CCA-1, EGL-19 and EXP-2 currents shape action potentials in the Caenorhabditis elegans pharynx
Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9148, USA
* Author for correspondence (e-mail: boris{at}eatworms.swmed.edu)
Accepted 19 February 2005
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Summary |
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We show that CCA-1 exhibits T-type calcium channel properties: activation at -40 mV and rapid inactivation. Our results suggest that CCA-1's role is to accelerate the action potential upstroke in the pharyngeal muscle in response to excitatory inputs. Similarly to other L-type channels, EGL-19 activates at high voltages and inactivates slowly; thus it may maintain the plateau phase of the action potential. EXP-2 is a potassium channel of the kV family that shows inward rectifier properties when expressed in Xenopus laevis oocytes. We show that endogenous EXP-2 is not a true inward rectifier - it conducts large outward currents at potentials up to +20 mV and is therefore well suited to trigger rapid repolarization at the end of the action potential plateau phase. Our results suggest that EXP-2 is a potassium channel with unusual properties that uses a hyperpolarization threshold to activate a regenerative hyperpolarizing current.
Key words: calcium channel, L-type/T-type/potassium channel, Caenorhabditis elegans
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Introduction |
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To observe the electrical activity of the pharynx, Raizen and Avery
(1994) invented pharyngeal
extracellular recordings - electropharyngeograms - and Davis and Avery
(Davis, 1995
) developed sharp
electrode voltage recording from the pharyngeal muscle. Methods for voltage
clamp recordings on C. elegans neurons
(Goodman et al., 1998
) and
body wall muscle (Richmond and Jorgensen,
1999
) have also been established. Using these preparations, a
variety of ligand and voltage-gated channels have been studied
(Francis et al., 2003
; Jospin
et al.,
2002a
,b
;
Mellem et al., 2002
;
Pierce-Shimomura et al.,
2001
). In this study, we develop a voltage clamp technique for the
C. elegans pharynx and look at the in vivo function of
pharyngeal ion channels.
The following model for the pharyngeal muscle is best supported by the
previous work. MC fires, causing an excitatory post-synaptic potential (EPSP),
which activates the EGL-19 L-type calcium channel
(Lee et al., 1997). Calcium
entry through EGL-19 further depolarizes the pharynx and causes contraction.
During contraction, M3 fires, causing small notch hyperpolarizations (IPSPs).
At some point, possibly triggered by M3 IPSPs, the potassium channel EXP-2
recovers from inactivation, causing a full rapid repolarization
(Davis et al., 1999
). Here we
show, using the voltage clamp, that this model is generally correct with
respect to EGL-19 and EXP-2, but that one player, a T-type calcium channel
CCA-1, had been overlooked. In the pharyngeal muscle, CCA-1 mediates a large
inward depolarizing current, which is the first demonstrated role of the
T-type calcium channel in C. elegans. This finding, along with the
accompanying work (Steger et al.,
2005
), suggest that CCA-1 activates in response to excitatory
inputs from the MC neuron and accelerates the action potential upstroke.
Our second finding concerns the native behavior of the EXP-2 potassium channel. When expressed in a heterologous system, Xenopus oocytes, EXP-2 behaves as an inward rectifier: it conducts very little current at potentials more positive than the equilibrium potential for potassium. This is due to its peculiar kinetics: the inactivation is much faster than the recovery from inactivation at positive potentials. Surprisingly, we show that in its native environment, in the pharynx, EXP-2 conducts large outward currents at positive potentials. Thus, the property of ultrafast inactivation in response to hyperpolarization is not observed in the native system. EXP-2 currents are effectively triggered by the hyperpolarization threshold, an unusual mechanism among ionic channels.
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Materials and methods |
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Recording and dissection chambers
For dissection and voltage clamp recordings, disposable chambers were made.
A square piece of Parafilm with a 10 mm circle embossed with a Sharpie pen cap
was placed on a clean 50 mm x30 mm coverslip (Fisher Scientific,
Pittsburgh, PA, USA). It was covered with another coverslip, moistened by
breathing on it to prevent sticking. This assembly was placed on a dry heating
block at 70-90°C, thumb pressed for 3 s, and air-cooled. The upper slide
was pried away with a razor blade and removed; the Parafilm stuck firmly to
the bottom slide. Chambers were stored like this to keep the glass clean. The
Parafilm circle was excised immediately before use, leaving a circle of clean
glass surrounded by Parafilm. The chamber held up to 150 µl of
solution.
Dissection procedure
Early gravid adult animals (2-2.5 days old) were used for experiments. They
were transferred to a 100 µl drop of low calcium Dent's solution (see
Solutions and Chemicals for all solutions) on a cooled dissection chamber. (A
flat tissue culture flask filled with ice-cold water was used for cooling.)
Under a dissection microscope, worms' heads were cut off with a hand-held
25-gauge syringe needle (Fig.
1B). The corpses were removed with 50 µl of buffer, then 50
µl of digestion mix 1 was added. The slide was placed on an Axiovert 35
inverted microscope (Zeiss, Germany). 10-15 pharynxes were processed in each
batch.
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While the pharynxes were digesting in mix 1, body wall removal (skinning) was performed as shown in Fig. 1C. The terminal bulb of the pharynx was sucked into the larger pipette. Then, the smaller pipette was attached to the front end of the pharynx and strong suction was applied to the small pipette by locking the piston of the 30 ml syringe as far out as it would go. The small pipette was then advanced into the big one, inverting the cuticle and the body wall covering the pharynx. At this point, the pressure in the small pipette was switched to atmospheric. By moving the small pipette back and fourth, the cuticle was torn off the pharynx; in cases when the inverted cuticle stayed attached we cut it off later with a hand-held 25-gauge syringe needle. Finally the pharynx was expelled from the big pipette. Cuticles and dead pharynxes were removed in 50 µl of solution, and 50 µl of digestion mix 2 was added. After 15-20 min digestion at room temperature, pharynxes were transferred with a pipette to a 100 µl drop of Dent's solution on a clean recording chamber.
Using the same small pipette that was used for skinning, pharynxes were positioned in the center of the chamber and attached to the glass by gently pressing them with the pipette. Dissection pipettes were removed to allow patch pipette access. After 2-3 min perfusion with the Dent's solution, recordings were started. All recordings were done at room temperature (22-25°C).
