1Fakultät für Biologie, Universität Konstanz, D-78457 Konstanz, Germany; 2Stazione Zoologica "Anton Dohrn," I-80121 Naples, Italy; and 3Department of Biology, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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Wenning, Angela,
Christian F. J. Erxleben, and
Ronald L. Calabrese.
Indirectly Gated Cl-Dependent Cl
Channels Sense Physiological Changes of Extracellular Chloride in the
Leech.
J. Neurophysiol. 86: 1826-1838, 2001.
The maintenance of ion
homeostasis requires adequate ion sensors. In leeches, 34 nephridial
nerve cells (NNCs) monitor the Cl
concentration
of the blood. After a blood meal, the Cl
concentration of leech blood triples and is gradually restored to its
normal value within 48 h after feeding. As previously shown in
voltage-clamp experiments, the Cl
sensitivity
of the NNCs relies on a persistent depolarizing
Cl
current that is turned off by an increase of
the extracellular Cl
concentration. The
activation of this Cl
-dependent
Cl
current is independent of voltage and of
extra- and intracellular Ca2+. The transduction
mechanism is now characterized on the single-channel level. The NNC's
sensitivity to Cl
is mediated by a slowly
gating Cl
-dependent Cl
channel with a mean conductance of 50 pS in the cell-attached configuration. Gating of the Cl
channel is
independent of voltage, and channel activity is independent of extra-
and intracellular Ca2+. Channel activity and the
macroscopic current are reversibly blocked by bumetanide. In
outside-out patches, changes of the extracellular
Cl
concentration do not affect channel
activity, indicating that channel gating is not via direct interaction
of extracellular Cl
with the channel. As shown
by recordings in the cell-attached configuration, the activity of the
channels under the patch is instead governed by the
Cl
concentration sensed by the rest of the
cell. We postulate a membrane-bound Cl
-sensing
receptor, which
on the increase of the extracellular Cl
concentration
closes the
Cl
channel via a yet unidentified signaling pathway.
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INTRODUCTION |
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The maintenance of stable
extracellular osmotic and ionic concentrations requires gathering and
processing sensory information about these quantities (Wenning
1999). The discovery of mechanosensitive channels
(Guharay and Sachs 1984
) provides a mechanism for
transducing changes in extracellular osmolality into electrical
activity as shown for the hypothalamic osmoreceptors (Oliet and
Bourque 1993
). In comparison, the sensing of extracellular ion
concentrations is as yet poorly understood. One example is the
Ca2+ sensor of the parathyroid glands, which
maintain the systemic Ca2+ concentration
([Ca2+]o) (Brown
et al. 1993
; Hebert et al. 1997
). The molecular
sensor, the Ca2+ sensing receptor (CaSR), belongs
to the superfamily of G-protein-coupled receptors. The CaSRs also sense
changes of other cations, e.g., La3+ and
Mg2+, as well as pH, ionic strength, and L-amino
acids (Conigrave et al. 2000
). Similarly, the
aforementioned hypothalamic magnocellular neurosecretory neurons are
also multimodal. In addition to sensing osmolality, they might also
sense physiological variations of the extracellular
Na+ concentration
([Na+]o) (Voisin
et al. 1999
). Changes in
[Na+]o cause proportional
changes in the relative permeability to Na+ of
the mechanosensitive channels thereby modulating the osmoreceptive response. We report here on the transduction mechanism of a
Cl
receptor neuron, the nephridial nerve cell,
which monitors the Cl
concentration of leech blood.
The nephridial nerve cells (NNCs), 34 peripheral neurons, innervate the
34 nephridia, the leech excretory organs (Fig.
1, top left) (Wenning
1983). Due to a large fraction of divalent organic anions
present in leech blood (~40 mM), its Cl
concentration is rather low (36 ± 6 mM; mean ± SD)
(Hoeger et al. 1989
; Zerbst-Boroffka
1970
). Feeding on mammalian blood causes the
Cl
concentration of leech blood to increase
from 36 to 88 mM (±8) within 15 min, while the
Na+ concentration increases by only 10 mM from
125 to 135 mM (Hildebrandt and Zerbst-Boroffka 1988
;
Wenning et al. 1980
; Zerbst-Boroffka 1973
). A Cl
receptor is advantageous
for an animal with a low blood Cl
concentration
and high Cl
diet. Long-term extracellular
recordings, such as shown in Fig. 1 (top right) demonstrated
that the NNC's electrical activity changes with, but does not adapt
to, changes of the external Cl
concentration
([Cl
]o) and, by
changing the composition of the bathing medium, that the NNCs are
insensitive to changes of extracellular osmolality (Wenning
1989
). More importantly, long-term recordings from semi-intact leeches before and after artificial meals confirm that the NNC's activity correlates with the physiological changes of the blood Cl
concentration (Wenning 1989
).
Intracellular recordings show that in normal, low
[Cl
]o, the NNC is
depolarized and fires spontaneously while it hyperpolarizes and stops
firing on the increase of
[Cl
]o (Wenning
and Calabrese 1991
). Voltage-clamp experiments (Wenning and Calabrese 1991
) reveal that the NNC has a persistent high resting conductance for Cl
that determines the
membrane potential in normal, low
[Cl
]o (Fig. 1, model).
