldicarb,
wild-type animals became paralyzed, and the body wall muscles hypercontracted,
causing eggs to be extruded from the uterus. In contrast, animals for the four
unc-2 alleles tested were not noticeably affected after 1 hr of
exposure (Fig. 3A).
After 1 week of aldicarb exposure, the wild-type animals had died without
successfully reproducing. Again, in contrast to wild-type animals, the four
unc-2 strains examined were hypercontracted by this point, but the
original animals plated remained alive, and living progeny were also present.
Differences among the four unc-2 alleles tested became apparent after
3 weeks of aldicarb exposure. Whereas all four strains produced some viable
progeny, there were 33-50% more animals on the e55, ra605, and
ra609 plates than on the ra612 plates
(Fig. 3B).
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Figure 3. Behavioral analysis of unc-2 mutants. A, B, unc-2 mutants
are resistant to the ACh esterase inhibitor aldicarb. Incubation of wild-type
worms (filled diamonds) on 0.5 mM aldicarb (A) resulted in
complete paralysis within 120 min. In contrast, the unc-2
mutants e55, ra605, ra609, and ra612 showed resistance to
aldicarb over the 3 hr period of observation. B, Continued growth of
wild-type worms on aldicarb resulted in both a failure to survive and to
produce progeny. The various unc-2 mutants were able to survive
continued growth on aldicarb for at least 3 weeks, even though the animals
became hypercontracted. In addition, viable progeny were produced by the
unc-2 mutants, although differences in the number of progeny were
observed. The nonsense mutants e55, ra605, and ra609
segregated more progeny than the ra612 mutant, suggesting that ACh
release and, hence, viability was more impaired by aldicarb for the
ra612 animals. C, Thrashing assays were performed to
characterize the affect of the unc-2 lesions on worm body movement in
fluid (for a definition of thrashing see Materials and Methods). Thrashing was
reduced in ra612 animals as compared with wild-type but was
significantly less severe than that displayed by alleles e55, ra605,
and ra609, the unc-36 mutant, and the
unc-36;unc-2(ra612) double mutant. D, Defects in
defecation were scored as failure of EMCs during the defecation process. The
percentage of failure of EMCs was compared with that of wild-type and the
severe defecation mutant unc-25. ra612 animals, although deficient in
EMCs, were significantly less severe than e55, ra605, ra609, unc-36,
and unc-25 mutant animals. As predicted, the unc-36;
unc-2(ra612) double mutant shows a defecation defect
comparable with unc-36 and severe unc-2 alleles.
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The addition of the acetylcholine agonist nicotine to C. elegans
stimulates postsynaptic acetylcholine receptors causing contraction of the
body wall muscles. Treatment of wild-type and the four unc-2 strains
with nicotine caused both wild-type and mutant animals to hypercontract (data
not shown). Because nicotine acts postsynaptically, these results suggest that
mutations in unc-2 do not directly affect the postsynaptic
transmission machinery. Taken together with the effects of aldicarb, the data
also suggest that mutations in unc-2 most likely reduce ACh release
at the neuromuscular junction, consistent with a role for UNC-2 as a
presynaptic Ca channel that contributes to triggering neurotransmitter
release.
For a more quantitative assessment of the neurotransmission defects in
unc-2, we focused on the behaviors of locomotion and defecation.
These behaviors are chiefly mediated by two different neurotransmitters, ACh
and GABA (for review, see Rand and Nonet,
1997). As previously noted, unc-2 mutants are slow moving
and resistant to the AChE inhibitor aldicarb, suggesting that cholinergic
transmission is impaired (Miller et al.,
1996). Using a thrashing assay in liquid media, we quantified the
extent of the impairment in various unc-2 alleles. Thrashing was
significantly reduced in e55, ra605, ra609, and ra612
mutants compared with wild-type animals.
