The C. elegans T-type calcium channel CCA-1 boosts neuromuscular transmission
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
Author for correspondence (e-mail:
leon{at}eatworms.swmed.edu)
Accepted 19 February 2005
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Summary |
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Key words: calcium channel, T-type channel, neuromuscular junction, Caenorhabditis elegans, action potential
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Introduction |
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While T-type calcium currents have been observed in a wide variety of cell
types and tissues, their physiological relevance is not completely understood.
Studies have implicated T-type conductances in the pacemaker current of the
cardiac sino-atrial node (Hagiwara et al.,
1988), contraction of vascular smooth muscle
(Hansen et al., 2001
),
secretion of aldosterone from the adrenal glomerulosa
(Cohen et al., 1988
), bursting
and rhythmic firing behavior in a variety of central neurons (reviewed by
Huguenard, 1996
), the
amplification of excitatory post-synaptic potentials (EPSPs) in pyramidal
neurons (Gillessen and Alzheimer,
1997
; Urban et al.,
1998
) and neurotransmitter release from retinal bipolar cells
(Pan et al., 2001
). None of
these functions has been directly linked to the loss of function of a
particular
1 subunit-encoding gene. Rather, the
physiological roles of T-type channels are largely inferred from
electrophysiological characterization and from the effects of a limited set of
pharmacological blocking agents. The lack of specificity of these blocking
agents, as well as the difficulty of isolating T-type calcium currents from
more-robust high voltage activated currents makes the inference of function
difficult. In one instance, the
1G (Cav3.1) subunit has been
knocked out in a mouse model, generating a specific defect in thalamocortical
relay neurons (Kim et al.,
2001
). However, the physiological functions of specific T-type
channels in many tissues remain uncertain.
When reporting the cloning of the 1G T-type channel
subunit, the authors noted that the GenBank entry with the highest homology to
1G was C54D2.5, a predicted protein from the genome of the
free-living nematode C. elegans
(Perez-Reyes et al., 1998
). In
the present study, we characterize the gene cca-1 encoding that
predicted protein, and its role in the feeding behavior of the worm.
C. elegans subsists on bacteria encountered in the soil, and feeds
by rhythmically contracting and relaxing a neuromuscular pharynx, thus sucking
food in. The pharynx consists of three regions: the corpus is closest to the
mouth of the worm, the terminal bulb connects to the intestine, and the two
are linked by a slender isthmus (Albertson
and Thomson, 1976). Contraction and relaxation of the corpus and
anterior isthmus muscles pulls in and traps bacteria
(Avery, 1993b
;
Seymour et al., 1983
).
Contractions of the terminal bulb, which occur in synchrony with corpus
contractions (Avery, 1993a
),
grind up the bacteria and push the food through a valve into the intestine
(Doncaster, 1962
). The
near-simultaneous contraction of the corpus, anterior isthmus and terminal
bulb is referred to as a `pump'. Bacteria are transported from the corpus to
the terminal bulb by a second motion, called an isthmus peristalsis
(Avery and Horvitz, 1987
).
The C. elegans pharynx contains a small nervous system that seems
to play a modulatory role in the control of pharyngeal activity. Pumping
continues when all the pharyngeal neurons are ablated with a laser
(Avery and Horvitz, 1989), but
the cholinergic motor neuron MC is necessary for the rapid pumping normally
exhibited by worms and seems to act as a pacemaker. Worms lacking MC function
are viable, but pump at a much-reduced rate
(Avery and Horvitz, 1989
;
Raizen et al., 1995
). Thus,
the pharyngeal muscle is capable of rhythmic depolarization and contraction in
the absence of pharyngeal neuronal activity, although the nervous system plays
an important role in modulating the rate of pharyngeal pumping.
We have found that the cca-1gene product is intimately involved in
the process of depolarization and action potential initiation in the C.
elegans pharynx. In the accompanying paper, Shtonda and Avery describe a
current in pharyngeal muscle that displays T-type kinetics
(Shtonda and Avery, 2005).