In high [Cl
]o, this
inwardly directed, depolarizing Cl
current is
gated off. This "Cl
-dependent
Cl
current" is halogen-specific, independent
of voltage, and of extracellular Ca2+ and
Na+ (Wenning and Calabrese 1991
).
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At 33 mM [Cl]o, the
reversal potential of the Cl
-dependent
Cl
current
(ECl) was measured to be
16 mV and
the membrane potential (Vm) was
between
30 and
37 mV (Wenning and Calabrese 1991
). These observations indicate that the intracellular
Cl
concentration
([Cl
]i) is maintained
well above the Cl
equilibrium concentration
(9.3 mM in low [Cl
]o)
and is estimated to be around 17 mM (Fig. 1, model).
In the NNC, which receives no peripheral synaptic input, the
depolarizing Cl current provides continuous
excitation at the normal, low
[Cl
]o, keeping the NNC
firing (Wenning and Calabrese 1991
, 1995
). The natural
changes of the blood Cl
concentration make the
NNC of the leech a good model for studying the neural basis of ion
homeostasis and the mechanism of ion sensing. To further characterize
sensory transduction, we investigated the single-channel currents
underlying the NNC's sensitivity to Cl
. We
describe a slowly gating anion channel whose open probability decreases
in high [Cl
]o and which
has an indirect gating mechanism. Preliminary results appeared in
abstract form (Erxleben et al. 1997
; Wenning et
al. 1996
).
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METHODS |
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Leeches (Hirudo medicinalis L.) were obtained from a commercial supplier (Leeches USA, Westbury, NY) and kept in artificial pond water at 16°C. Experiments were carried out at room temperature (20-22°C).
Isolation of the nephridial nerve cell (NNC) is described in
Wenning and Calabrese (1991). Briefly, a single
nephridium and the dorsal part of its urinary bladder were dissected
out and transferred to a silicone elastomer (Sylgard)-lined dish. The bladder wall was pinned inside out and a small cut through the bladder
wall exposed the NNC. Dissection time per cell was 30 min.
The preparation was constantly superfused allowing complete changes of
the bathing solution within 1-2 min. We used various Cl concentrations of the bathing medium (Table
1). "High
[Cl
]o " refers to
leech saline with Cl
concentrations between 108 and 123 mM. "Low
[Cl
]o " refers to
artificial leech blood with Cl
concentrations
between 33 and 43 mM. Intermediate concentrations were obtained by
mixing solutions with low and high Cl
concentrations. Actual Cl
concentrations are
stated in the text and Figures. Dissections were done in high
[Cl
]o saline.
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Intracellular recordings
Intracellular recordings from the NNC began in leech saline.
Glass microelectrodes were filled with a mixture of 4 M potassium acetate and 20 mM KCl and had resistances of 20-30 M. To lower their capacitance, they were dipped into dimethyl-polysiloxane (Sigma,
St. Louis, MO) prior to use. Measurements in discontinuous current
clamp were made either with a NPI SEC-05 l amplifier (NPI electronic
GMBH, Tamm, Germany) using a switch frequency between 12 and 14 kHz or with an Axoclamp-2A (Axon Instruments, Foster City, CA) using a
switch frequency of 2.5 kHz. Single-electrode voltage-clamp experiments
were carried out in discontinuous mode (switch frequency of 2.5 kHz)
with an Axoclamp-2A using a gain setting of ~0.8 nA/mV. We used 0 Ca2+/5 mM Co2+ saline
(Table 1) in voltage-clamp experiments to reduce spiking and to
eliminate Ca2+-mediated currents. To chelate
intracellular Ca2+, glass microelectrodes were
filled with 200 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA; Sigma) in 20 mM KCl. To increase the intracellular Cl
concentration of the NNC, glass
microelectrodes were filled with 3 M KCl (Schmidt and Calabrese
1992
).
Single-channel recordings
Giga-ohm seals on the soma or distal neurites of the NNC (Fig.
1, top left) were obtained following 50-70 min of enzymatic digestion with a mixture of collagenase (5 wt/vol) Type IA (Sigma) and Trypsin (0.25%; Life Technologies, NY) at 22°C with agitation. Preparations were rinsed several times in leech saline and, if not
immediately used, stored at 10°C for up to 3 h.
The pipette solutions for the single-channel recordings in the
cell-attached configuration were either leech saline (high [Cl]) or artificial leech blood (low
[Cl
]) diluted by 10% with
H2O to make them hypo-osmotic with respect to the
bathing solutions. A holding potential of nominally
20 mV (see
junction potential corrections) from the cell's membrane potential was
routinely used to increase the driving force on Cl
and hence the amplitude of single-channel currents.
For the outside-out patches, we used two different
Cl concentrations for the pipette solution: a
Cl
-free solution containing (in mM) 50 CsOH, 50 citric acid, 10 tetraethylammonium acetate (TEA), 10 HEPES, 5 EGTA, and
1 Mg-ATP or a 14 mM Cl
solution containing (in
mM) 50 CsOH, 50 citric acid, 10 tetraethylammonium-Cl (TEA-Cl), 10 HEPES, 5 EGTA, 2 MgCl2, and 1 Na2-ATP (all from Sigma). Pipette solutions were
adjusted to pH 7.4.