Figure 3C shows that
thrashing of e55, ra605, and ra609 mutants was reduced to
<3% of wild-type rates measured under similar conditions. Thrashing in
ra612 mutants was reduced to 6.5% of thrashing rates compared with
wild-type, a value that is significantly different from both wild-type animals
and the unc-2 mutants examined
(Fig. 3C).
The results of the thrashing assay provide further support for a defect in
cholinergic transmission in unc-2 animals. To assess the effects of
mutations in unc-2 on the release of another neurotransmitter, we
examined the expulsion step of the defecation process [enteric muscle
contraction (EMC)]. EMC is mediated by GABA, and failure of EMC is indicative
of a defect in GABA neurotransmission (McIntire et al.,
1993a,b).
As shown in Figure 3D,
the expulsion failure rates of the unc-2 mutants were significantly
different from that of wild-type. Wild-type animals failed to defecate in
<2% of cycles, whereas ra612 mutants had an 44% failure rate,
and e55, ra605, and ra609 animals failed 70% of the
time. Interestingly, the failure rates of the latter unc-2 mutants
were similar to those of unc-25(e265) animals, which are completely
defective in GABA biosynthesis (Jin et
al., 1999). Overall, the data are consistent with the notion that
UNC-2 functions presynaptically to affect the release of the neurotransmitters
ACh and GABA.
The unc-36 gene encodes a homolog of the mammalian Ca channel
2 subunit, and genetic analyses suggest that
unc-2 and unc-36 may function as part of the same protein
complex in vivo (Schafer and
Ken-yon, 1995; Schafer et al.,
1996). A prediction of this hypothesis is that
unc-36(null); unc-2(null) double mutants should have no
worse a phenotype than either single mutant alone. In addition, it would be
expected that unc-36; unc-2(ra612) double mutants should be
more severe than unc-2(ra612) single mutants, but no worse than
either unc-36 or unc-2 null mutants. To test this notion we
constructed unc-36(e251); unc-2(ra605), and
unc-36(e251); unc-2(ra612) double mutants. In all cases, the double
mutants were indistinguishable from either the unc-36(e251) or
unc-2(ra605) single mutants in their thrashing and defecation
behaviors (Fig. 3C,D).
Taken together, the data are consistent with the hypothesis that UNC-36 and
UNC-2 function as part of the same Ca channel complex in vivo.
unc-2 gene expression pattern
The results of the behavioral studies suggested that UNC-2 functions
primarily in the nervous system and plays a prominent role in synaptic
release. To confirm this hypothesis, we constructed an unc-2::GFP
reporter construct to determine the expression pattern of the unc-2
gene in transformed animals. GFP fluorescence was found to be primarily
localized within the nervous system (Fig.
4). Expression was first observed late in embryogenesis at
450 min of development when most neurons have been generated, and
continued throughout development to the adult stage. Of particular note, in
contrast to previously reported in situ hybridization results
(Schafer and Kenyon, 1995),
GFP expression was observed only a subset of pharyngeal muscle cells but not
in other types of muscle tissues.
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Figure 4. Expression of an unc-2::GFP fusion construct in transgenic
animals. A, GFP fluorescence exhibited by a typical transgenic L3
larva (left side view of animal). Expression is observed in most sensory
neurons in the head (filled arrowhead), as well as a subset of motor neurons
in the ventral nerve cord (VNC, double arrowhead). Expression of the GFP
reporter is also observed in the interneurons SDQL and SDQR (not in focus) and
the touch receptors including ALM and PVM. B, Right side view showing
expression in the SDQR interneuron and the touch cells AVM and ALM.