Wild-type cca-1 is necessary for the expression of this current,
suggesting that cca-1 encodes a T-type
1 subunit
that functions in the pharyngeal muscle. Cloning and characterization of
cca-1 gene products confirms a strong similarity to vertebrate T-type
channels, but analysis of cca-1 mutants through electrophysiological
techniques reveals a previously undescribed role for T-type calcium channels
at the neuromuscular junction, where they aid in triggering action potentials
in response to stimulation by motor neurons. Because the function of CCA-1
resembles those inferred from pharmacological blockade of T-type currents in
vertebrates, yet can be traced to the loss of function of a single gene
product, our description of CCA-1 provides significant insight into the roles
of vertebrate T-type channels.
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Materials and methods |
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cDNA cloning
C. elegans poly(A)+ RNA was converted to cDNA using the
RNase H- MMLV reverse transcriptase, SuperScript II (Invitrogen,
Carlsbad, CA, USA) and the primer T22V (oligo(dT)22V, where V=A, G
or C), following the method of Regan et al.
(2000). Then, PCR was
performed using an SL1-specific primer containing a NotI restriction
site (5'-ATAAGAATGCGGCGCGGTTTAATTACCCAAGTTTG-3') in combination
with the antisense primer cca-30
(5'-GGGGGTACCGTAGAGGAAACATGGACCGGA-3'), which anneals within the
cca-1 3' UTR and introduces a KpnI restriction site.
The resulting PCR products were digested with NotI-KpnI, and
cloned into pBluescript SKII. Because full-length clones were difficult to
propagate in E. coli, RT-PCR products were also digested with
appropriate restriction enzymes to clone overlapping cDNA fragments. The use
of this procedure prevented us from making an exact assessment of the relative
frequency of different cca-1 splice variants. Clones were isolated
and sequenced on both strands. In some cases the RT-PCR products were
sequenced directly.
Sequence alignments
Sequences were aligned using the ClustalW program, and the shaded alignment
was generated with GeneDoc (Pittsburgh Supercomputing Center;
http://www.psc.edu/biomed/genedoc).
The phylogenetic tree in Fig. S2 in supplementary material was constructed by
aligning the sequences with the ClustalX program and plotting the alignments
using TreeView.
Analysis of cca-1 expression
Two different cca-1::GFP fusion constructs were used to examine
the expression pattern of the calcium channel gene. The first construct fused
an 8074 base pair (bp) fragment of the cca-1 gene comprising 5.4 kb
of sequence upstream of the putative cca-1 initiation codon and three
coding exons to GFP. The cca-1 fragment was amplified by PCR using
the primers cca-11 (5'-TGATGATAGGACGCTGGTCA-3') and cca-12
(5'-TGTTCCGACAGCTGCAGAGT-3'). The PCR product was digested with
PvuII and PstI and cloned into pBluescript SK. The GFP gene
and unc-54 3' UTR regions of pPD95.69 (a gift from A. Fire,
Carnegie Institution of Washington, USA) were isolated by digestion with
PstI and EagI and the resulting 1.9 kb fragment was inserted
into pBluescript, 3' to the cca-1 sequences, to generate an
in-frame cca-1::GFP fusion (pcca-1::GFP). Transgenic worms were
generated by injecting 20 ng/µl of pcca-1::GFP with 50 ng/µl of
plin-15(+) and 50 ng/µl of pBluescript SK into lin-15(n765ts) X
worms. Transformants were isolated by rescue of the Lin-15 multivulval
phenotype.
The second cca-1::GFP construct was generated using a PCR based
fusion approach similar to that described by
(Hobert, 2002). A total of
three PCR products were used. The first two products (overlapping fragments
encompassing the entire cca-1 genomic locus) were amplified using
wild-type DNA as template (Fig.