Voltage clamp recording
An Axoclamp 2B amplifier (Axon Instruments, Union City, CA, USA) equipped
with an HS-2A 0.1LU recording headstage was used in the cSEVC
(continuous single electrode
voltage clamp) recording mode. The headstage was mounted
on an MHW-4 (Narishige, Japan) one-axis water hydraulic manipulator, which was
fixed on a UMM-3FC manipulator. The amplifier was interfaced with a Pentium 3
Windows NT computer via a PCI-6035E E-Series DAQ-200 board (National
Instruments, Austin, TX, USA) and controlled by custom-designed software
developed in the Labview 6 environment (National Instruments). Amplifier
settings were as follows: gain 3 nA/mV, phase lag 0.07 ms, multiplier 100,
output bandwidth 3 kHz. Sampling rate was 4 kHz. Patch pipettes were produced
from 1/0.58 mm borosilicate capillaries (A-M systems) on a P-2000 puller and
heat-polished; they had resistances of 5.5-7 M when filled with
intracellular solution. After break-in to the whole-cell configuration by
suction or mild buzzing, series resistance Rseries was
10-15 M
. Series resistance compensation ('BRIDGE' knob in the cSEVC
mode) and capacitance compensation were used as stability allowed.
Preparations with initial Rseries>15 M
could not
be clamped. The bath solution was perfused by gravity flow at approx. 0.1 ml
min-1 during recordings. During pulse protocols, we used 5 ms
voltage ramps in informative voltage steps, or 40 ms ramps for non-informative
steps instead of instantaneous (square) voltage steps. This greatly increased
the survival of the pharynxes and overall success rate; square pulses to above
0 mV usually killed pharynxes.
The holding potential was -80 mV. The linear current component (leak current) was measured using 20 mV hyperpolarizing test pulses (from -80 to -100 mV) and subtracted during recordings.
In addition to inward currents (Fig.
2), in some experiments, depolarizing pulses evoked an outward
current similar to a delayed rectifier Kv current (data not shown).
This outward current was highly variable, from non-existent to very
noticeable. In these experiments we observed strong contractions in response
to depolarizing pulses. We tend to distrust these experiments, because we
believe that in these experiments intracellular Ca2+ was poorly
buffered, causing non-physiological changes during recording. Such
preparations were discarded. We only chose preparations in which the
intracellular calcium concentration [Ca2+]i was
apparently well buffered, as judged by the complete absence or hardly
noticeable muscle contraction in response to the depolarization. Buffering
[Ca2+]i could certainly block some currents, for example
calcium-activated K channels. However, in Ascaris lumbricoides
pharynx no delayed rectifier current was observed, even when no attempt to
buffer [Ca2+]i was made
(Byerly and Masuda, 1979).
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Sharp electrode voltage recordings
Sharp electrode voltage recordings were performed as described by Steger et
al. (2005). To determine the
slope of the action potential plateau phase, 30 action potentials from five
pharynxes (six from each) were analyzed. For each action potential, the
coordinates of the start and the end of the plateau phase were manually
determined using Igor Pro software (Wavemetrics, Lake Oswego, OR, USA). The
slope of these segments was averaged to obtain the average plateau phase
slope.
Solutions and chemicals
Digestion mix 1 was prepared by mixing collagenases F and H (Sigma, cat.
nos. C-7926 and C-8051) to adjust collagenase activity to 20 U ml
l-1 and protease activity to 45 U ml l-1 in low calcium
Dent's solution [same as Dent's solution (see below) except the
Ca2+ concentration was 10-5 mmol l-1]. Mix 2
contained (in U ml l-1; all from Sigma): 20 collagenase, 600
protease (adjusted by mixing collagenases F and H), 13 protease X (cat. no.
P-1512), 1300 trypsin (T-0303), 1 chitinase (C-6137) in low calcium Dent's
solution. Modified Dent's solution (Dent
and Avery, 1993) was used as an extracellular solution (in mmol
l-1): 140 NaCl, 6 KCl, 1 MgCl2, 3 CaCl2, 10
Na-Hepes, pH 7.3, osmolarity adjusted to 345 mOsm kg l-1 with
xylitol. Intracellular solution contained (in mmol l-1): 130
potassium gluconate, 10 NaCl, 5 K-EGTA, 0.5 CaCl2, 1
MgCl2, 10 K-Hepes, pH 7.3, osmolarity adjusted to 325 mOsm kg
l-1 with xylitol. Nifedipine was from Sigma.
Pharynx capacitance calculation
The physical dimensions of the pharynx of an adult worm were taken from
table 1 in Avery and Shtonda
(2003). The total length of the
pharynx is 144.7 µm. The perimeter of the interior lumen is 25 µm in the
corpus and 20 µm in the isthmus and in the terminal bulb; the lengths of
the corpus, isthmus and terminal bulb are 76.6, 35.8 and 32.3 µm.
Therefore, the area of the internal lumen is 76.6 x25+35.8
x20+32.3 x20= 3277 µm2. (We assumed the lumen
section in the isthmus and terminal bulb to be the same.) Using a pharynx
micrograph, we found the outer radius of the pharynx at 100 points along its
length (r1-r100). The area of the
outer surface is a sum
{
(ri+ri+1)
x[(ri-ri+1)2+L2]}=8939
µm2 (from i=1 to i=99), where L is a
step along the x axis (1.447 µm).
Thus, the total surface membrane area is 12216 µm2. In
various studies, the specific membrane capacitance was measured in the range
0.7-1.3 µF cm-2 (Curtis and
Cole, 1938; Gentet et al.,
2000
). Assuming the specific membrane capacitance of 1 µF
cm-2 (0.01 pF µm-2), the surface capacitance is 122
pF. Next, we estimated the contribution of some internal membranes to the
total capacitance. In the corpus and isthmus, there is an invagination in each
muscle cell. Assuming that the depth of these invaginations is equal to the
radius of the pharynx (figs 5 and 6 in
Albertson and Thomson, 1976
),
the total area of invaginations is
6
[(ri+ri+1)L/2]= 5226
µm2; they would contribute 52 pF of capacitance.
Three marginal cells run the length of the pharynx; and each marginal cell
has two lateral membranes that face pharyngeal muscle cells. It is not known
whether marginal cells contract, but most likely they are charged because they
appear to receive neuronal input (Albertson
and Thomson, 1976), and gap junctions have been observed
connecting them to the muscle cells (L.A., unpublished data). The total area
M of lateral muscle cell and marginal cell membranes is
12
[(Mi+Mi+1)L/2]=7231
µm2 where Mi=0.7 ri in
the isthmus and 0.6 ri in the corpus, as estimated from
figs 5 and 6 in Albertson and Thomson
(1976
). These membranes would
add 72 pF to the total capacitance.