C, Confocal images of the head region of an L1 larva. The left image
is a composite confocal z-series of the entire head showing all GFP
fluorescent cells, whereas the single slice on right shows expression of the
reporter construct in a single AWC sensory neuron cell body. Expression of
unc-2::GFP was observed in both sister AWC cells, although for
clarity only one side of the head is shown. D, Expression of
unc-2::GFPin the HSN (arrow) and VC neurons that control
egg-laying. The cell bodies of the VC neurons are located in the ventral nerve
cord and are not in the same focal plane. Processes extending from the more
anterior VCs that innervate the vulval muscles are indicated by the star
(*). E, Expression of unc-2::GFP in the
pre-anal, dorsal-rectal, and lumbar ganglia of the tail. GFP fluorescence in
the DVB neuron (arrowhead) which is required for defecation is indicated. The
barbed arrow in A and the triangle in D represent the
position of the vulva. In all panels, the anterior of the animals is
positioned to the left, and the dorsal side is topmost.
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The unc-2 reporter gene was expressed in most motor neurons in the
ventral nerve cord, nerve ring motoneurons—interneurons, and the touch
cells (Fig. 4A).
Postembryonic cell migration of the touch cell AVM and its sister, the
interneuron SDQR, are often perturbed in unc-2 mutant animals
(Tam et al., 2000).
Examination of the unc-2::GFP transgenic animals showed expression of
the reporter gene in these two cells (Fig.
4B), suggesting that UNC-2 activity is required within
AVM and SDQR to control their migration. A number of neurons in the head and
tail regions were also observed to express the unc-2::GFP reporter
gene. Of particular note, neurons identified in the head included the
olfactory sensory cells AWC (Fig.
4C). UNC-2 has been shown to function in a Ca-dependent
pathway that controls the asymmetric expression of the str-2
olfactory receptor gene between the two sister AWC cells that reside on
opposite sides of the animal. GFP expression was also observed in both the HSN
and VC neurons (Fig.
4D) which together control egg laying in the
hermaphrodite and support a previously proposed role for UNC-2 in this
process. Cells expressing GFP in the tail region included the GABAergic neuron
DVB (Fig. 4E), which
together with AVL controls the enteric muscle contractions required for proper
defecation (McIntire et al.,
1993b). The impaired expression of UNC-2 in DVB may explain the
defecation defects exhibited by the unc-2 mutants.
Biophysical consequences of the e55 and ra612
mutations
As described above, the e55, ra605, ra606, ra609, and
ra610 alleles would all result in premature truncation of the
unc-2 gene product and thus would be predicted to result in no
channel function. Of particular interest among these nonsense alleles, the
e55 premature stop mutation resides in the I-II loop and might allow
expression of a single domain truncated product that could lead to the
formation of functional voltage-gated channels with a structure similar to
that for certain types of single domain voltage-gated potassium channels. To
test this possibility, the corresponding e55 mutation (R477stop) was
introduced into the rat brain 1A/Cav2.1
(P/Q-type) cDNA and transfected into HEKtsa201 cells (together with rat brain
1b and 2 subunits). As shown in
Figure 5A, cells
transfected with the plasmid did not result in detectable currents using
conditions identical to that for cells transfected with wild-type rat brain
1A/Cav2.1 cDNA. These results, together with the
genetic analysis of e55, suggest that the nonsense mutation fails to
produce functional voltage-gated Ca channels.
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Figure 5. Functional consequences of the e55 and ra612 mutations on
properties of the rat brain P/Q-type channel. A, Transfection of a
rat 1A cDNA containing the corresponding e55
nonsense mutation failed to produce any currents (solid line) compared with
those obtained from wild-type 1A cDNA (dashed line).