Single-channel currents were recorded with an Axopatch 1 D or 200 (Axon Instruments) or an EPC9 (HEKA, Lambrecht, Germany). Inward currents are shown as downward deflections.
Drugs
Drugs were bath applied and were obtained from Sigma or
Calbiochem (San Diego, CA). Channel blockers: DIDS (Sigma),
SITS (Sigma), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid
(NPPB, Calbiochem), ZnCl2 (Sigma), Bumetanide
(Sigma). Ca2+-signaling: BAPTA,
CdCl2 (Sigma). DIDS, SITS and NPPB were dissolved in DMSO (obtained as 1 ml aliquots from Sigma). DMSO caused slight depolarization and a drop in input resistance at concentrations 2 × 103 M (unpublished observations).
Its final concentration was therefore kept
2 × 10
3 M.
Junction potential corrections
The junction potential at the reference electrode changes in
response to changes in
[Cl]o. In intracellular
recordings and voltage-clamp experiments, the two functions of the
reference electrode (current return and stable reference potential)
were assigned to separate electrodes using a bath probe thereby
compensating for these potential changes (Wenning and Calabrese
1991
). In addition, the bath probe potential provided a
convenient way to indicate the actual time course of changes of
[Cl
]o.
For single-channel recordings, potential changes at the reference
electrode were compensated by also using a bath probe (Axopatch 1D) or
were minimized by using an agar bridge (Axopatch 200 and EPC9, both of
which do not provide an input for a separate bath probe). The liquid
junction potentials at the patch electrode and potential changes at the
agar bridge were calculated using the "junction potential" module
of AxoScope (Axon Instruments). The principal anions of leech blood,
malate and succinate (Hoeger et al. 1989), are not in
the library of the junction Potential module. Therefore the liquid
junction potential difference between high and low
[Cl
]o was measured
(Neher 1992
). The potential difference was
9.6 ± 0.7 mV (n = 8). Holding potentials (cell-attached
patches) or absolute membrane potentials (excised patches) used for
reversal potential measurements and current-voltage plots were
corrected accordingly. The membrane potential of four enzyme-treated
cells was measured in the whole cell configuration. It was
48.6 ± 7.5 mV in high [Cl
]o
and
32.5 ± 11.5 mV in low
[Cl
]o, respectively.
These values were used to estimate the NNC's membrane potential in the
cell-attached patches.
The Goldman-Hodgkin-Katz constant field equation (Hodgkin and
Katz 1949) was used to describe rectification due to asymmetric [Cl
] distribution
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Data acquisition, storage, and analysis
Data from intracellular recordings and voltage-clamp experiments were acquired at 2 kHz and stored using Clampex (Axon Instruments). Data reduction was necessary for display purposes of long-term recordings and spike amplitudes are therefore attenuated.
Single-channel data were acquired at 1 or 2 kHz using a Digidata 1200 (Axon) or EPC9 (HEKA) and low-pass filtered at 200 Hz. Data was analyzed using the pClamp software suite (Axon) or the Pulse software (HEKA) in combination with a personal computer. The open probability (Po) of channels was determined by summing open times during 1-5 min of channel activity (depending on the frequency of openings) and dividing through the number of channels in the patch. To access the variability in Po, the activity for each experimental condition was divided in 10-s segments, which were than averaged. The standard deviation of this average is shown in the figures.
All values are expressed as means ± SD. Statistical significance was assessed by using a two-tailed Student's t-test.
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RESULTS |
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A Cl-dependent, indirectly gated anion
channel mediates the NNC's sensitivity to the external
Cl
concentration
The NNC's tonic sensitivity to the extracellular
Cl concentration
([Cl
]o) relies on a
high resting conductance for Cl
and the active
maintenance of its nonequilibrium distribution. ECl is estimated to be about
21 mV
in low (38 mM) [Cl
]o,
while the membrane potential is
36.9 ± 10 mV under these conditions (Fig. 1; model). As previously shown in voltage-clamp measurements (Wenning and Calabrese 1991
), the high
resting conductance for Cl
is gated off by
Cl
, it is inwardly directed and is carried by
Cl
leaving the cell (Fig. 1; model). This
current is therefore referred to as
Cl
-dependent Cl
current. It was found to be independent of voltage and extracellular Ca2+
([Ca2+]o) (Wenning
and Calabrese 1991
). These properties were used to identify the
Cl
-dependent Cl
current
on the single-channel level. By using bathing solutions of different
Cl
concentrations (Table 1), we mimicked the
physiological changes of the Cl
concentration
that occur in leech blood after feeding on mammalian blood
(Wenning et al. 1980
; Zerbst-Boroffka
1973
).