Finally, we included membranes that connect different layers of muscle
cells, as deduced from fig. 21 in Albertson and Thomson
(1976). These membranes would
contribute at least 30 pF (their invaginations were not included).
Thus, the total membrane capacitance of the pharynx is 122+52+72+30=276 pF. We assumed capacitances of pharyngeal cells to be in parallel, so that they add up to produce the maximum possible total capacitance. If some capacitances are in series, they would reduce the total capacitance. However, some membranes that lie within the pharynx, such as those lining cavities in the terminal bulb where gland cells reside or those of the terminal bulb marginal cells, were not included in this calculation because of their convoluted shape, and some internal membranes that were included are not flat and contain invaginations. These simplifications could result in underestimation of the capacitance. Because of the uncertainties in membrane area and the lack of a direct measurement of the specific capacitance of pharyngeal muscle membrane, this is a very rough estimate, probably only reliable to within a factor of two.
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Results |
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Functionally and anatomically, the pharynx can be divided into three major
compartments: the corpus, the isthmus and the terminal bulb (listed from
anterior to posterior; Fig.
1A). When a worm's head is cut off, the body wall muscles
underlying the cuticle contract and the terminal bulb falls out, while the
corpus remains covered (Fig.
1B). When we and others
(Davis, 1999) attempted to
voltage clamp the pharynx via the terminal bulb, the corpus
apparently could not be clamped and fired action potentials in response to
depolarizing voltage pulses. Empirically, we found that the pharynx could be
clamped via the corpus if a patch electrode seals on the pm4 muscle
cell (Fig. 1A, area of patching
is shaded). To make the corpus accessible to a patch electrode, we devised a
microdissection procedure. Using two glass pipettes, the cuticle covering the
pharynx was inverted and torn off (Fig.
1C). Then, the pharyngeal basement membrane was digested with
enzymes, and the pharynx was attached to the glass slide
(Fig. 1D).
We do not fully understand why it is possible to voltage clamp the pharynx
via one, but not the other compartment. It is probable that the
corpus has lower resistance than the terminal bulb and is a more powerful
current source, which is suggested by the fact that the relaxation transients
of the terminal bulb, recorded by electropharyngeogram, are about six to
tenfold smaller than those of corpus
(Raizen and Avery, 1994). In
this case, currents that leak into the terminal bulb from the corpus will be
large compared to the currents measured in the terminal bulb, whereas currents
that leak into the corpus from the terminal bulb will be small compared to the
currents measured in the corpus. When we clamped pharynxes via the
corpus, we did not see interference that looked like current injections from
neighboring poorly clamped cells. Therefore, either the terminal bulb is
clamped well from the corpus, so it cannot interfere, or, it is clamped poorly
but it does not have the ability to interfere, because it is not as
electrically active as corpus. We could not test how well the terminal bulb is
clamped, because we could not measure its membrane potential with another
electrode.
Mechanically, the pharynx is a very rigid structure - during contractions its inner lumen opens, while its outer shape does not change. Because the pharynx is attached to other tissues only at its very front and back ends, this mechanical rigidity is entirely conferred by the pharyngeal basement membrane. In order to be able to form gigaseals, we had to use rather harsh digestion with a mixture of collagenases, proteases and chitinase. It is certainly possible that as a result of this digestion, the physiology of the pharynx changes, and we cannot test this because we cannot record from undigested pharynxes. One of the reasons why we think these changes are not too dramatic is because digested pharynxes still contract spontaneously and fire trains of action potentials in response to current injection (data not shown).
Electrical parameters of the wild-type pharynx are as follows: input
resistance 22.8±5.2 M, capacitance 362.6±36.8 pF,
equilibrium potential for the leakage current -53.6±4.1 mV
(N=29). Current densities (normalized to the cell capacitance) are
comparable to those in body wall muscle. For example, EGL-19 currents in the
body wall have a peak density of 7 A/F
(Jospin et al., 2002a
). In the
pharynx, the maximal density of the high voltage-activated current is about 12
A/F (see Fig. 3A, see text
below). For CCA-1 and especially EXP-2 currents, peak densities are much
higher; up to 50 A/F (up to 18 nA in current amplitude). Our results (see
below) suggest that CCA-1 and EXP-2 currents function at the start and at the
end of the action potential; thus, they must be large to rapidly charge the
membrane capacitance. In comparison with EGL-19 currents recorded in the body
wall muscle, which have peak amplitudes of 0.5 nA
(Jospin et al., 2002a
),
pharyngeal currents are very large in amplitude.
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In order to see whether or not we fully charge the pharynx in our voltage clamp, we compared its measured capacitance with a crude estimate based on geometry (see Materials and methods). The calculated capacitance, deduced from its membrane area, is approximately 276 pF. The calculated capacitance is smaller than the measured capacitance (363 pF), suggesting that the former is an underestimate that cannot be used as the only way to estimate the voltage clamp efficiency. But within the possible uncertainty of the estimate, this result is at least consistent with the hypothesis that most of the capacitance is charged.
The large CCA-1 and EXP-2 currents evidently function to rapidly charge
membrane capacitance at the start and at the end of the action potential
upstroke. Thus, the physiologically relevant pharyngeal capacitance can be
estimated. In voltage recordings, the peak rate of the voltage change during
upstroke reaches 20 V s-1
(Steger et al., 2005). The
peak CCA-1 current amplitude is about 8 nA
(Fig. 2A). Because for
capacitive current I=C(dV/dt), this active
current can charge a capacitance of about 400 pF, which is close to the
measured capacitance of 363 pF. Therefore, crude estimates of the pharyngeal
capacitance, based either on its structure or on the size of active currents,
are consistent with the experimentally measured capacitance. This suggests
that most of the pharyngeal capacitance is charged in our voltage clamp
experiments.
CCA-1 is a T-type calcium channel that mediates the low voltage-activated current in the pharynx
In response to step depolarizations from a holding potential of -80 mV, an
inward current is activated (Fig.
2). In wild-type pharynxes, there is a low-voltage activated
(LVA), quickly inactivating component in this inward current, which looks like
a conventional T-type calcium channel current (N=29;
Hille, 2001). CCA-1 is a
calcium channel alpha subunit in C. elegans homologous to mammalian
T-type calcium channel alpha 1 subunits
(Steger et al., 2005
). In
worms cca-1::GFP fusion protein is expressed in a variety of
tissues, including the pharyngeal muscle
(Steger et al., 2005
).
Pharynxes of a cca-1(ad1650) null mutant did not exhibit the LVA
current (N=19; Fig.