B, Representative traces of barium currents obtained from HEK cells
transfected with either wild-type rat brain 1A or
1A-612. Currents were evoked by stepping membrane potential
for 200 msec to voltages between -60 and 25 mV in 5 mV increments from a
Vh = -120 mV. The ra612 missense mutation
(G1477R) at the proximal region of the 1A C tail modifies
the kinetics of macroscopic inactivation of inward currents recorded in 5
mM external barium. C, ra612 shifts the voltage dependence
of channel activation to more depolarized potentials. Normalized peak current
amplitude was plotted as a function of membrane potential to obtain the
I-V relationship. Values obtained from the best fits of data (smooth
curves) are: wild-type 1A V0.5 = -12.27
± 0.17 mV, k = 3.97 ± 0.11 mV (n = 5),
1A-612 V0.5 = -4.64 ± 0.22 mV,
k = 4.03 ± 0.12 mV (n = 13). D, ra612 causes
a negative shift in the steady-state inactivation curves. Smooth-curve fits to
data corresponding to 1A (filled circles) and
1A-612 (hollow circles) showed a shift in the midpoint
voltage and the slope factor (ki) for steady-state
inactivation: 1A-612 V0.5= -73.64
± 0.37 mV,Ki=7.6 ± 0.3 mV
(n=6).1A V0.5= -52.47 ±
0.18 mV, Ki = 5.6 ± 0.1 mV (n = 4). Note
that the error bars for the wild-type 1A channel are smaller
than the symbol size. E, Current densities for wild-type and
ra612 mutant channels showed no significant difference when measured
using either Ba or Ca as the charge carrier (n = 13, wild-type in Ca;
n = 11, mutant in Ca; n = 8, wild-type in Ba; n =
13, mutant in Ba). All 1 subunits constructs were
coexpressed with rat brain 1b and 2 subunits.
Data are presented as mean ± SEM. Significant differences were
determined using Student's t test with the significance value set at
p < 0.01.
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Of the three missense mutations examined, the ra612 allele with a
lesion in the conserved C terminal domain, appeared to be the most interesting
to pursue regarding functional analyses. The glycine residue at position 1477
is conserved among all high voltage-activated Ca channels and is located
within a region flanked by an upstream Ca-binding EF-hand motif and a
downstream IQ calmodulin binding motif. To test the effect of the G1477R
mutation on channel physiological properties, we introduced the corresponding
change (G1817R) into the 1A P/Q-type channel and expressed
the resulting construct in HEKtsa201 cells.
Currents recorded from HEK cells expressing wild-type
1A/Cav2.1
(Fig. 5B) inactivated
slowly during a test pulse to 0 mV (
inact =200 msec;
n = 5). In contrast, 1A-612 currents decayed
rapidly, with a biexponential time course (1 = 14 msec,
2 = 50 msec; n = 5). At the end of a 200 msec test
pulse, wild-type and mutant currents had decayed to 43 ± 4% (n
= 5) and 94 ± 1% (n = 5) of the peak current, respectively.
Comparison of the extent of inactivation measured as the magnitude of residual
current as a function of membrane potential showed that the degree of
inactivation of 1A-612 decreased monotonically with
depolarization and reached a plateau at 20% of the maximum current.
The current-voltage relationship for the 1A-612 channel
was shifted 10 mV more positive than that of the wild-type channel
(Fig. 5C). In 5
mM Ba, currents through the wild-type
1A/Cav2.1 channels first activated at
approximately -30 mV and peaked at approximately -10 mV. In contrast,
1A-612 channels activated and peaked after depolarizations
to -20 and 0 mV, respectively. No significant differences were detected in the
voltage dependence of activation (Ka 1A
= 3.97 ± 1.1, n = 5; Ka
1A-612 = 4.03 ± 1.2; n = 13).
Comparison of the steady-state inactivation properties of the wild-type
1A/Cav2.1 and 1A-612 mutant
channels demonstrated significant differences in the potential at which half
of the current was inactivated (V0.5[inact])
(Fig. 5D). Compared
with 1A/Cav2.1, the voltage dependence of
inactivation of 1A-612 was shifted by 20 mV to more
hyperpolarized potentials (V0.5[inact] for:
1A/Cav2.1 = -52.5 ± 0.2 mV, n =
4; 1A-612 = -73.6 ± 0.4 mV, n = 6).
Figure 5E shows that
the current densities for the wild-type mutant ra612 mutant channels
were comparable using either Ba or Ca as the charge carriers (n = 13,
wild-type in Ca; n = 11, mutant in Ca; n = 8, wild-type in
Ba; n = 13, mutant in Ba). The fraction of noninactivating current
was not affected by using Ca as the charge carrier, and kinetics of
inactivation did not depend on the nature of the permeant ion
(Fig. 6), suggesting that the
inactivation mechanism produced by the G1447R mutation is unlikely caused by
to alteration of a typical Ca-dependent process.