The simplest mechanism to account for the macroscopic observation of a
Cl current turned off in the presence of high
[Cl
]o would be a block
of a Cl
channel by extracellular
Cl
itself. Consequently, channel activity in
the cell-attached configuration should depend on the
Cl
concentration of the pipette solution. We
therefore used a pipette solution of either high or low
Cl
concentration to study the activity of
Cl
channels in the cell-attached configuration
but did not see any consistent difference. A change of the
Cl
concentration of the bathing medium,
however, had pronounced effects on the open probability of a slowly
gating channel (Figs. 2 and
3). Regardless of the
Cl
concentration in the pipette, channel
activity was low in high (123 mM)
[Cl
]o, with brief and
infrequent openings. When changing to low (43 mM)
[Cl
]o, the open
probability increased due to an increase of the burst duration. Channel
activity subsided on changing the bathing solution back to high
[Cl
]o (Fig. 2). In nine
patches, which contained one or two active channels, judging from the
maximum number of simultaneous openings at high activity, the mean
activity (Po) in high (123-128 mM) [Cl
]o was 0.027 (±0.03) and increased to Po = 0.31 (±0.34) on change to low
(38-43 mM) [Cl
]o. This
increase in Po was determined to be statistically significant using a
paired t-test (P < 0.013).
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With low (39 mM) [Cl] in the recording
pipette, currents of this slowly gating
Cl
-dependent channel were inward with
amplitudes of about 1-2 pA at the cell's membrane potential (Fig. 4,
A and C). The
current amplitude increased with hyperpolarization and decreased with depolarization as expected for an anion channel. The channel is subsequently referred to as Cl
-dependent
Cl
channel because Cl
is the permeant anion under our experimental conditions. Two other
classes of anion channels were occasionally observed (for example the
channels marked with asterisks in Fig. 3). They had conductances of
5-10 and 90 pS, respectively, but their activity was
Cl
independent, and these channels were
therefore not further characterized.
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A total of 140 patches were examined. In 43 of these we recorded
Cl channels. In 24 of these 43, we also
recorded spikes (Fig. 5). The activity of
the Cl
-dependent Cl
channel preceded the characteristic bursts of action potentials of the
NNC, indicating that opening of these channels initiate, rather than
follow, the depolarization caused by turning on the Cl
-dependent Cl
current
in low [Cl
]o. The
activity of the channels under the patch is governed by the
Cl
concentration of the bathing medium, which
suggests that gating is not by direct interaction of
Cl
with the channel but rather via an
intracellular signaling pathway.
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Since the macroscopic Cl-dependent
Cl
current is voltage independent
(Wenning and Calabrese 1991
), the underlying
Cl
-dependent Cl
channel
should also be voltage insensitive. The voltage dependence of the
Cl
-dependent Cl
channel
was examined using two different approaches. First, we measured the
open probability (Po) during steady polarization at different membrane
potentials (Fig. 4, B and C). In terms of a
Boltzmann fit, the voltage sensitivity of the example shown in Fig. 4
and a second patch was such that 200 mV were needed for an
e-fold change in Po of the channel, indicating that Po was
largely voltage independent. Second, we examined channel activity during voltage ramps. Channels opened at random with no preference at
de- or hyperpolarized potentials (Fig.
6A), further confirming that
the Po of the Cl
-dependent
Cl
channel is voltage independent.
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The mean conductance of the Cl-dependent
Cl
channel in the cell-attached configuration
with low (39 mM) [Cl
] in the pipette was
50 ± 7.4 pS with a reversal potential of +29 ± 6 mV from
the cells' membrane potential (n = 4). An example is
shown in Fig. 4B (123 mM
[Cl
]o). In the
cell-attached configuration, the Cl
-dependent
Cl
channel shows characteristic subconductance
levels at ~50% and 75% of the fully open channel (see for example
Fig. 6A, *).
Both the Cl-dependent
Cl
channel observed in the cell-attached
configuration and the Cl
-dependent
Cl
current characterized previously in
voltage-clamp experiments (Wenning and Calabrese 1991
)
(see also following text) are gated off on the change from low to high
Cl
concentration of the bathing medium.
Furthermore, the macroscopic current and the single-channel open
probability are voltage independent, strongly indicating that the
identified Cl
-dependent
Cl
channels mediate the NNC's sensitivity to
[Cl
]o.
Characterization of the channels in excised patches
We further characterized the Cl-dependent
Cl
channel in excised patches. If gating is
indeed indirect
as suggested by the experiments in the cell-attached
configuration
changes of
[Cl
]o should
not affect channel activity when recorded in (outside-out) excised patches, while the open probability would change with changes
of [Cl
]o if gating were
direct. In outside-out patches, we identified a slowly gating
Cl
channel with ~1 pA outward current at 0-mV
holding potential. Current amplitudes increased with depolarization and
decreased on hyperpolarization (Fig.
7A). With 0 mM
Cl
in the pipette solution, we did not observe
inward currents. The nonreversal of currents confirms that the channel
is Cl
selective and impermeable to citrate, the
organic anion present in the pipette solution (METHODS).
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Gating of the Cl channel in the outside-out
configuration was not affected by changes of the extracellular
Cl
concentration (Fig.
8). The average ratio of the open
probability in low (43 mM) versus high (128 mM)
[Cl
]o (Po low/Po high
[Cl
]o) was near 1 (0.93 ± 0.36; n = 6). The slope conductance was determined from outward currents 0 mV. As expected for a
Cl
channel, the slope conductance of outward
currents in the outside-out patches increased with increasing
[Cl
]o (Fig.