2A). In order to isolate the non-LVA component of the inward
current in the wild type, we used a prepulse voltage protocol. A 300 ms
prepulse to -40 mV activates and inactivates the LVA current, revealing a high
voltage-activated (HVA), slowly inactivating component
(Fig. 2B). The HVA current is
the same in the wild type and in cca-1. Consistent with the
pharmacology of calcium channels, both HVA and LVA currents are blocked by 2
mmol l-1 Ni2+ (Fig.
2C). In our system T- and L-type currents seemed to be equally
sensitive to Ni2+ and we only saw an effect at rather high
concentrations, starting with 0.5 mmol l-1. Both T-type and HVA
vertebrate channels are also sensitive to these concentrations of
Ni2+ (Hille, 2001
).
Among vertebrate T-type alpha subunits, only
1H is highly
sensitive to block by low Ni2+ concentrations, with
IC50=13 µmol l-1
(Lee et al., 1999
), so the
Ni2+ sensitivity of the pharyngeal LVA current is consistent with
most known T-type channels. The HVA but not the LVA current is partially
blocked by the L-type calcium channel blocker nifedipine
(Fig. 2D).
Current-voltage dependencies of LVA and HVA currents for wild type (N2) and
cca-1 are shown in Fig.
3. The LVA current activates starting from -40 mV, reaching a
maximum at -30 mV. The HVA current starts to activate at about -10 mV,
reaching a maximum at about +20 mV. Finally, it is interesting to note that
the pharyngeal muscle of C. elegans, unlike the body wall muscle
(Richmond and Jorgensen,
1999), does not show a noticeable delayed rectification, except
under special circumstances (see Materials and methods). This is also true for
another nematode, Ascaris lumbricoides
(Byerly and Masuda, 1979
).
Our results suggest that CCA-1 is a true worm T-type calcium channel ortholog with typical T-type kinetics, voltage dependency and pharmacology. We have not, however, determined whether CCA-1 is selective for Ca2+, because of the sensitivity of the pharynx preparation to low sodium or EGTA in the bath solution.
EGL-19 mediates the high-voltage activated current in the pharynx
By their high voltage activation and slow inactivation, HVA currents
recorded from the pharynx look very similar to L-type calcium channel currents
(Hille, 2001). They are also
similar to L-type currents recorded in C. elegans body wall muscle
(Jospin et al., 2002a
).
Nifedipine, a dihydropyridine L-type calcium channel antagonist, blocks the
HVA current (Fig. 2D). (In
contrast to the body wall L-type current, which is blocked by 1 µmol
l-1 nifedipine, we only saw an effect starting from 5 µmol
l-1.) Other channels may also be affected by these rather high
nifedipine concentrations, but in our system it only affected the HVA, not the
LVA current.
EGL-19, a C. elegans L-type calcium channel alpha subunit, has
previously been implicated in pharyngeal physiology: hypomorphic mutations in
egl-19 cause feeble pumping, whereas gain-of-function mutations cause
extended terminal bulb contractions. In hypomorphic mutants such as
egl-19(n582) (Trent et al.,
1983) the slope of the rising phase of the action potential is
smaller than in the wild type (Lee et al.,
1997
). n582 is an arginine to histidine substitution in
the S4 voltage sensor segment of the channel, but it is not a null mutation;
unfortunately, egl-19 null mutants are embryonic lethal
(Lee et al., 1997
). We found
that in the egl-19(n582) hypomorphic mutant the activation of the HVA
current is clearly delayed (Fig.
4A,B). This is observed both in the cca-1 background
where LVA currents are conveniently absent
(Fig. 4A) and in the wild type
with a prepulse voltage protocol (Fig.
4B). Activation-time constants in n582 are significantly
larger than in the wild type at pulses to 30, 40 and 50 mV
(Fig. 4D). Consistent with the
study of the same mutation in the body wall muscle
(Jospin et al., 2002a
), the
current-voltage dependence in n582 is shifted by about 10 mV to the
right (Fig. 4C); however, in
contrast to the body wall, the peak current amplitude is not decreased
compared to the wild type. This could be explained by a compensatory increase
of mutant EGL-19 expression in the pharynx. For example
(Steger et al., 2005
) have
shown that compensatory changes in pharyngeal currents occur if MC
neurotransmission is lost, suggesting that pharyngeal excitability is tightly
feedback regulated. Alternatively, the effect of n582 on the
pharyngeal EGL-19 could be different because of different non-alpha
subunits.
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A certain component of the depolarization-activated current is not blocked by either Ni2+ or nifedipine. This component shows almost no inactivation (Fig. 2C,D) and has very shallow voltage dependence (Fig. 3A,B). It is unlikely to be a residual L-type current because its voltage dependence is very shallow and totally different. It is also unlikely to be a T-type current because it is unchanged in cca-1. Most probably, this is a weakly voltage-dependent, leakage-like conductance.
EXP-2 generates outward current in response to hyperpolarization
Byerly and Masuda (1979)
described a negative spike potassium current in the pharyngeal (also called
esophagus) muscle of the parasitic nematode Ascaris lumbricoides.
This current activates in response to step hyperpolarization of a depolarized
muscle. In studies by Wayne Davis and colleagues
(Davis, 1999
;
Davis et al., 1999
), the
potassium channel EXP-2 has been proposed to be the negative spike channel in
the pharyngeal muscle of C. elegans. According to the amino acid
sequence, EXP-2 is a potassium channel of the kV family, and a single member
of its own subfamily (Fleischhauer et al.,
2000
). By analyzing mutants, Davis
(1999
) showed that EXP-2
regulates action potential duration in the C. elegans pharynx by
initiating rapid repolarization at the end of the plateau phase of an action
potential. Normally, action potentials last for about 100-250 ms, and in
exp-2 null mutants they are extended up to 6 s. In contrast, in
exp-2 gain-of-function mutants action potentials are shortened to
about 50 ms.
When it is expressed in Xenopus laevis oocytes, EXP-2 behaves as
an inward rectifier with properties similar to mammalian HERG
(Fleischhauer et al., 2000).
Kinetic measurements on EXP-2 expressed in Xenopus laevis oocytes
have shown that the inward rectification of this channel results from its
ultrafast inactivation (Fleischhauer et
al., 2000
), similarly to mammalian HERG
(Spector et al., 1996
). While
the channel activation and deactivation are relatively slow in a wide voltage
range (apparent time constants are of the order of 100 ms; fig. 5 in
Fleischhauer et al., 2000
),
the inactivation rate is extremely fast: time constants are of the order of 1
ms. Because inactivation is faster than activation, EXP-2 does not conduct in
response to depolarization (fig. 2 in
Fleischhauer et al., 2000
).
