Department of Physiology, University of Utah School of Medicine, Salt Lake City, Utah 84108
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ABSTRACT |
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Petty, Christopher N. and
Mary T. Lucero.
Characterization of a Na+-dependent betaine transporter
with Cl channel properties in squid motor neurons.
Most marine invertebrates, including squids, use transporters
to accumulate organic osmolytes such as betaine, to prevent water loss
when exposed to elevated salinity. Although a limited number of flux
studies have shown the Na+ dependence of betaine transport,
nothing is known about the electrogenic properties of osmolyte
transporters. We used whole cell and perforated-patch voltage-clamp
techniques to characterize the electrical properties of the betaine
transporter in giant fiber lobe motor neurons of the squid
Lolliguncula brevis. Betaine activated a large,
Cl
-selective current that was reversibly blocked by 100 µM niflumic acid (97 ± 2% block after 40 s, SD;
n = 7) and partially inhibited by 500 µM SITS (29 ± 11%; n = 5). The Cl
current was
Na+ dependent and was virtually eliminated by isotonic
replacement of Na+ with Li+, NMDG+,
or Tris+. Concentration-response data revealed an
EC50 in a physiologically relevant range for these animals
of 5.1 ± 0.9 mM (n = 11). In vertebrates, the
betaine transporter is structurally related to the GABA transporter,
and although GABA did not directly activate the betaine-induced
current, it reversibly reduced betaine responses by 34 ± 14%
(n = 8). Short-term changes in osmolality alone did not
activate the Cl
current, but when combined with betaine,
Cl
currents increased in hypertonic solutions and
decreased in hypotonic solutions. Activation of the betaine transporter
and Cl
current in hypertonic conditions may affect both
volume regulation and excitability in L. brevis motor
neurons. This study is the first report of a novel betaine transporter
in neurons that can act as a Cl
channel.
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INTRODUCTION |
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Transporters are integral membrane proteins that
facilitate the movement of neurotransmitters, osmolytes, sugars, amino
acids, and ions across the plasma membrane. The model of transporter function has changed over the years from that of a shuttle for coupled
movement of substrate to something similar to an ion channel (Lester et al. 1994). The line between ion channel and
transporter has become even less distinct, with recent findings that
transporters can go into an uncoupled mode where transmitter-activated
ionic currents occur, exceeding those predicted by stoichiometric
models (DeFelice and Blakely 1996
). Examples of the
latter include transporters for serotonin (Galli et al.
1997
; Mager et al. 1994
), glutamate (Fairman et al. 1995
; Larsson et al.
1996
; Picaud et al. 1995
; Wadiche
1995
), GABA (Cammack et al. 1994
, 1996
;
Mager et al. 1993
, 1996
), dopamine (Sonders et
al. 1997
), and norepinephrine (Galli et al.
1995
). In this paper we report on a Na+-dependent,
betaine transporter that is coupled to or contains a Cl
channel.
Glycine-betaine (betaine) is a trimethylamine produced by the oxidation
of choline. Betaine is used as a nonperturbing osmolyte by plants,
bacteria, invertebrates, and vertebrates to compensate for hypertonic
stress (Burg 1995; Yancey et al. 1982
).
The vertebrate betaine transporter is part of the
Na+/Cl
-dependent carrier superfamily, with
the closest homology to the GABA transporter (Yamauchi et al.
1992
). The betaine transporter has been cloned from a number of
vertebrate cell types, including canine Madin-Darby kidney cells
(Yamauchi et al. 1992
), rabbit renal papilla cells
(Ferraris et al. 1996
), and human brain (Borden et al. 1995
). Northern blot studies show that it is distributed in kidney, liver, lung, spleen, brain (Takenaka et al.
1994
), and macrophages (Warskulat et al. 1995
;
Zhang et al. 1996
). An invertebrate betaine transporter
has not been cloned.
In vertebrates, betaine transport requires Na+ and
Cl and is modulated by changes in osmolarity
(Nakanishi et al. 1990
). The invertebrate betaine
transporter found in the gills of marine mussels (Mytilus
californianus) is also dependent on Na+, and transport
activity decreases with decreased osmolarity (Wright et al.
1992
).
We studied the electrical properties of the Na+-dependent
betaine transporter in giant fiber lobe (GFL) motor neurons of the squid Lolliguncula brevis, a euryhaline cephalopod. These
osmoconforming squids rapidly adjust their blood osmolality to 2% of
the ambient sea water, allowing them to cross osmotic gradients that
range from 525 to 1,089 mOsm without harm (Hendrix et al.