7A). On average, the slope conductance was 25.5 ± 8 pS in low versus 37 ± 12 pS in high
[Cl
]o
(n = 5). With Cl
-free solution
in the recording pipette and low outside
[Cl
], the single-channel conductance in the
outside-out patches was on average half that of the
Cl
channel identified in the cell-attached
configuration (compare Figs. 4A and 7A). This
difference in slope conductance raises the possibility that we are
looking at different channels in these two configurations.
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To resolve this issue, we first identified the
Cl channel based on its
Cl
sensitivity in the cell-attached mode and
then excised the patch into the inside-out configuration measuring the
conductance in both configuration. Under these conditions, the
single-channel conductance for outward currents carried by a high
Cl
concentration in the pipette did not change
(Fig. 6, B and C). Next we examined whether the
difference in the Cl
concentration on the
intracellular side of the membrane, that is, the bathing medium in the
inside-out configuration and the pipette solution in the outside-out
configuration, respectively, affected single-channel conductance. When
the pipette solution for the outside-out patches contained 14 mM
Cl
(METHODS), the average
conductance for outward currents was 69 ± 3 pS (n = 3), which is similar to the single-channel conductance measured in
the cell-attached and inside-out configuration (compare Figs.
4A and 6, B and C, to 6D).
To facilitate a comparison of the single-channel conductance measured
under different conditions, we fitted the currents of Fig. 6 with the
Goldman-Hodgkin-Katz constant field equation (Hodgkin and Katz
1949) that describes the rectification due to asymmetric ion
distribution. A fit to the currents in the cellattached
configuration gives a permeability PCl
of 2.2 × 10
13 cm3/s
and an internal chloride concentration
[Cl
]i of 18 mM, which
is in good agreement with previous estimates from voltage-clamp
investigations of the NNC's macroscopic chloride currents (Fig. 1,
model) (Wenning and Calabrese 1991
). Since the resting
potential for cell-attached patches was estimated and absolute membrane
potentials were subject to errors despite numerical corrections of
junction potentials, a correction factor was used to shift the fitted
curve along the voltage axis. Corrections were
8 mV in B,
4.4 and 18 mV for C for high and low
[Cl
]i, respectively,
and 18 mV for D. On excision of the patch into a saline with
a [Cl
]i close to
physiological concentration (33 mM
[Cl
]I, Fig.
6C), the I-V relationship for both outward as
well as inward currents is well described by the GHK equation with the same PCl of 2.2 × 10
13 cm3/s and 33 mM
Cl
in the bathing solution. With a high
(nonphysiological)
[Cl
]i of 123 mM;
however, the I-V relationship in the range of inward currents markedly deviates from the GHK prediction (123 mM
[Cl
]i, Fig.
6C). With
[Cl
]i in the
physiological range (14 mM, Fig. 6D), the I-V
relationship and permeability for outside-out patches is also correctly
predicted by the GHK equation.
By fitting a Boltzmann curve to the data, the voltage sensitivity of
the Cl channel's open probability in the
outside-out configuration was assessed. Of the five patches analyzed,
the example shown in Fig. 7B had the highest voltage
sensitivity (+54 mV in 128 mM
[Cl
]o and +101 mV in 43 mM [Cl
]o for an
e-fold change in Po). Of the other four patches, only one
showed comparable voltage sensitivity (+121 mV for an e-fold change in Po). From the remaining three patches, two had a positive and
one a negative voltage dependence (>200-mV polarization for an
e-fold change in Po) similar to that found in the
cell-attached patches. We consider this low voltage sensitivity
insignificant for the physiological response.
Another characteristic of the Cl-dependent
Cl
current is its independence of
[Ca2+]o, suggesting that
no Ca2+ entry is required for the response to
changes of [Cl
]o
(Wenning and Calabrese 1991
). To test whether the
macroscopic response of the NNC depends on the intracellular
Ca2+ concentration
([Ca2+]i), we used
electrodes filled with BAPTA to chelate
[Ca2+]i and, in addition,
CdCl2 (10
4 M) in the
extracellular solution to block influx of Ca2+
through Ca2+ channels. The sensitivity of the NNC
to [Cl
]o was not
diminished
if anything, the depolarization on change from high to low
[Cl
]o was even stronger
(Fig. 9). Gating of the
Cl
channel in the outside-out patches was found
to be independent of the intracellular Ca2+
concentration ([Ca2+]i)
since channel activity persisted in Ca2+-free,
EGTA-buffered pipette solution.
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Gating of some Cl channels depends on the
intracellular Cl
concentration
([Cl
]i) (reviewed in
Foskett 1998
). We tested whether manipulating [Cl
]i of the NNC would
affect the macroscopic response to
[Cl
]o. Leech neurons
can be loaded conveniently with Cl
by using
microelectrodes filled with 3 M KCl (Schmidt and Calabrese 1992
). When loading the NNC with Cl
,
Vm settled at
45.4 ± 11.5 mV
(n = 11) in 123 mM
[Cl
]o, which is
somewhat depolarized from the control value of
58.7 ± 8.8 mV
[n = 49; P < 0.001 (unpaired
t-test)] (Wenning and Calabrese 1991
). On
the change to 38 mM
[Cl
]o, the cells
depolarized further to
18.7 ± 15.3 mV (n = 11) as compared with
35.8 ± 12.3 mV [n = 11;
P < 0.01 (unpaired t-test)] (Wenning and Calabrese 1991
), and more importantly,
showed a threefold increase of the input conductance. Thus an increase
of [Cl
]i shifts
Vm but does not change the
Cl
sensitivity of the NNC.