Depolarization-activated outward currents in oocytes are small and very
transient, lasting at most for 10 ms (fig. 4 in
Fleischhauer et al.,
2000
).
More significantly, EXP-2 expressed in oocytes fails to conduct upon
hyperpolarization to voltages more positive than the equilibrium potential for
K+, since at those potentials it remains inactivated. Yet in the
pharynx, it must generate an outward, hyperpolarizing current, causing a
negative spike, when the membrane potential is about 0 mV, much more positive
than EK+. This function of EXP-2 is predicted by studies in both
Ascaris (Byerly and Masuda,
1979; del Castillo and
Morales, 1967
) and C. elegans
(Davis, 1999
;
Davis et al., 1999
). Thus
Fleischhauer et al. (2000
)
concluded that the properties of oocyte-expressed EXP-2 are not well suited
for its suggested role. We find, however, that in the pharynx, its native
environment, EXP-2 is not an inward rectifier. It conducts large outward
currents at potentials far more positive than the EK+
(Fig. 5A). These currents are
absent in the exp-2(ad1426) null mutant. (Note that an inward current
in exp-2(ad1426) is most likely an EGL-19 tail current.) Starting
from +20 mV and going more negative, the current-voltage dependence of EXP-2
is almost linear, with no signs of inward rectification
(Fig. 5B). This is similar to
the voltage dependence of the negative spike current in Ascaris
pharynx, except that in Ascaris this current starts from the
hyperpolarization to -15 mV (Byerly and
Masuda, 1979
). The reversal potential of EXP-2 current is close to
the predicted EK+ under these conditions (-82 mV). As
expected, currents rise faster at more negative potentials, indicating strong
voltage dependence of recovery from inactivation.
|
Action potential duration is controlled by the slope of the plateau phase
During the action potential plateau phase, the membrane slowly repolarizes
(Davis et al., 1995),
presumably because of slow EGL-19 inactivation. During this time, EXP-2 falls
into a `primed' activated, but inactivated state. At some point, rapid
recovery from inactivation occurs and an outward current spike arises, causing
rapid repolarization. We were interested in how the shape of an action
potential, particularly of a plateau phase, affects the timing of the EXP-2
spike. Using voltage recordings, we determined that in the wild type, the peak
membrane potential during upstroke was 33±4 mV and the slope of the
plateau phase was -0.22±0.06 V s-1 (30 action potentials
from five pharynxes; see Materials and methods). Then, we voltage clamped
pharynxes under two pulse protocols. To test the ramp effect, we depolarized
pharynxes from the holding potential of -80 mV to 33 mV and then applied
varying negative voltage ramps (Fig.
6A). To test the effect of the action potential amplitude, we
depolarized pharynxes to different potentials and then applied the same ramp
of -0.22 V s-1 (Fig.
6B). Under the varying ramp protocol, current transients appear
starting from -30 mV at fast ramps and then at 0 mV
(Fig. 6B). These are EXP-2
currents, since they are absent in the exp-2 null mutant
(N=2, data not shown). Under the varying depolarization/constant ramp
protocol, currents start at peak depolarizations to +20 mV, but,
interestingly, with increasing depolarization, the potential at which the
EXP-2 current transient develops increases by the same increment in such a
manner that the time when the current develops is almost unchanged (about 200
ms). These results suggest that EXP-2 is tuned to generate a current spike
once the membrane potential has dropped by a certain value, approximately 30
mV, from the peak depolarization. Thus, the latency of the EXP-2 current and
the timing of the repolarization onset is effectively regulated by the ramp of
the action potential plateau phase; the action potential amplitude has very
little effect once some minimum depolarization has been reached (about +20 mV
in our recordings). How could such regulation work? Most probably, it is
explained by the voltage dependence of EXP-2 activation: at more positive
voltages, the activation occurs faster, so channels more quickly become
available for the recovery from inactivation
(Fleischhauer et al., 2000
).
Because EXP-2 activation is relatively slow
(Fleischhauer et al., 2000
),
it is limiting in determining the EXP-2 current timing under these conditions.
Activation also limits the number of available channels, as seen from larger
EXP-2 current amplitudes following larger peak depolarizations. As seen in
Fig. 6A, the voltage at which
EXP-2 current develops is lower with very fast ramps, probably because under
these conditions activation is again limiting - even though EXP-2
deinactivates rapidly, there is no current until enough activation is
achieved. When activation reaches saturation, the potential at which current
develops does not change, it stays at about 0 mV, and its onset is completely
determined by the recovery from inactivation voltage dependence. It is indeed
remarkable how nicely EXP-2 kinetic properties are tuned to control the action
potential duration.
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Discussion |
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Our model does not provide a quantitative description of the voltage change
during the action potential. We were unable to achieve the high-compliance
voltage clamp required for kinetic measurements. We can, however, identify
pharyngeal ion channels by the effect of mutations. CCA-1 and EXP-2 currents
were identified by their complete absence in the respective null mutants,
which was very obvious despite the imperfect clamp. The effect of an EGL-19
hypomorphic mutation is less striking but significant and consistent with
previous reports. Furthermore, this model makes predictions about the effects
of mutations on pharyngeal behavior, some verified by previous work
(Davis et al., 1999;
Lee et al., 1997
), and some
confirmed in the accompanying paper
(Steger et al., 2005
).
CCA-1 functions in the action potential rising phase
We here report one in vivo function of the C. elegans
T-type calcium channel ortholog CCA-1. We show that CCA-1 generates an inward
current in response to the depolarization. In the accompanying paper
(Steger et al., 2005), we
present evidence that CCA-1 aids in neurotransmission from MC to the
pharyngeal muscle, and is necessary for the MC EPSP (excitatory post-synaptic
potential) to rapidly and reliably trigger a muscle action potential. This is
similar to the role of the T-type calcium channel in other systems. In the
mammalian sino-atrial node, T-type current triggers an action potential after
the hyperpolarization-activated inward current (If) brings
the membrane potential to its activation threshold of about -50 mV
(DiFrancesco, 1993
;
Hagiwara et al., 1988
).
Starting from -20 mV, L-type currents are activated. Similarly to the heart
pacemaking tissue, T-type and L-type calcium channels function in concert in
the pharyngeal muscle.