1A). The first product (10676 bp) was generated using the sense
primer cca-35 (5'-GAGTCTAGATGAGACGCACA-3'), which annealed
approximately 5.8 kb upstream of the first exon, and antisense primer cca-40
(5'-GCAAGTGTGTAACCCGTTG-3'), located in exon nine. The second
product extended from intron six up to the predicted stop codon and was
amplified using the sense primer cca-38
(5'-TTGCTCGTTCAACACCACTC-3'), and antisense oligo cca-34
(5'-CTTTGGCCAATCCCGGGGATCTAAAGCAGACTTGTGTGATCCA-3'). Next, GFP
sequences from the plasmid pPD95.75 (a gift from A. Fire, Carnegie Institution
of Washington, USA) were amplified using the sense primer cca-39
(5'-TGGATCACACAAGTCTGCTTTAGATCCCCGGGATTGGCCAAAG-3') and oligo
GFP3' UTR (5'-TTCACCGTCATCACCGAAAC-3'). Equimolar amounts of
the 3' cca-1 and GFP PCR products were then fused by PCR
resulting in an in-frame carboxyl-terminal translational fusion. The sequences
of the internal oligos used for the PCR fusion were cca-36
(5'-CATCCGCGTATTTCACGTTG-3') and GFP3'
(5'-ATCCGCTTACAGACAAGCTG-3'). Transgenics were generated by
injecting 20 ng/µl of the 10676 bp 5' cca-1 PCR product with
20 ng/µl of the 3' cca-1::GFP fusion product, and 60
ng/µl of pBluescript SK into cca-1(ad1650) X worms. GFP expression
is contingent on recombination of the 5' cca-1 PCR product with
the overlapping 3' cca-1::GFP fusion. After transformed lines
carrying each of the constructs were established, the extrachromosomal arrays
were integrated using
-irradiation. The resulting strains were
backcrossed to wild-type worms, generating strain TS33 carrying the
pcca-1::GFP construct and TS321 carrying the full-length PCR-fusion based
construct.
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Measurement of pumping rate
Motions of the terminal bulb grinder plates were used to count pumps.
Twenty gravid adult worms of approximately the same size and age were placed
on a lawn of E. coli HB101 and allowed to acclimate for at least 1 h.
Counts were made at room temperature (approximately 23°C). Each worm was
observed for 30 s and each recorded number of pumps was doubled to generate a
`pumps per minute' value. Therefore, N=20 for each calculation of
pumping rate. We note, however, that each experiment was performed at least
three separate times, and data shown are representative. To determine the
statistical significance of a difference in pumping rate between two strains,
we used the two-tailed heteroscedastic t-test.
Extracellular recordings and statistical analysis
Extracellular recordings (electropharyngeograms) were recorded on dissected
pharynxes as previously described (Avery et
al., 1995). For all recordings, the bath included 1 µmol
l-1 serotonin. Recordings were made with a Patch PC-501A amplifier
(Warner Instruments, Hamden, CT, USA), a Digidata 1322A acquisition system
(Axon Instruments Inc, Union City CA, USA) and Axoscope 8.1 software (Axon
Instruments). All recordings were filtered with a low-pass 5 Hz filter and a
high-pass 20 Hz filter. Traces shown in figures underwent an additional
low-pass filter of 350 Hz using Clampfit 8.1 software (Axon Instruments). The
sampling rate was 10 KHz.
The heights of E1 and E2 peaks, the time intervals between them and the log of the ratio of the E1 and E2 heights were recorded for each of the first 12 action potentials from five cca-1 mutant worms and eight wild-type worms. The data were analyzed by two-level nested ANOVA, followed by an F-test of the ratio of the mean square between genotypes to the mean square within genotypes.
The ratio of I-phase spikes to action potentials was calculated by direct counting of spikes in a 1 min recording for each of six to eight worms of each genotype. Any sharp, brief depolarizing spike that was not immediately followed by an action potential was assumed to be an I-phase spike. In snt-1, snt-1; cca-1 and unc-17; cca-1 worms, action potentials are sometimes preceded by a series of depolarizations, the last of which triggers an action potential. All but the last of the series were counted as I-phase spikes. Because the occurrence of I-phase spikes is more common in some individual worms than others, we have summed the data from all the worms of each genotype.
Intracellular voltage recordings
Intracellular voltage recordings were performed using the Axoclamp 2B with
the HS-2A 0.1LU headstage as described by Davis et al.
(1999), with the following
exceptions. The recording chambers and extracellular solution were the same as
used by Shtonda and Avery
(2005
), and pipette solution
was 3 mmol l-1 potassium acetate, 10 mmol l-1 KCl.