1981
). Betaine concentrations in cephalopod axoplasm can be
very high (70 mM) compared with blood (4 mM) (Deffner
1961
), implicating betaine as an important osmolyte for squids.
We found that the betaine transporter in L. brevis is
capable of moving into an uncoupled mode with large ion channel-like
fluxes. Interestingly, for the betaine transporter that we studied, the
fluxes are carried by Cl
, similar to the Cl
fluxes in glutamate transporters (Fairman et al. 1995
)
rather than the cationic fluxes of GABA transporters (Mager et
al. 1996
). In addition, activity of the Cl
current is dependent on changes in osmolarity, and this may be one
mechanism used by euryhaline invertebrates to rapidly equilibrate to
changing salinities of their natural environment.
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METHODS |
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Cell preparation and culture conditions
The well-known squid giant motor axons are formed by the fusion
of hundreds of normal sized axons that emanate from cell bodies located
in the posterior tip of the GFL of the stellate ganglia (Young
1939). The animals used in this study, L. brevis,
were obtained from the National Resource Center for Cephalopods
(Galveston, TX). Isolation and culture of GFL cell bodies was similar
to that described by Gilly et al. (1990)
. Briefly, the animals were
decapitated, and the GFLs of the squid L. brevis were
excised under a dissecting microscope and treated with nonspecific
protease (10 mg/ml Sigma type XIV) in sterile filtered artificial
seawater (ASW) for 40 min. After a 3- to 5-min rinse in ASW, cell
bodies (40-60 µm diam) were teased from the GFL with glass
micropipettes and plated onto a drop of culture media on concanavalin
A-coated (10 mg/ml; Sigma type IV) glass coverslips and allowed to
settle for 15 min before adding 2 ml media and placing in the incubator
at 22°C. In the majority of cells, the axon was either cutoff at the
level of the cell body or reabsorbed, and all recordings were made from round cells. Culture media was changed daily, and cells were used for
experiments between 1 and 3 days in culture.
Solutions
The external bath solutions and internal pipette solutions are listed in Tables 1 and 2, respectively. The osmolality of 780 mOsm/kg H2O was chosen for our standard recording conditions because it is in the midrange of osmolalities of the seawater that this osmoconforming species of squid inhabits. The culture media consisted of Leibovitz's L-15 (Gibco, Grand Island, NY) supplemented with salts to bring the osmolality to 780 mOsm/kg H2O, 2 mM HEPES (pH 7.6), 4 mM 1-glutamine, 200 units/ml penicillin G, and 200 mg/ml streptomycin sulfate (Irvine Scientific, Santa Ana, CA). The media was set to pH 7.6 with NaOH and immediately sterile filtered. All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless stated otherwise. A 20-mM stock solution of the chloride channel blocker niflumic acid was made by sonication in DMSO and then diluted to 100 µM in ASW. Control application of DMSO had no effect.
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Gramicidin and whole cell recordings
Gramicidin perforated-patch recordings were used to preserve
[Cl]i (Myers and Hayden
1972
) and internal second messengers, and were made according
to the technique of Abe et al. (1994)
. The gramicidin stock solution
was made fresh daily and consisted of 1 mg gramicidin/100 µl
methanol; 10 µl of this stock solution was added to 1 ml internal
solution (Table 2) and was kept in the dark and on ice.
Three to 6 M resistance electrodes were pulled from thick-walled
borosilicate filament glass (Sutter Instrument, San Rafael, CA) to make
both whole cell (Hamill et al. 1981
) and gramicidin perforated-patch recordings. Bath solutions (21-23°C, the
temperature at which these animals live) were perfused through the
chamber at a rate of 1-2 ml/min. Test solutions were delivered with a Warner SF-77 rapid solution changer (Warner Instrument, Hamden, CT).
Measuring the time course of current responses during solution changes
to ASW in which the NaCl was replaced by KCl revealed that it took
200-300 ms to completely change the solution on the GFL cells. A 3-M
KCl-agar bridge was used to ground the bath solutions. The 1- to 13-mV
liquid junction potential between bath and pipette solutions was
calculated with Axoscope (Axon Instruments, Foster City, CA), and all
reversal potentials reported in the text and in Fig.
1E were appropriately
corrected. Calculated reversal potentials were based on ionic
concentrations.