The macroscopic Cl-dependent
Cl
current and the Cl
channel in the outside-out patches are independent of
[Ca2+]i and largely
voltage-insensitive, suggesting that the Cl
channel mediates the NNC's sensitivity to
[Cl
]o. The
Cl
channel in the outside-out patches shares
its gating characteristics
slow gating
with the
Cl
-dependent Cl
channel
identified in the cell-attached configuration. The
Cl
-dependent Cl
channel
in the cell-attached configuration responds to changes of the
Cl
concentration of the bathing medium rather
than to changes of the pipette solution, suggesting indirect gating.
Consistent with indirect gating is that the Cl
channels in the outside-out patches are insensitive to changes in
[Cl
]o (Figs. 7 and 8).
Pharmacological characterization of the
Cl-dependent Cl- current and
the Cl
-dependent Cl
channel
We next compared pharmacological characteristics of the
Cl-dependent Cl
current
and the Cl
channel identified in the
outside-outside patches to provide further independent evidence
for their identity. We tested known Cl
channel blockers for their ability to reduce both channel activity in
the outside-out patches as well as the macroscopic
Cl
-dependent Cl
current.
To characterize the macroscopic Cl-dependent
Cl
response, we used voltage- or current-clamp
experiments to screen for pharmacological agents. Recordings started in
high [Cl
]o (108-128
mM). Under these conditions, the input resistance is high and
recordings stabilize more rapidly than in low (38-43 mM)
[Cl
]o (Wenning
and Calabrese 1991
). The NNC was voltage-clamped at
60 mV,
its membrane potential in high
[Cl
]o (Fig. 1, model).
On the change to normal, low (38-43 mM)
[Cl
]o, the NNC develops
an inward current of ~4-5 nA (Wenning and Calabrese
1991
). Since these large inward currents tended to compromise voltage control, we used intermediate Cl
concentration between 60 and 85 mM to decrease the inward currents associated with the decrease in
[Cl
]o.
Five known Cl channel blockers were screened
for their effectiveness on the macroscopic current: the two stilbenes
DIDS and SIDS, NPPB, bumetanide, and Zn2+. Of
those, only bumetanide reliably blocked both the macroscopic Cl
-dependent Cl
response and single-channel Cl
currents.
Bumetanide, best known to block the
Na+/K+/2Cl
co-transporter present in epithelial and nonepithelial cells, has been
shown to also block the CFTR-Cl
channel (cystic
fibrosis transmembrane conductance regulator) (Reddy and Quinton
1999
) at higher concentrations. Strong and reversible
inhibition of the NNC's response to change of
[Cl
]o was observed at
10
3 M (n = 7). At
10
4 M, the inhibitory effect of bumetanide was
less pronounced (n = 4). In three preparations, we
tested the effect of bumetanide at 10
3 M under
voltage clamp, asking whether it is able to abolish an existing, or
prevent the onset of a developing, inward current and whether it
changes the input conductance of the NNC. In the example shown in Fig.
10, the response to changes of
[Cl
]o was first tested
in bumetanide-free solution. On the change from high (114 mM) to low
(70 mM) [Cl
]o
(A,
), the NNC turned on an inward current (
2.3 nA)
with a concomitant three- to fourfold increase in input conductance. The time course of the changes in
[Cl
]o, and hence fluid
exchange rate, are indicated by the bath probe potential. On the height
of the response to low
[Cl
]o, bumetanide was
added to the superfusate (A, bar). Bumetanide reversibly
decreased the inward current to 25% (to
0.6 nA) and the input
conductance by 50%. Washout (B) restored the inward current
and the high-input conductance characteristic for low [Cl
]o. The change to
high [Cl
]o
(B,
) decreased the inward current to almost 0 nA with a
concomitant decrease of the input conductance. When returning to low
[Cl
]o, now with
bumetanide present (C, bar and
), the NNC developed a
much smaller inward current (
0.6 nA) with a less pronounced increase
of the input conductance. Washout yielded the full response to low
[Cl
]o, namely the large
inward current (3 nA) and a further increase of the input conductance
(D). With the change back to high
[Cl
]o (D,
), the inward current was turned off, the input conductance decreased and the NNC returned to its initial 0-nA holding current and
small input conductance (D, end). The swift and rather
dramatic changes of both the inward current and input conductance due
to the change of [Cl
]o
and its block by bumetanide are emphasized in the top panel. In four preparations, the effect of bumetanide was tested in
discontinuous current clamp (data not shown). As expected, application
of bumetanide (10
3 M) in low
[Cl
]o led to
hyperpolarization and an increase in the input resistance. Again, the
response was reversible. When tested at the same concentration (10
3 M) on the Cl
channels in the outside-out configuration (n = 6), with
low or high [Cl
]o,
bumetanide completely and reversibly blocked channel openings (Fig.