Micropipettes were pulled from 1/0.58 mm borosilicate capillaries (A-M
Systems, Carlsborg, WA, USA) on the P-2000 puller; they had resistance of
50-100 M
when filled with the pipette solution. Data were acquired
using custom-designed Labview software
(Raizen and Avery, 1994
) at a
sampling rate of 2 KHz. Another Labview program was used to algorithmically
extract, align, differentiate and average action potentials from recorded
voltage traces
For analysis, regions of recorded traces were chosen in which the resting membrane potential had completely stabilized (normally 2-3 min after electrode insertion). The micropipette tip potential was measured after electrode removal and subtracted from the trace in order to compensate for the small drift of the tip potential that usually occurred during penetration into the cell. The average tip potential change was 4.3±5.8 mV in the positive or negative direction (N=48, mean ± S.D.). Action potentials were recognized when the following conditions were met in this order: (1) the analyzed point in the trace was within the specified baseline (between -90 and -50 mV for the wild type, adjusted for mutants with higher resting potential); (2) the analyzed point was followed within 200 ms by at least one point at voltage higher by 50 mV or more, and (3) the average rate of voltage change in the 50 ms following the analyzed point exceeded 0.5 V s-1. The end of the action potential was determined as the first point within the baseline where the voltage slope over the next 25 ms was positive. To align action potentials, we minimized the sum of squared differences between the rising phase segment from -20 to 10 mV (this segment was chosen because it was the least variable) and a vertical line at 0 ms.
The resting membrane potential was extracted as all points in the analyzed region of the trace within the baseline for which the average rate of change of voltage over the next 100 ms was between -0.02 and +0.02 V s-1. These points were averaged over a 1-3 min interval to calculate the resting membrane potential for a given pharynx.
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Results |
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All of the full-length cDNAs we isolated were trans-spliced to the
22 nucleotide base pair (bp) SL1 leader RNA found on the 5' ends of many
C. elegans mRNAs (Blumenthal and
Steward, 1997) and were similar in structure except for variations
involving alternative splicing of exons 18, 19 and 24. The most commonly
isolated transcript, cca-1A (6129 base pairs in length) was composed
of all exons except exon 19 (Fig.
1A), and encodes a predicted product of 1837 amino acids. The
cca-1B transcript uses an alternative splice donor site within exon
18, which is spliced to exon 19 resulting in a 6273 bp transcript encoding an
1885 amino acid product. The cca-1D transcript differs in that exon
17 is spliced directly to exon 19 generating a transcript of 6150 bp and a
predicted 1844 amino acid polypeptide. The alternative splicing found in the
A, B and D variants is predicted to generate
1 subunits that
only differ in the IIS5-IIS6 loop, which includes the P-loop region for domain
II. Finally, we have found that the cca-1C transcript is identical in
exon composition to the cca-1A variant except for the use of an
alternative splice acceptor for exon 24 (exon 24a in
Fig. 1A), which results in the
incorporation of an additional 15 amino acids in the III-IV loop of the
encoded gene product.
The membrane spanning regions of the most abundant cca-1 splice
variant (CCA-1A) show significant sequence conservation with the corresponding
regions of the vertebrate subunits 1G-
1H
and
1I. CCA-1 resembles the vertebrate T-type
1 subunits in containing aspartate residues in the P-loops
of the third and fourth domains (alignment presented in Fig. S1 in
supplementary material). Calcium channel
1 subunits that
contribute to high voltage-activated (HVA) channels contain glutamate residues
at these positions (Perez-Reyes,
2003
). Furthermore, in contrast to HVA calcium channels, the
domain I-II linker of the CCA-1 protein lacks a ß subunit-binding site,
and the carboxyl region lacks both an E-F hand region and a calmodulin-binding
domain (data not shown). Construction of a phylogenetic tree including all the
putative calcium channel
1 subunits from human,
Drosophila melanogaster and C. elegans demonstrates that
CCA-1 is more closely related to vertebrate T-type
1
subunits than to other
1 subunits (Fig. S2 in supplementary
material). CCA-1 is most similar to vertebrate
1I (42%
identity), and is also closely related to
1G (39% identity)
and
1H (37% identity). These similarities suggest that CCA-1
is likely to function as a T-type calcium channel
1
subunit.
In order to determine the physiological roles of CCA-1, we examined two
cca-1 deletion alleles that are likely to severely compromise
cca-1 function. ad1650, which we isolated from a deletion
library by PCR screening, is a 2.5 kb deletion that removes exons encoding
half of the second and most of the third repeated domains, and also causes a
frameshift (Fig. 1A; see S1 in
supplementary material). gk30, a gift from the C. elegans
Reverse Genetics Core Facility (University of British Columbia, Canada),
contains a smaller deletion that removes a portion of the P-loop of domain II
along with alternatively spliced exon 19, causes a frameshift and results in a
prematurely truncated protein (Fig.