Interestingly, the T-type current has not been recorded in Ascaris
(fig. 3 in Byerly and Masuda,
1979). But of course T-type calcium channels were not known at
that time, so the authors of that study did not attempt to look for them and
clamped cells at the holding potential of -37 mV, at which the T-type calcium
channel is probably inactivated. So the depolarization-activated inward
current in Ascaris pharynx looks much like the cca-1 mutant
response (compare Fig. 2 in our
paper and fig. 3 in Byerly and Masuda,
1979
).
EXP-2 displays unique properties in its native environment
In studies of the pharyngeal muscle of the parasitic nematode Ascaris
lumbricoides it was noted that in addition to positive-going action
potentials evoked by the depolarizing pulses, negative spike action potentials
in the depolarized membrane can be evoked by small hyperpolarizing pulses
(del Castillo et al., 1964;
del Castillo and Morales,
1967
). (The name `negative spike' reflects the fact that these
events are similar to the `positive spike', conventional action potentials
resulting from the Na+ channel opening, but they are opposite in
direction.) Byerly and Masuda
(1979
) determined that this
current is carried by potassium and measured its voltage dependence and
kinetics. Finally, work of Wayne Davis and collaborators
(Davis, 1999
;
Davis et al., 1999
) have shown
that C. elegans EXP-2 potassium channel is likely to be the negative
spike channel.
We show that EXP-2 conducts an outward current in response to
hyperpolarization (Fig. 4). Our
results suggest that properties of endogenous EXP-2 are similar, yet different
from those observed when EXP-2 is expressed in Xenopus oocytes
(Fleischhauer et al., 2000).
Similarly to the oocyte-expressed channel, pharyngeal EXP-2 does not conduct
upon depolarization, thus the property of ultrafast inactivation upon
depolarization observed in oocytes is maintained in the native system. Also,
the inactivation/deinactivation equilibrium is voltage-dependent in both
oocytes and pharyngeal muscle, with positive membrane potential favoring
inactivation. In oocytes the inactivation/deinactivation equilibrium is far
towards inactivation even at 0 mV. But surprisingly, the deinactivation
threshold appears to be much more positive in the pharynx, allowing for large
outward currents lasting up to 80 ms at depolarized potentials as high as +20
mV (Fig. 5B). Thus, in contrast
to the oocyte-expressed channel, pharyngeal EXP-2 does not inwardly rectify.
EXP-2 current is triggered by membrane hyperpolarization to below the
deinactivation threshold, which makes EXP-2 a channel with unusual properties,
somewhat resembling those of the mammalian channel HERG
(Spector et al., 1996
) but
otherwise unique. These properties are ideally suited for the EXP-2 function
in the pharyngeal muscle, which is to control the action potential duration by
initiating rapid repolarization when the membrane potential is about 0 mV
during the plateau phase.
That the EXP-2 properties in the heterologous system are different from the
ones in the native environment is not very surprising. Other C.
elegans ion channels require accessory subunits for their proper
function, counterparts of which have not yet been found in vertebrates. For
example, EAT-18 is required for the function of the pharyngeal nicotinic
acetylcholine receptor (McKay et al.,
2004), and SOL-1 protein is required for the function of the
C. elegans GLR-1 glutamate receptor
(Zheng et al., 2004
). It is
possible that other unknown subunits are needed for the wild-type function of
EXP-2 as well.
Regulation of the pharyngeal muscle action potential duration
We show that the ramp of the action potential plateau phase determines the
onset of the EXP-2 current and thereby regulates the action potential duration
(Fig. 6A). It is the peculiar
kinetic properties of EXP-2 that make this regulation possible. EXP-2 is tuned
to generate a current spike once the membrane has hyperpolarized by
approximately 30 mV from the peak depolarization.
The slope of the plateau phase might be regulated by the inactivation
kinetics of the EGL-19 inward current. Mutations in a region of EGL-19 known
to be important for channel inactivation cause dramatic elongation of
pharyngeal action potentials (Lee et al.,
1997), suggesting that EGL-19 inactivation is important in shaping
the plateau phase. Extracellular Ca2+ concentration affects action
potential duration: in lower extracellular Ca2+, duration is
extended (Dent and Avery,
1993
). Barium at 1 mmol l-1 concentration causes
extension of pharyngeal action potentials to over 1 s
(Franks et al., 2002
).
Substitution of 6 mmol l-1 Ca2+ with 6 mmol
l-1 Ba2+ slows the inactivation of the EGL-19 current in
the body wall muscle (Jospin et al.,
2002a
). These observations are consistent with calcium-dependent
inactivation mechanism of EGL-19. EGL-19 inactivation kinetics are critical
for the shape of the plateau phase; thus, the amount of Ca2+ entry
is likely to be the key factor in regulating the plateau phase slope, and, by
affecting the timing of EXP-2 current, the action potential duration.
It is also possible that calcium-dependent potassium channels are involved
in shaping the plateau phase, but this possibility was not tested because we
had to buffer intracellular Ca2+ (see Materials and methods). In
Ascaris esophagus, delayed rectifier potassium currents have not been
observed, even though [Ca2+]I was not buffered
(Byerly and Masuda, 1979),
which is in agreement with our results. Expression of the C. elegans
calcium-dependent potassium channel gene slo-1
(Wang et al., 2001
) has not
been detected in pharyngeal muscle, and slo-1 mutations have no
detectable effect on muscle function (Alan Chiang and L. A., unpublished
results). The gene encoding a second calcium-activated potassium channel,
slo-2, may be expressed in pharyngeal muscle
(Yuan et al., 2000
).
Another important factor that regulates action potential duration is the
inhibitory motor neuron M3. The M3 neuron fires inhibitory postsynaptic
potentials during the plateau phase of the action potential
(Raizen and Avery, 1994),
which presumably trigger the EXP-2 recovery from inactivation. But M3 does not
play a major role in terminating the pharyngeal action potential: if this
neuron is ablated, the pharyngeal contraction is only slightly extended
(Avery, 1993b
). Probably, it is
the coordinated activity of the inward EGL-19 current and M3 that cause the
EXP-2 recovery from inactivation, leading to the rapid membrane
repolarization.
The unusual electrophysiology of the pharyngeal muscle is dictated by its function
If viewed as a single unit, the pharynx is likely the most active excitable
structure in C. elegans. It is also a very large structure relative
to the rest of the worm body, which is evidently dictated by the size of
bacterial food and the physics of food intake. Hence there is a need for a
powerful excitation mechanism.