11).
|
|
The effect of bumetanide on the Cl channels was
tested in excised patches (o/o) with one or two channels present. The
macroscopic response was tested on the whole cell with the peripheral
arborizations of the NNC in the nephridium intact (Fig. 1, top
left). Penetration of the drug in the periphery might be slower
than onto the exposed cell body, which might explain the incomplete
block of the macroscopic current.
The macroscopic current and the Cl channel
identified in outside-out patches were reliably blocked by bumetanide.
We therefore conclude that the Cl
channel of
the outside-out patches are the same as the
Cl
-dependent Cl
channels identified in cell-attached patches and are indeed those that
mediate the NNC's sensitivity to
[Cl
]o.
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DISCUSSION |
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The NNCs of the leech, peripheral neurons close to the nephridium
(Fig. 1, top left), monitor blood Cl
concentration by a novel slowly gating Cl
channel whose activity is inversely correlated to the external Cl
concentration. In principle, a
Cl
channel with positive voltage dependence
could explain the observed depolarization on the change from high to
low [Cl
]o, simply due
to a more depolarized ECl. A key
feature of the macroscopic Cl
-dependent
Cl
current, however, characterized previously
in voltage-clamp experiments (Wenning and Calabrese
1991
) is its voltage independence. To account for the threefold
increase in Cl
conductance that accompanies the
23-mV depolarization on the change from high (133 mM) to low (38 mM)
[Cl
]o (Fig. 1, model),
voltage sensitivity of a Cl
channel would have
to be >23 mV for an e-fold change in membrane potential.
This is far more than we ever observed in cell-attached or excised
patches (Figs. 4B, 6A, and 7B).
Alternatively, the shift in ECl, and
the concomitant depolarization, could initiate a
Ca2+ influx. Increase in
[Ca2+]i could in turn
activate a Cl current and lead to further
depolarization. The Cl
-dependent
Cl
current of the NNC is, however, independent
of extracellular [Ca2+] (Wenning and
Calabrese 1991
), and, as shown here, the macroscopic response
to changes of the extracellular Cl
concentration persisted when
[Ca2+]i was chelated with
BAPTA (Fig. 9). Consistent with the macroscopic current being
independent of [Ca2+]i,
gating of the Cl
channel identified in the
outside-out configuration is also independent of
[Ca2+]i because the
pipette solution was Ca-free and contained EGTA as a
Ca2+-chelator.
The Cl-dependent Cl
channel identified in the cell-attached patches shares both the inverse
correlation of channel activity with
[Cl
]o and the voltage
independence with the macroscopic Cl
-dependent
Cl
current (Figs. 4 and 6) (Wenning and
Calabrese 1991
). We therefore conclude that the
Cl
-dependent Cl
channel
identified in cell-attached patches accounts for the Cl
sensitivity of the NNC. The
Cl
channel identified in the outside-out
configuration shares the independence of voltage and
[Ca2+]i with the
macroscopic Cl
-dependent
Cl
current. To further confirm that the
Cl
channels identified in the outside-out
patches
which are insensitive to changes of
[Cl
]o (Fig. 8), see
following text
underlie the macroscopic current, we used
pharmacological tools.
Bumetanide provided an efficient tool to block both the macroscopic
Cl current and the Cl
channels identified in the outside-out configuration (Figs. 10 and 11).
Bumetanide is structurally related to NPPB, a widely used Cl
channel blocker. Bumetanide is best known to
block the
Na+/K+/2Cl
co-transporter in epithelial cells. The
Na+/K+/2Cl
co-transporter elevates
[Cl
]i above
thermodynamic equilibrium and thereby yields Cl
outflow through the appropriate channels on the opposite cell membrane. In the leech nephridium, for example, bumetanide blocks urine
flow completely at 10
4 M
(Zerbst-Boroffka et al. 1997
). At higher concentrations
(10
3 M), however, as shown in native sweat
ducts, bumetanide also blocks the cystic fibrosis transmembrane
conductance regulator Cl
channel when applied
to the apical side of the epithelium (Reddy and Quentin
1999
). When tested on the NNC at the same high concentration (10
3 M), bumetanide reversibly decreased the
inward current and the input conductance in voltage clamp developing on
the change to low [Cl
]o
in the NNC (Fig. 10), increased the input resistance and hyperpolarized the NNC when administered in low
[Cl
]o (data not shown),
and blocked the Cl
channels identified in the
outside-out configuration (Fig. 11). The similar pharmacology indicates
that the Cl
channels identified in the
outside-out patches underlie the NNC's response to
[Cl
]o and are the same
as the Cl
-dependent Cl
channels identified in the cell-attached configuration.
The NNC's resting conductance is three times larger in low than
in high [Cl]o (90 vs.
30 nS) (Wenning and Calabrese 1991
), indicating that the
depolarizing Cl
-dependent
Cl
current dominates the membrane conductance
under normal conditions (Figs. 1, model; 10, change to low
[Cl
]o in the 1st
trace). However, from 140 patches in the cell-attached configuration
that lasted long enough to test channel activity at different
[Cl
]o (
5 min), only
43 contained the Cl
-dependent
Cl
channel. This suggests that the channel
density might be lower in the cell body than in the sensory projections
themselves. This suggestion is corroborated by earlier findings that
the Cl
-sensitivity of the NNC is abolished when
the peripheral projections are cut near the cell body (Wenning
1989
).