1A; see S1 in supplementary material). The gk30 allele
also contains a complex rearrangement outside the cca-1 coding
region. Shtonda and Avery
(2005) recently demonstrated
that cca-1(ad1650) mutants lack the T-type current observed in
wild-type pharyngeal muscle, providing further evidence that cca-1
encodes the C. elegans T-type calcium channel.
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EPGs reveal that the depolarization of the pharyngeal muscle is abnormal in cca-1 mutants. While EPGs from wild-type worms display a small EPSP before each large E-phase spike, cca-1(ad1650) and cca-1(gk30) deletion mutants often display two peaks of almost equal size in the excitation phase (Fig. 3C). cca-1(ad1650)/cca-1(gk30) heterozygotes have a similar phenotype, while heterozygotes carrying one wild-type and one mutant copy of cca-1 are unaffected (data not shown). We suspect that the first spike in these M-shaped segments represents an EPSP, and the second spike is a small, delayed E-phase spike. Because the EPG trace represents the time derivative of the action potential, a small, late E-spike reflects a delay and a reduced slope in the rise in the pharyngeal muscle membrane potential in response to an EPSP. In wild-type pharynxes, E-phase spikes are, on average, 3.4 times the size of the EPSPs that precede them (Table 1). The average time interval between the peaks of the EPSP and E spikes is seven milliseconds (ms). In cca-1(ad1650) mutant worms, E-phase spikes average 1.3 times the size of their paired EPSPs and the time between peaks averages 27 ms (Table 1).
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In addition to abnormalities related to E-phase spikes, EPGs from
cca-1(ad1650) mutant worms contain more inter-pump phase (I-phase)
spikes than wild-type EPGs (Fig.
3C, Table 1).
I-phase spikes are small depolarizations occurring between action potentials
and represent EPSPs that fail to trigger action potentials. They appear
frequently in EPGs from worms with defects in synaptic transmission, and are
not found in EPGs from worms whose MC neurons have been ablated
(Raizen et al., 1995). For
example, Fig. 3D shows EPGs
recorded from two different mutant strains with defects in MC
neurotransmission. snt-1 encodes synaptotagmin
(Nonet et al., 1993
), a
vesicle-associated protein necessary for effective calcium-stimulated release
of neurotransmitter, while unc-17 encodes a transporter that packs
acetylcholine into synaptic vesicles
(Alfonso et al., 1993
). Both
snt-1(md290) and unc-17(e245) mutants pump more slowly than
wild-type worms (Fig. 3A), and
their EPGs have many I-phase spikes (Fig.
3D and Table 1).
The frequent I-phase spikes in cca-1 mutants combined with the
altered relationship of E-phase spikes to EPSPs suggest that cca-1
mutants are defective in their ability to trigger pharyngeal muscle action
potentials in response to MC stimulation. To further characterize the
depolarization defect in cca-1 mutant animals, we therefore employed
intracellular voltage recordings of pharyngeal muscle activity.
Loss of cca-1 alters the shape of the pharyngeal muscle action potential
Recordings from wild-type pharynxes show a steeply rising slope throughout
the depolarization phase of the action potential. In contrast, recordings from
cca-1 mutant worms often contain exaggerated flattened regions or
notches in the early phase of the muscle depolarization.
Fig. 4A compares representative
single traces from a wild-type worm and from a cca-1(ad1650) deletion
mutant animal. Fig. 4C,D
compares large numbers of action potentials from two worms of each genotype,
and shows that pauses or dips in membrane potential are common in
cca-1(ad1650) mutants, but rare in wild-type worms. (The traces shown
are consistent with traces recorded from a total of 19 wild-type and 16
cca-1 mutant animals.) Membrane potential in cca-1 mutants
consistently stalls at around -30 mV, the potential at which the CCA-1 channel
is expected to activate (Shtonda and
Avery, 2005). If we calculate and plot the time derivatives of
intracellular recordings from cca-1 mutants, the plateau we observe
in membrane potential becomes a valley between the initial MC-stimulated
depolarization of the muscle membrane (the EPSP) and the fast upstroke of the
action potential. Thus, plotting the time derivative of the intracellular
voltage recording trace recreates the phenotype observed in cca-1
mutant EPGs (Fig. 4C,D, lower
panels). As a result of the notches and dips early in the rising phase of the
action potential, the maximal speed of the action potential upstroke is
decreased by about 2.5 times in cca-1(ad1650) mutants compared to
wild-type worms (Fig. 4B).