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Discussion
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Structural characterization of unc-2
The initial description of the unc-2 gene structure and predicted
UNC-2 protein were derived from the partial sequence from the nematode
genome-sequencing project (Schafer and
Kenyon, 1995). The present study extends the initial
unc-2 gene characterization and provides a more complete and detailed
understanding of the intron/exon boundaries and the UNC-2 protein. The first
two introns of unc-2 are quite large, spanning 5.4 and 4.9 kb,
respectively. Although introns in C. elegans are mostly <60 bp in
size, a number of genes contain large introns near their 5' ends. In
some cases, these large introns have been shown to contain regulatory elements
such as alternative promoters or transcriptional enhancers
(Blumenthal and Steward, 1997).
There does not appear to be any obvious relationship between the intron/exon
structure of the unc-2 gene and the structural domains of the UNC-2
protein. For example, the putative transmembrane regions of domain I are
encoded by five exons with no distinct boundaries that define any particular
transmembrane region. By comparison, the majority of domain IV is encoded by
only two exons. Furthermore, some exons encode portions of adjacent domains,
such as with exon 17 that contains the last two transmembrane segments from
domain III as well as the first two transmembrane segments of domain IV.
The predicted UNC-2 protein is structurally similar to other HVA Ca channel
1 subunits and is most closely related to the
DHP-insensitive 1 subunit classes (N- and P/Q-type
channels). Although the overall structure of UNC-2 is well conserved with its
mammalian counterparts, the predicted intracellular domain II-III loop is
considerably shorter and consists of only 97 residues, as compared with the
500 residues encoded by the mammalian 1A P/Q-type and
1B N-type subunits. In this respect, UNC-2 is more similar
in structure to the mammalian L-type and the Drosophila Dmca1A
1 subunits. In the mammalian P/Q-type and N-type channel
domain II-III linkers, the Synprint site has shown to be required for the
physical interaction of SNARE proteins involved in eliciting presynaptic
neurotransmitter release (Sheng et al.,
1994,
1996; Rettig et al.,
1996,
1997). The physical binding of
the SNARE protein syntaxin to the Synprint site results in modulation of
channel steady-state inactivation properties
(Bezprozvanny et al., 1995) and
also a negative feedback between Ca channel function and Ca-dependent gene
regulation (Sutton et al.,
1999). The lack of a readily identifiable Synprint site in UNC-2
suggests that the mechanisms of neurotransmitter release and the control of Ca
channel regulation in C. elegans may differ from those in
vertebrates.
UNC-2 contributes to both cholinergic and GABAergic
neurotransmission
The unc-2 mutant phenotype is primarily similar to that of mutants
known to be defective in neurotransmission
(Rand and Nonet, 1997).
Furthermore, unc-2 animals are aldicarb-resistant
(Miller et al., 1996),
implicating UNC-2 specifically in cholinergic neurotransmission. The wild-type
response of unc-2 animals to levamisole
(Miller et al., 1996) and
nicotine indicates that the aldicarb resistance is not caused by an abnormal
response to ACh by the postsynaptic cell. Furthermore, expression of an
unc-2 promoter::GFP fusion revealed fluorescence in the nervous
system, but not the body wall, uterine, or vulval musculature. These
observations suggest that UNC-2 primarily functions in the nervous system to
regulate neurotransmitter release.
In addition to aberrant locomotor behavior, unc-2 mutants are
defective in EMC of the defecation motor program
(Miller et al., 1996). The AVL
and DVB neurons that stimulate the contraction of the enteric muscles are
GABAergic. Thus, the unc-2 gene product appears to play a role in the
release of GABA as well as ACh and may have a generalized role in
neurotransmission in C. elegans.