Gating of the Cl-dependent
Cl
channel is not through interaction of
Cl
with the channel itself. In cell-attached
recordings, its activity increases markedly on change from high to low
Cl
in the bathing medium rather than to changes
of the pipette solution (Figs. 2, 3, and 5), suggesting indirect
gating. Consistent with an intracellular signaling pathway is the fact
that the Cl
channel identified in the
outside-out configuration is insensitive to changes in
[Cl
]o (Fig. 8).
The properties of the leech Cl-dependent
Cl
channel differ fundamentally from other
known Cl
channels. One feature of the leech
Cl
-dependent Cl
channel, a dependence of the open probability on the permeating ion
itself, has been described for ClC-type Cl
channels of Torpedo electroplaque. However, the open
probability of ClC-type Cl
channels increases
with increasing [Cl
]o
(Chen and Miller 1996
; Pusch et al. 1995
;
Richard and Miller 1990
), while it decreases in the
leech Cl
-dependent Cl
channel. Moreover, gating of the ClC-type channels is direct while it
is indirect in the Cl
-dependent
Cl
channel of the leech NNC.
The Cl-dependent Cl
channel is modulated by the NNC's endogenous peptide,
FMRF-NH2 (Wenning et al. 1993b
).
Voltage-clamp experiments showed that FMRF-NH2
turns off the Cl
-dependent Cl- current
(Wenning and Calabrese 1995
), suggesting a second,
probably also indirect, gating mechanism.
The observation that the conductance of the
Cl-dependent Cl
channel
is only 50% when Cl
is absent on the
cytoplasmic side compared with the case when Cl
is present (compare Figs. 6 and 8) might indicate that the channel is
"locked" into a subconductance state with no cytoplasmic
Cl
(outside-out patches). Subconductance states
were frequently observed in cell-attached recordings (Fig.
6A, asterisk) and in outside-out patches with
Cl
present on the cytoplasmic side but never in
outside-out patches with no cytoplasmic Cl
. A
dependence of channel gating on intracellular
Cl
has been reported for other
Cl
channels (see Foskett 1998
)
and for SLO-2 K+ channels from
Caenorhabditis elegans (Yuan et al. 2000
). In
the intact NNC, however, we expect only small changes in
[Cl
]i, and the reduced
conductance of the channels seen in the absence of
[Cl
] seems thus irrelevant under
physiological conditions. Also, loading of the cell with
Cl
shifted Vm
but did not abolish the macroscopic response.
The NNC and, as shown more recently, a growing number of
vertebrate neurons (Ben-Ari et al. 1997; Enz et
al. 1999
; Reuter et al. 1998
; Rohrbough
and Spitzer 1996
), belong to the type of neurons that maintain
ECl well above
Vm so that activation of any
Cl
currents will lead to depolarization. In the
NNC, the Cl
-dependent
Cl
current dominates
Vm in the normal ionic environment
(36-42 mM [Cl
]o)
(Wenning et al. 1980
; Zerbst-Boroffka
1970
) providing continuous excitation and keeping the cell
firing bursts of action potentials (Fig. 1, top right)
(Wenning 1989
; Wenning and Calabrese
1991
). The combination of an
ECl above
Vm and second-messenger-gated Cl
channels drives fluid transport across many
vertebrate epithelia. Neurons that maintain
ECl above
Vm, and hence have depolarizing Cl
currents, serve a variety of purposes. A
depolarizing Ca2+-dependent
Cl
current was found to contribute to
chemo-electrical transduction in olfactory sensory neurons
(Reuter et al. 1998
). In immature vertebrate neurons, a
nonpassive distribution of Cl
with
ECl positive from the membrane
potential provides excitation in response to GABAergic innervation
(Ben-Ari et al. 1997
; Rohrbough and Spitzer
1996
). The leech NNC's ability to monitor extracellular Cl
concentration, without adapting to it (Fig.
1, top right), relies also on the active maintenance of its
nonequilibrium distribution and a high resting conductance for
Cl
. This Cl
conductance
is turned off when the Cl
concentration in
leech blood increases as it does, for example, after a blood meal. We
show here that the underlying Cl
channel is a
novel, slowly gating, voltage- and
Ca2+-independent Cl
channel that is indirectly gated and whose activity is inversely correlated to the external Cl
concentration.
Future experiments will focus on the signaling mechanism of the
Cl
-dependent Cl
channel.
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ACKNOWLEDGMENTS |
---|
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-24072 (R. L. Calabrese), the Stazione Zoologica "Anton Dohrn" (C.F.J. Erxleben), and Deutsche Forschungsgemeinschaft Grant We 745/4 (A. Wenning).
Present address of C.F.J. Erxleben: NIEHS, F210, 111 Alexander Dr., Research Triangle Park, NC 27709.
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FOOTNOTES |
---|
Present address and address for reprint requests: A. Wenning, Dept. of Biology, Emory University, 1510 Clifton Rd., Atlanta, GA 30322 (E-mail: awenning{at}biology.emory.edu).
Received 26 February 2001; accepted in final form 26 June 2001.
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REFERENCES |
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