C. elegans neurons are extremely small and have a very high
phenomenological input resistance; most probably, they conduct excitation
passively and do not need regenerative action potentials
(Goodman et al., 1998). In the
body wall, spontaneous activity is slow; and the action potential upstroke is
more than 50 ms long (Jospin et al.,
2002a
). The pharynx is different: the membrane depolarization and
repolarization during the action potential are both very rapid, less than 10
ms long. No close homolog of the voltage-gated sodium channel that mediates
rapid events in vertebrates has been found in the C. elegans genome
(Bargmann, 1998
). Its role is
played instead by CCA-1 in the pharyngeal muscle. Compared to currents
recorded in other C. elegans excitable cells, CCA-1 current is huge -
of the order of 10 nA, which evidently allows a very rapid charging of the
membrane capacitance and drives a rapid action potential upstroke. Similarly
to the role of CCA-1 in the upstroke, EXP-2 functions in the downstroke to
rapidly terminate action potentials; EXP-2 currents reach 18 nA in amplitude.
Presumably, large CCA-1 and EXP-2 currents allow fast, precisely timed muscle
contractions. Indeed, pharyngeal muscle motions are much faster than those of
body wall muscle, especially the relaxation. As del Castillo and Morales
(1967
) proposed, the
"efficiency of the esophagus as a pumping device" depends
on "the sudden onset and relatively high speed of the relaxation
process".
Another possible reason why EXP-2 is needed is the apparent absence of
substantial delayed rectifier-like potassium current in the pharynx. Most
likely, the EXP-2 negative spike is a more precise and more advanced mechanism
to control the end of the action potential than the delayed rectifier. The key
difference between the negative spike repolarization mechanism and the delayed
rectifier is that the latter is initiated by the depolarization during the
action potential upstroke, so the onset of repolarization is inherently linked
to the start of the action potential. Negative spike current, in contrast, is
activated by hyperpolarizations more negative than the deinactivation
threshold, and is independent of the upstroke (except for very short
durations, when channel activation becomes limiting, see
Fig. 6). Therefore, the
negative spike mechanism allows regulation of action potential duration over a
very wide range (in C. elegans, from about 50 ms to more than 500 ms;
B.S. and L.A., unpublished data), while keeping the repolarization rapid at
any duration. Consistent with this hypothesis, EXP-2 current was not recorded
in the body wall muscle (Jospin et al.,
2002b; Richmond and Jorgensen,
1999
), while the delayed rectifier current was not recorded in the
pharynx (Fig. 2 in this paper
and fig. 3 in Byerly and Masuda,
1979
).
The pharynx possesses redundant mechanisms of excitation
(Steger et al., 2005), which
ensure its proper function and a wide range of adaptation. The rate of
pharyngeal contractions is tightly regulated by various factors, such as
developmental stage and mechanical stimuli
(Keane and Avery, 2003
), food
availability (Avery and Horvitz,
1990
) and food quality
(Steger, 2003
). Even extreme
perturbations, such as laser ablation of the whole pharyngeal nervous system
or getting rid of both MC and CCA-1 excitation mechanisms do not completely
abolish pharyngeal function. In the latter case, the pharynx adapts by raising
its resting membrane potential and by upregulating the leakage current
(Steger et al., 2005
).
In this study, we have treated the pharynx as a single functional unit.
This is not totally appropriate: the timing and nature of electrical activity
is different in different pharyngeal compartments, and precise control of
these differences is critical for efficient food transport
(Avery and Shtonda, 2003). For
example, the relaxation of anterior isthmus has to slightly lag the corpus
relaxation. Corpus and terminal bulb movements are rapid, whereas posterior
isthmus contractions are slow and peristaltic and do not occur in synchrony
with other compartments. Undoubtedly, differences in ion channel expression
and regulation underlie some differences in the function of pharyngeal
compartments. We predict, for example, that in the posterior isthmus,
electrical activity is graded and CCA-1 and EXP-2 currents are absent; the
isthmus may function similarly to body wall muscle. As electrophysiological
techniques for the pharynx are improved, it will be possible to uncover how
ion channels encode this amazingly precise regulation of electrical and
contractile activity in different compartments of the pharynx.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albertson, D. G. and Thomson, J. N. (1976). The pharynx of Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 275,299 -325.[Medline]
Avery, L. (1993a). The genetics of feeding in
Caenorhabditis elegans. Genetics
133,897
-917.
Avery, L. (1993b). Motor neuron M3 controls pharyngeal muscle relaxation timing in Caenorhabditis elegans. J. Exp. Zool. 175,283 -297.
Avery, L. and Horvitz, H. R. (1989). Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C. elegans. Neuron. 3, 473-485.[CrossRef][Medline]
Avery, L. and Horvitz, H. R. (1990). Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans. J. Exp. Zool. 253,263 -270.[Medline]
Avery, L. and Shtonda, B. B. (2003). Food
transport in the C. elegans pharynx. J. Exp.
Biol. 206,2441
-2457.
Bargmann, C. I. (1998). Neurobiology of the
Caenorhabditis elegans genome. Science
282,2028
-2033.
Byerly, L. and Masuda, M. O. (1979). Voltage-clamp analysis of the potassium current that produces a negative-going action potential in Ascaris muscle. J. Physiol. 288,263 -284.[Abstract]
Curtis, H. J. and Cole, K. S. (1938).
Transverse electric impedance of the squid giant axon. J. Gen.
Physiol. 21,757
-765.
Davis, M. W. (1995). Intracellular recording from pharyngeal muscles. Worm Breeder's Gazette 13, 34.
Davis, M. W. (1999). Regulation of the relaxation phase of the C. elegans pharyngeal muscle action potential.PhD dissertation , The University of Texas Southwestern Medical Center at Dallas.
Davis, M. W., Fleischhauer, R., Dent, J. A., Joho, R. H. and
Avery, L. (1999). A mutation in the C. elegans EXP-2
potassium channel that alters feeding behavior.
Science 286,2501
-2504.
Davis, M. W., Somerville, D., Lee, R. Y., Lockery, S., Avery, L. and Fambrough, D. M. (1995). Mutations in the Caenorhabditis elegans Na,K-ATPase alpha-subunit gene, eat-6, disrupt excitable cell function. J. Neurosci. 15,8408 -8418.[Abstract]
del Castillo, J., de Mello, W. C. and Morales, T. (1964). Hyperpolarizing action potentials recorded from the esophagus of the Ascaris lumbricoides. Nature 203,530 -531.[Medline]
del Castillo, J. and Morales, T. (1967). The
electrical and mechanical activity of the esophageal cell of Ascaris
lumbricoides. J. Gen. Physiol.
50,603
-629.
Dent, J. A. and Avery, L. (1993). A defined medium for the pharynx. Worm Breeder's Gazette 13, 44.
