First published online May 24, 2005
Journal of Experimental Biology 208, 2191-2203 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01616
The C. elegans T-type calcium channel CCA-1 boosts neuromuscular transmission
Katherine A. Steger1,*,
Boris B. Shtonda1,*,
Colin Thacker2,
Terrance P. Snutch2 and
Leon Avery1,
1 Department of Molecular Biology, University of Texas, Southwestern Medical
Center, Dallas, TX 75390, USA
2 Department of Biotechnology Laboratory, University of British Columbia,
Vancouver, BC, Canada
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Fig. 1. Structure of the cca-1 gene and CCA-1 calcium channel. (A)
Intron-exon structure of cca-1. Coding exons are represented by
filled rectangles, non-coding exons by open rectangles. The ad1650
and gk30 deletions are indicated by bars. All cDNAs characterized
were found to be trans-spliced at the 5' end to the SL1 leader
RNA (SL1). The most-abundant transcript, cca-1A, is composed of all
exons except exon 19. Splice variant cca-1B uses an alternative
splice donor in exon 18, which is spliced to exon 19. The cca-1D
transcript splices exon 17 directly to exon 19. The inclusion of exon 19 would
add amino acid residues to the pore loop region of domain II, and may have
significant effects on the activity of the channel. Splice variant
cca-1C uses an alternative 5' splice donor for exon 24, which
is predicted to result in the incorporation of an additional 15 amino acids in
the III-IV loop. Sequences of the four splice variants have been submitted to
GenBank under accession numbers AY313898 (cca-1A), AY313899
(cca-1B), AY313900 (cca-1C) and AY322480 (cca-1D).
The PCR products used to generate the cca-1::GFP fusion constructs
are diagrammed and labeled with the names of the primers used for
amplification (cca35/40 and cca38/34). (B) Domain structure of CCA-1. Open
cylinders indicate transmembrane domains, and the positions of the alternative
exons found in splice variants B, C and D are shown.
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Fig. 2. A cca-1::GFP gene fusion is expressed in the pharyngeal muscle.
(A) Transgenic animals carrying an extra-chromosomal array including a
full-length cca-1::GFP fusion (strain TS321) exhibit GFP fluorescence
in all pharyngeal muscle including cells of the procorpus (pc), metacorpus,
the isthmus (i) and in the terminal bulb (tb). The intense staining in the
posterior of the terminal bulb is localized to the pm8 muscle cell. The
asterisk indicates expression in extrapharyngeal neurons (n) and sheath cells
(s) in the head. A differential interference contrast (DIC) image of the same
worm is shown in B for comparison. Labeling as in A.
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Fig. 3. Loss of cca-1 function or reduction of MC neurotransmission
compromises action potential initiation and reduces pharyngeal pumping rates.
(A) Effect of cca-1 mutations on the pumping rates of intact worms in
the presence of E. coli HB101. Results are shown as the mean number
of pumps per minute. Values are means ± standard error of the mean
(S.E.M.). N=20 worms for each data
point. See Materials and methods for additional details. (B) In
electropharyngeograms (EPGs) from wild-type worms, a small EPSP (excitatory
post-synaptic potential) precedes each excitation (E-phase) spike. An EPSP is
marked with a large black arrow while an E-spike is marked with a small black
arrow. The action potential terminates with a large negative repolarization
(R-phase) spike (large gray arrow). Smaller negative spikes between E and R
represent inhibitory post-synaptic potentials (IPSPs) from the inhibitory
motor neuron M3 (Dent et al.,
1997 ; Raizen and Avery,
1994). (C) In EPGs from cca-1 mutants homozygous for
either of two loss-of-function alleles (ad1650 or gk30),
small, delayed E-phase spikes (small arrows) follow the EPSPs. EPGs from
cca-1 mutants also contain interpump phase (I-phase) spikes between
pumps (arrowheads). (D) Effect on EPG traces of defects in MC
neurotransmission. unc-17 encodes a membrane transporter that loads
acetylcholine into synaptic vesicles within the cholinergic neurons, including
the MC motor neuron. e245 is a viable missense allele of
unc-17 (Alfonso et al.,
1993). Null mutations of unc-17 are lethal. EPGs from
unc-17(e245) mutants contain occasional I-phase spikes (arrowheads),
because the low acetylcholine content of synaptic vesicles reduces the success
rate of MC neurotransmission. snt-1 encodes synaptotagmin, a
vesicle-associated protein necessary for effective calcium-stimulated release
of neurotransmitter. snt-1(md290) is a putative null allele of
synaptotagmin (Nonet et al.,
1993). EPGs from snt-1(md290) mutant worms contain many
I-phase spikes (arrowheads), often occurring in clusters, as a result of
uncoordinated neurotransmitter release. Because EPGs recorded from different
individual worms show some variation, we have annotated the features that
consistently differ between worms of different genotypes. Unmarked differences
are likely to be due to individual variation.