The observed behavioral, pharmacological, and cellular expression studies
suggest that UNC-2 functions in neurons in C. elegans in a manner
analogous to that of the presynaptic N- and P/Q-type channels in vertebrate
CNS. Null mutations in cha-1 and unc-17, which encode
choline acetyltransferase and a synaptic vesicle ACh transporter,
respectively, completely abrogate ACh release. These mutants are not viable,
arresting at the L1 stage of development
(Nonet et al., 1993). In
contrast, the unc-2 null phenotype predicted by mutants e55,
ra605, ra606, ra609, and ra610 are all significantly milder than
that of cha-1 and unc-17, suggesting that even in the
absence of unc-2, there is some residual release of ACh from motor
neurons. This notion is substantiated by the observation that unc-2
animals become hypercontracted after prolonged exposure to aldicarb and
suggests the release of ACh from at least some motor terminals.
Several mechanisms could account for residual neurotransmitter release in
unc-2 animals. As shown in Figure
1B, there are two other predicted Ca channel
1 subunits in C. elegans. One of these, the
putative L-type channel encoded by egl-19, is thought to act
predominantly in muscle, however it is also expressed in a subset of neurons
(Lee et al., 1997), and thus
could play a role in neurotransmission at some terminals. There are also two
novel four domain Ca channel-like 1 subunits encoded by
nca-1 and nca-2 (Fig.
1B) (T. P. Snutch and K. Hamming, unpublished results),
and a T-type-like Ca channel encoded by cca-1 that may play a role in
regulating synaptic activity, although no mutations that affect
neurotransmission have been mapped to these parts of the genome. The
possibility of either overlapping or redundant roles in synaptic transmission
could be examined in the future by the analysis of double and triple mutants
of these genes. Alternatively, it is possible that the viability of
unc-2 null mutants is due, at least in part, to the spontaneous
release of ACh from the motor neurons. The presence of spontaneous miniature
postsynaptic currents has been demonstrated in the absence of Ca
(Richmond and Jorgensen,
1999), and it is possible that even in the absence of UNC-2
protein, sufficient ACh release occurs to maintain viability.
UNC-2 and UNC-36 likely function in the same channel complex
The unc-36 gene encodes a polypeptide that is highly similar to
the Ca channel 2/ subunits expressed in mammals
(Lee et al., 1997). Several
observations suggest that UNC-2 and UNC-36 function together in vivo.
First, mutations in the unc-36 gene result in movement and egg-laying
defects that are similar to those exhibited by unc-2(null) mutants.
Second, unc-2 and unc-36 mutants also exhibit very similar
responses to the AChE inhibitor aldicarb
(Nguyen et al., 1995) and also
similar defects in adaptation to dopamine and serotonin
(Schafer and Kenyon, 1995;
Schafer et al., 1996). Third,
mutations in these genes also exhibit similar genetic interactions;
unc-36; egl-19 and unc-2; egl-19 double mutants are both
essentially paralyzed and are indistinguishable in this respect
(Schafer et al., 1996).
Finally, unc-36; unc-2 double mutants are indistinguishable from
either single mutant in most behavioral assays, suggesting that these genes
function in many of the same pathways
(Schafer et al., 1996).
We also find suggestive evidence that UNC-36 activity is required for the
functional expression of the UNC-2 Ca channel complex. For example, the
unc-2(ra612) allele reduces, but does not eliminate, unc-2
function. However, unc-36; unc-2(ra612) double mutant phenotype is
identical to the unc-2(null) phenotype. Because the absence of
unc-36 (2 subunit) function has the same
effect as the absence of unc-2 (1 subunit)
function, the 2 subunit must be essential for the
activity of the complex. This conclusion is consistent with results from a
number of surrogate expression systems wherein co-expression of a cloned
2 subunit is necessary for the expression of
detectable whole-cell Ca currents.