Dent, J. A., Davis, M. W. and Avery, L. (1997).
avr-15 encodes a chloride channel subunit that mediates inhibitory
glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis
elegans. EMBO J. 16,5867
-5879.
DiFrancesco, D. (1993). Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55,455 -472.[CrossRef][Medline]
Doncaster, C. C. (1962). Nematode feeding mechanisms. I. Observations on Rhabditis and Pelodera. Nematologica 8,313 -320.
Fleischhauer, R., Davis, M. W., Dzhura, I., Neely, A., Avery, L.
and Joho, R. H. (2000). Ultrafast inactivation causes inward
rectification in a voltage-gated K(+) channel from Caenorhabditis
elegans. J. Neurosci.
20,511
-520.
Francis, M. M., Mellem, J. E. and Maricq, A. V. (2003). Bridging the gap between genes and behavior: recent advances in the electrophysiological analysis of neural function in Caenorhabditis elegans. Trends Neurosci. 26, 90-99.[CrossRef][Medline]
Franks, C. J., Pemberton, D., Vinogradova, I., Cook, A., Walker,
R. J. and Holden-Dye, L. (2002). Ionic basis of the resting
membrane potential and action potential in the pharyngeal muscle of
Caenorhabditis elegans. J. Neurophysiol.
87,954
-961.
Gentet, L. J., Stuart, G. J. and Clements, J. D.
(2000). Direct measurement of specific membrane capacitance in
neurons. Biophys. J. 79,314
-320.
Goodman, M. B., Hall, D. H., Avery, L. and Lockery, S. R. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron 20,763 -772.[CrossRef][Medline]
Hagiwara, N., Irisawa, H. and Kameyama, M. (1988). Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J. Physiol. (Lond). 395,233 -253.[Abstract]
Hille, B. (2001). Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer.
Jospin, M., Jacquemond, V., Mariol, M. C., Segalat, L. and
Allard, B. (2002a). The L-type voltage-dependent
Ca2+ channel EGL-19 controls body wall muscle function in
Caenorhabditis elegans. J. Cell Biol.
159,337
-348.
Jospin, M., Mariol, M. C., Segalat, L. and Allard, B.
(2002b). Characterization of K(+) currents using an in situ patch
clamp technique in body wall muscle cells from Caenorhabditis
elegans. J. Physiol.
544,373
-384.
Keane, J. and Avery, L. (2003). Mechanosensory
inputs influence Caenorhabditis elegans pharyngeal activity
via ivermectin sensitivity genes. Genetics
164,153
-162.
Lee, J. H., Gomora, J. C., Cribbs, L. L. and Perez-Reyes, E.
(1999). Nickel block of three cloned T-type calcium channels: low
concentrations selectively block alpha1H. Biophys. J.
77,3034
-3042.
Lee, R. Y., Lobel, L., Hengartner, M., Horvitz, H. R. and Avery,
L. (1997). Mutations in the alpha1 subunit of an L-type
voltage-activated Ca2+ channel cause myotonia in Caenorhabditis
elegans. EMBO J.
16,6066
-6076.
Maupas, E. (1900). Modes et formes de reproduction dés nematodes. Arch. Zool. Exp. Genet. 8,463 -624.
McKay, J. P., Raizen, D. M., Gottschalk, A., Schafer, W. R. and
Avery, L. (2004). eat-2 and eat-18 are required for nicotinic
neurotransmission in the Caenorhabditis elegans pharynx.
Genetics 166,161
-169.
Mellem, J. E., Brockie, P. J., Zheng, Y., Madsen, D. M. and Maricq, A. V. (2002). Decoding of polymodal sensory stimuli by postsynaptic glutamate receptors in C. elegans. Neuron 36,933 -944.[CrossRef][Medline]
Pierce-Shimomura, J. T., Faumont, S., Gaston, M. R., Pearson, B. J. and Lockery, S. R. (2001). The homeobox gene lim-6 is required for distinct chemosensory representations in C. elegans. Nature 410,694 -698.[CrossRef][Medline]
Raizen, D. M. and Avery, L. (1994). Electrical activity and behavior in the pharynx of Caenorhabditis elegans. Neuron 12,483 -495.[CrossRef][Medline]
Raizen, D. M., Lee, R. Y. and Avery, L. (1995).
Interacting genes required for pharyngeal excitation by motor neuron MC in
Caenorhabditis elegans. Genetics
141,1365
-1382.
Richmond, J. E. and Jorgensen, E. M. (1999). One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 2, 791-797.[CrossRef][Medline]
Seymour, M. K., Wright, K. A. and Doncaster, C. C. (1983). The action of the anterior feeding apparatus of Caenorhabditis elegans (Nematoda: Rhabditida). J. Zool. (Lond.) 201,527 -539.
Sherman-Gold, R. (1993). The Axon Guide For Electrophysiology and Biophysics Laboratory Techniques. Union City, CA: Axon Instruments, Inc.
Spector, P. S., Curran, M. E., Zou, A., Keating, M. T. and Sanguinetti, M. C. (1996). Fast inactivation causes rectification of the IKr channel. J. Gen. Physiol. 107,611 -619.[Abstract]
Steger, K. A. (2003). Cholinergic regulation of feeding in C. elegans: studies of a T-type calcium channel and three muscarinic acetylcholine receptors. PhD dissertation, The University of Texas Southwestern Medical Center at Dallas.
Steger, K. A., Shtonda, B. B., Thacker, C., Snutch, T. P. and Avery, L. (2005). The Caenorhabditis elegans T-type calcium channel CCA-1 boosts neuromuscular transmission. J. Exp. Biol. 208,2191 -2203.[CrossRef]
Sulston, J. and Hodgkin, J. (1988). Methods. InThe Nematode C. elegans (ed. Wood W), pp.587 -606. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Trent, C., Tsuing, N. and Horvitz, H. R.
(1983). Egg-laying defective mutants of the nematode
Caenorhabditis elegans. Genetics
104,619
-647.
Wang, Z. W., Saifee, O., Nonet, M. L. and Salkoff, L. (2001). SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction. Neuron 32,867 -881.[CrossRef][Medline]
Yuan, A., Dourado, M., Butler, A., Walton, N., Wei, A. and Salkoff, L. (2000). SLO-2, a K+ channel with an unusual Cl- dependence. Nat. Neurosci. 3, 771-779.[CrossRef][Medline]
Zheng, Y., Mellem, J. E., Brockie, P. J., Madsen, D. M. and Maricq, A. V. (2004). SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans. Nature 427,451 -457.[CrossRef][Medline]