Thus, comparing the shapes of pharyngeal muscle action potentials in wild-type
and cca-1 mutant worms explains the abnormality we observed in the
relationship of MC EPSPs to the pharyngeal muscle in cca-1 mutants:
the CCA-1 channel is necessary for a smooth, steep rise of membrane potential
after an EPSP. CCA-1, a low-voltage activated channel, may serve as a bridge
between the small membrane depolarization achieved by an EPSP and the EGL-19
L-type channel that activates at more-depolarized potentials.
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Loss of cca-1 enhances phenotypes caused by defects in synaptic transmission
Because wild-type cca-1 seems to be necessary for the efficient
initiation of action potentials in response to MC EPSPs, we hypothesized that
loss of cca-1 function would increase the severity of phenotypes
caused by abnormal synaptic release from MC. We therefore constructed double
mutant strains of cca-1(ad1650) with mutations in unc-17 or
snt-1. As shown in Fig.
5 and Table 1, the
combination of mutations in cca-1 with mutations that decrease the
effectiveness of MC neurotransmission severely compromises pharyngeal pumping.
Both unc-17; cca-1 and snt-1; cca-1 mutant strains exhibit
severely reduced pumping rates and high frequencies of I-phase spikes on EPGs.
These data show that when synaptic release from MC is weakened, wild-type
cca-1 is crucial to the efficient generation of action potentials,
and support a role for cca-1 channels as a bridge between the MC EPSP
and full membrane depolarization.
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Double mutant animals carrying mutations in both eat-2 and cca-1 are almost indistinguishable from eat-2 single mutants in terms of viability and pumping rate (Fig. 3A). EPGs recorded from eat-2; cca-1 double mutants lack EPSPs, and resemble EPGs recorded from eat-2 single mutants (Fig. 6A,B). However, intracellular recordings reveal alterations in resting membrane potential that can explain the lack of interaction between eat-2 and cca-1 mutations: eat-2 single mutants display a resting potential that is elevated by about 13 mV over wild-type resting potential (compare Fig. 7A and C, and see Fig. 7E; this is a statistically significant difference). More strikingly, resting potential in eat-2 mutant pharynxes tends to be unstable and to spontaneously increase (Fig. 7D). Because this alteration in membrane potential cannot easily be explained as a direct result of the absence of the EAT-2 gene product, we suspect that it represents an adaptation employed by the pharynx to facilitate action potential generation. This adaptation may cause the membrane to spontaneously depolarize to a level at which the EGL-19 channel is activated, or may enhance the actions of some other current that brings the membrane to the EGL-19 threshold. In either case, the positive shift in membrane potential seems to allow the pharyngeal muscle to spontaneously depolarize without relying on calcium influx through CCA-1. Resting potential in eat-2; cca-1 double mutants is elevated, but not significantly more than in eat-2 single mutants (Fig. 7D,E). Yet, pumping is just as efficient in eat-2; cca-1 double mutants as in eat-2 single mutants (Fig. 3A). These results demonstrate that the pharynx adapts to the loss of nicotinic MC input by enhancing muscle excitability through changes in resting membrane potential. The process of adaptation allows the animal to preserve pharyngeal muscle activity and maintain feeding behavior, but makes the CCA-1 T-type channel less important.