The ra612 mutation alters channel biophysical
properties
The ra612 mutation alters a glycine residue that is located in the
C terminal region of the protein, nine residues downstream of a Ca binding
EF-hand motif and upstream of a calmodulin binding IQ motif, both implicated
in regulating Ca channel function (de Leon
et al., 1995; Lee et al.,
1999; DeMaria et al.,
2001). All HVA Ca channels cloned to date contain a glycine
residue at the homologous position, and it was of interest to determine
whether the alteration in this region of the channel affected biophysical
properties. Electrophysiological analysis of
1A/Cav2.1 channels harboring the ra612
(G1817R) mutation showed that compared with wild-type that a stronger
depolarization is required to open the mutant channels which, once open
inactivated more rapidly, thereby terminating Ca influx sooner. In addition, a
hyperpolarizing shift in the steady-state inactivation curve indicated a
significant reduction in the number of channels available for opening at a
given membrane potential.
Although we are cautious about inferring the exact in vivo
physiological implications of these results, they are consistent with the
phenotypic observations that suggest that the ra612 mutation does not
result in the complete elimination of channel function. For example, the
apparent decrease in neuronal activity in unc-2(ra612) animals could
be attributed to diminished Ca influx through the mutant channels. The
increased rate of inactivation together with the changes in current-voltage
properties would be predicted to reduce Ca influx resulting in decreased
neurotransmitter release.
The mechanism underlying the increased rate of inactivation does not appear
to be a direct alteration of Ca- or current-sensitive properties of the
channel. Ion substitution experiments illustrate that the increase in the rate
of inactivation of 1A-ra612 cannot be attributed to
Ca-dependent modulation previously reported for L-type or N-type calcium
channels (Cox and Dunlap, 1994;
Ferreira et al., 1997).
Potentially, the ra612 mutation might alter or occlude sites that
interact with modulatory proteins that may or may not be Ca-activated. For
example, a CaMKII-PKA consensus sequence is located near the altered glycine
and phosphorylation of this site, which may serve to activate or facilitate
the channel, might be inhibited by the ra612 alteration. This is just
one possible model among others that will require additional investigation to
determine the underlying mechanisms responsible for the altered biophysical
properties of the mutant channel.
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Footnotes
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|---|
Received Dec. 4, 2002;
revised May. 22, 2003;
accepted May. 22, 2003.
This work was supported by grants from the Canadian Institutes of Health
Research (CIHR) of Canada to T.P.S. and D.G.M., fellowship support from the
Human Frontiers Research Program to C.M.S., the British Columbia and Yukon
Heart and Stroke Foundation to E.A.M., and a CIHR Senior Scientist Award to
T.P.S. Some strains in this work were kindly provided by E. M. Jorgensen
(University of Utah, Salt Lake City, UT), J. B. Rand (Oklahoma Medical
Research Foundation, Oklahoma City, OK), and J. A. Hodgkin (University of
Oxford, Oxford, UK). Additional strains were provided by the
Caenorhabditis Genetics Center. We also thank A. Fire, G. Zamponi,
and Y. Kohara for providing clones, J. Thomas for details pertaining to the
defecation assay, B. Barstead for the ACT-RB2 cDNA library, and C.
Bargmann for help with the identification of GFP fluorescent cells. Excellent
technical assistance was provided by Tracy Evans and Daniel Malebranche.
Correspondence should be addressed to Terrance P.Snutch, Room 237-6174,
University Boulevard, University of British Columbia, Vancouver, British
Columbia, Canada V6T 1Z3. E-mail:
snutch{at}zoology.ubc.ca.
E. A. Mathews' and G. P. Mullen's present address: Program in Molecular and
Cellular Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK
73104.
C. M. Santi's present address: Department of Anatomy and Neurobiology,
Washington University School of Medicine, St. Louis, MO 63110.
E.García's present address: Centro de Investigaciones Biomedicas,
Universidad de Colima, A.P.199, Colima, 2800, Mexico.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236537-09$15.00/0
|
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Abstract
Introduction