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Fig. 4. Intracellular recordings reveal altered depolarization in action potentials
from cca-1 mutant pharynxes. (A) Sample intracellular voltage
recordings from wild-type and cca-1(ad1650) mutant worms. While the
depolarization phase of the wild-type action potential usually rises steeply
and steadily from resting potential to a peak, action potentials from
cca-1 mutant worms often display a notch or flattened area early in
depolarization. (B) Average maximal rising phase slopes for the wild-type,
cca-1, eat-2 and eat-2; cca-1. 50-100
differentiated and aligned action potentials were averaged for each individual
pharynx. A peak slope for each individual pharynx was obtained from the
averaged trace. This calculation was performed for 19 wild-type animals, 16
cca-1 mutant animals, 14 eat-2 mutant animals and 11
eat-2; cca-1 double mutant animals. From the peak slopes for each
individual, an average peak slope was calculated for each genotype, expressed
as mean ± S.E.M.
*Significantly different from the wild type;
significantly different from all other strains
(P<0.005 for all comparisons, t-test). (C) (Top)
Superimposed action potentials (with rising phases aligned) from two
representative wild-type pharynxes. (Bottom) Time derivatives of the action
potentials above. N=63 and 66 for left and right traces,
respectively. (D) Same as in C for two representative cca-1(ad1650)
worms; N=51 and 74 for left and right panels, respectively. While
wild-type action potentials generally display a smooth steep rising phase, the
majority of action potentials from cca-1 mutant worms contain an
initial depolarization, followed by a flattened area or notch. Plotting the
time derivative of each action potential trace reproduces the EPG phenotypes
of wild-type and cca-1 mutant worms (compare with
Fig. 3) with a small, delayed
depolarization spike following each initial rise in membrane potential. (E)
Action potentials from two representative eat-2(ad465); cca-1(ad1650)
double mutants, labeled as in C and D. These action potentials lack the
initial depolarization and plateau seen in cca-1 single mutants,
suggesting that early depolarization in a wild-type pharynx or cca-1
mutant pharynx is mediated by the EAT-2-containing nicotinic receptor.
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Fig. 5. CCA-1 activity is crucial for the efficient initiation of action potentials
in animals with defects in cholinergic neurotransmission. (A) EPGs from
unc-17(e245); cca-1(ad1650) double mutants contain many I-phase
spikes, reflecting the fact that MC is often unsuccessful in triggering a
pharyngeal muscle action potential. The frequency of I-phase spikes is far
greater in an unc-17(e245); cca-1(ad1650) double mutant than in an
unc-17(e245) single mutant (shown in
Fig. 3D). (B) EPGs from
snt-1(md290); cca-1(ad1650) mutant worms contain rare action
potentials separated by many clusters of I-phase spikes. Again, the rate of
failure of action potential generation is far greater in a snt-1(md290);
cca-1(ad1650) double mutant than in a snt-1(md290) single mutant
(shown in Fig. 3D). EPSPs are
marked with large arrows. E-phase spikes are marked with small arrows. I-phase
spikes are marked with arrowheads.
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Fig. 6. Worms lacking the EAT-2 nicotinic receptor subunit initiate action
potentials without MC-stimulated EPSPs. (A) EPGs from eat-2 mutants
lack EPSPs and I-phase spikes because eat-2 mutants are defective in
nicotinic neurotransmission from MC to the pharyngeal muscle. The
ad465 allele of eat-2 contains an early stop codon. (B) EPGs
from eat-2; cca-1 double mutants closely resemble those of
eat-2 single mutants. E-phase spikes are marked with arrows.
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Fig. 7. Worms adapt to the loss of nicotinic MC stimulation by altering resting
potential to preserve pharyngeal pumping. (A) Sample intracellular voltage
recordings from wild-type worms show that resting potential is about -73 mV.
(B) Resting potential in cca-1 single mutants matches that of
wild-type worms. (C) In contrast, a recording from an eat-2 mutant
reveals that resting potential is elevated by  13 mV over wild-type
resting potential, and has a slight tendency to rise between action
potentials. (D) A recording from an eat-2; cca-1 double mutant
reveals an elevated resting potential that drifts significantly towards
positive potentials between action potentials. (E) Average resting membrane
potential for above four strains. Mean ±
S.E.M.; N=12 (WT), 10
(cca-1), 15 (eat-2), 11 (eat-2; cca-1).
*Significantly different from both the wild type and cca-1
(P<0.005, t-test).
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Fig. 8. Proposed role of the CCA-1 T-type channel in the pharyngeal action
potential. In wild-type worms, action potentials are triggered by the influx
of cations through the EAT-2-containing acetylcholine gated channel. The small
rise in membrane potential resulting from cation influx is sufficient to
activate the CCA-1 T-type channel. Calcium influx through CCA-1 lifts membrane
potential further, until the EGL-19 L-type calcium channels can be activated.
In the absence of CCA-1 function, some EPSPs fail to activate EGL-19 channels,
or activate them more slowly than in wild-type worms. When both MC EPSPs and
CCA-1 function are absent, the pharynx employs an alternative method, possibly
a leak conductance and an elevation of resting membrane potential, to trigger
EGL-19 activation.
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© The Company of Biologists Ltd 2005