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Discussion |
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Unexpectedly, CCA-1 does not seem important when nicotinic signaling from
MC is specifically abolished: adding a cca-1 mutation to a strain
lacking the EAT-2 nicotinic receptor subunit has little overt effect on
pharyngeal pumping. Intracellular voltage recordings reveal that worms adapt
to the loss of nicotinic MC input by altering the resting membrane potential
of the pharyngeal muscle. Resting potential in both eat-2 and
eat-2; cca-1 mutants is more positive than in wild-type worms, and
tends to drift upwards in double mutant pharynxes. We speculate that this
unstable membrane potential results from the activation of a leakage
conductance that is normally obscured by frequent action potentials, but is
upregulated in eat-2; cca-1 double mutants to preserve pharyngeal
muscle activity. Shtonda and Avery
(2005) observed a weakly
voltage-dependent, Ni2+- and nifedipine-insensitive conductance
that may be involved. Depolarization by way of the leakage conductance may be
able to bring the membrane to the threshold for EGL-19 activation, thus
directly triggering an action potential, or may rely on additional
intermediate steps that we have not yet identified. It is interesting that
snt-1; cca-1 and unc-17; cca-1 double mutant worms do not
successfully employ the same adaptive strategy used by eat-2; cca-1
double mutants, and have severe feeding defects. This disparity suggests that
adaptation requires a different form of cholinergic input (either from a
source other than MC or through receptors other than nicotinic ones), and may
explain why acetylcholine is essential for pumping
(Avery and Horvitz, 1990
) but
EAT-2/EAT-18 pharyngeal nicotinic receptors are not. Additional
characterization of the relationship of membrane potential to the activity of
MC and other motor neurons will be required to clarify this issue.
The role we have described for CCA-1 channels in pharyngeal muscle
depolarization is similar to the function described for T-type currents in the
vertebrate heart. T-type currents in sino-atrial node and latent pacemaker
cells mediate a late diastolic segment of depolarization, and serve as a
bridge between the early pacemaker currents (If and Ik)
and the opening of voltage gated sodium channels that mediate rapid
depolarization (Hagiwara et al.,
1988; Zhou and Lipsius,
1994
). When T-type currents are blocked with nickel, the slope of
the late diastolic depolarization is reduced, the fast upstroke is delayed and
heart rate slows (Hagiwara et al.,
1988
; Satoh,
1995
), in much the same way that pharyngeal pumping rate slows
when cca-1 is mutated. However, CCA-1 activates in response to
neuronal input, rather than to muscle-intrinsic leak currents. It is therefore
interesting to speculate that C. elegans may retain a leak current
pacemaking mechanism, which becomes important when motor neuron input and
CCA-1 activity are both compromised.
Because we have direct electrophysiological evidence of a CCA-1-mediated
T-type current in the pharyngeal muscle, and because all the observed
pharyngeal function defects in cca-1 mutants can be explained by a
role for CCA-1 in the muscle membrane, we have not discussed possible
functions for CCA-1 outside the pharyngeal muscle. Perhaps the most likely
site of an additional CCA-1 effect on feeding is the MC motor neuron, which
expresses a cca-1 reporter construct (Fig. S3 in supplementary
material). Pan et al. (2001)
reported that T-type channels contribute to neurotransmitter release in
retinal bipolar cells, and the CCA-1 channel might play a similar role in MC.
Disruption of acetylcholine release from MC might contribute to the
inefficient feeding observed in cca-1 mutants, and may explain some
of the synergistic effects seen when cca-1 mutations are combined
with snt-1 or unc-17 mutations that reduce synaptic
transmission. (As noted above, the maintenance of efficient pumping in the
absence of nicotinic MC input may require other types of cholinergic
signaling, which might be compromised in cca-1 mutants.) However,
effects within MC would not explain the alterations we have observed in
voltage recordings from the pharyngeal muscle in cca-1 mutants.
Because we have not yet developed a rescuing cca-1 construct, we
cannot exclude a role for cca-1 function in MC. Similarly, we cannot
rule out the possibility that CCA-1 channels located outside the pharynx
influence feeding behavior. We have observed no obvious defects in locomotion,
egg-laying, defecation or mating in cca-1 mutants (data not shown).
However, loss of cca-1 function may cause subtle defects in the
activity of other excitable cells which are obscured by adaptive mechanisms
such as that observed in eat-2; cca-1 double mutants. The development
of additional double mutant strains and careful electrophysiological analysis
may therefore reveal roles for T-type conductances in other muscles and
neurons.
In summary, this study has provided an initial characterization of the cca-1 gene from C. elegans, and directly demonstrates a role for T-type currents in pharyngeal muscle depolarization. CCA-1 T-type channels participate in the initiation of action potentials, helping the membrane to reach the threshold for activation of L-type channels after an EPSP from the MC motor neuron. The importance of this function is suggested by both the impact on feeding behavior of loss of cca-1 function, and the fact that the pharyngeal muscle undergoes considerable adaptive changes - altering membrane potential - in order to preserve muscle function in the absence of CCA-1 and motor neuron activity.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work
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References |
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