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Data acquisition
Voltage-clamp recordings were made as described by Danaceau and
Lucero (1998). Cells were voltage clamped to a given voltage for 4,000 ms. To allow time for voltage-gated currents to stabilize, betaine and
other stimulants were applied 1,500 ms after the initiation of the
voltage step. An example of a raw current trace is shown in Fig.
1A. In all of the following figures, we eliminated the first
500 ms containing transient voltage-gated currents. We baseline subtracted the steady-state, voltage-dependent and linear-leak currents
by setting the current amplitudes recorded for 300 ms before a response
to 0 pA so that only the betaine-sensitive currents are shown (Fig.
1B).
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RESULTS |
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Betaine activates a Cl selective current
We applied 5 mM betaine to GFL neurons isolated from the squid
L. brevis that were whole cell voltage clamped to 90 mV
and internally perfused via the patch pipette with a high
Cl
(366 mM) internal solution. Under these conditions we
found that betaine activated inward currents as large as 15 nA, with
average current values of 6.7 ± 4.1 nA (SD, n = 14). When normalized to cell capacitances that averaged 119 ± 33 pF (n = 14), the current densities averaged 63.7 ± 50.5 pA/pF (n = 14).
To determine the ionic selectivity of betaine responses, we used
patch pipettes filled with various concentrations of Cl
(see Table 2) and measured the reversal potential
(Erev) of betaine responses. By using ion
substitution, the Erev of Na+,
K+, and Ca2+ were set to very positive values
(more than +60 mV) to measure the Cl
current reversal in
isolation. Betaine-induced currents reversed near the predicted
Cl
reversal potential (ECl) of
49 mV during whole cell recordings with a K+-free 51-mM
Cl
pipette solution (TMA-Glut) and a 330-mM
Cl
bath solution (Fig. 1, B and D,
, and E,
). Changing the Cl
concentrations shifted the reversal potential of betaine current responses. Figure 1C shows that betaine-induced currents
reversed at
10 mV in a different cell, with a K+-free 366 mM Cl
pipette solution (TMA-Cl) and ASW bath solution
(calculated ECl =
5 mV). The current-voltage
(I-V) relationships for the peak currents in Fig.
1, B and C, are shown in Fig. 1D. The
plot of the average Erev of betaine responses
obtained by using different [Cl
]i versus
the ECl, calculated with the Nernst equation and
the Cl
concentrations of the pipette and bath solutions,
is shown in Fig. 1E. The data closely follow the solid line,
which indicates the values for a perfectly selective Cl
channel. Changes in Ca2+concentration (data not shown) or
Na+ and K+ concentrations had no effect on the
Erev of betaine-induced currents (Fig.
2B).
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In comparing the I-V relationships in which
Erev was negative to 40 mV (n = 10) with those with Erev close to 0 mV
(n = 14), we observed a consistent nonlinearity (inward
rectification) when the ECl was set to less
negative potentials (Fig. 1D,
). This nonlinearity was
not observed when ECl was more negative (Fig. 1,
D,
, and F) and is likely due to the decrease
in membrane resistance at depolarized potentials rather than a voltage
dependence of the betaine-induced current.
Once we determined that the betaine responses were Cl
selective, we used the gramicidin perforated-patch technique, which allows whole cell measurement of current responses without perturbing internal Cl
concentration to determine the cell's
natural [Cl
]i. When 5 mM betaine was
applied to a cell in gramicidin perforated-patch mode, currents were
inward at voltages negative to
60 mV but outward at voltages positive
to
60 mV (Fig. 1F). On average, betaine responses recorded
in gramicidin perforated-patch mode reversed at
46 ± 6 mV
(n = 19), giving a calculated
[Cl
]i of ~71 mM. The calculated
[Cl
]i of L. brevis GFL neurons
is similar to the [Cl
]i of the axoplasm
from the squid Loligo (151 mM) (Deffner
1961
), considering that our measurements were made in reduced
salinity ASW (780 mOsm).
Betaine-Cl responses are Na+ dependent
To investigate the dependence of betaine-Cl
responses on external Na+, we made equimolar substitutions
of Li+ for Na+ in 470 mM Na ASW (the
Na+ concentration of normal 1,000-mOsm seawater). We found
that external Na+ is required for betaine responses. Figure
2A shows a plot of normalized and averaged betaine-induced
current responses recorded at
70 mV in whole cell mode with different
[Na+]o. A Hill equation fit to the data gave
an EC50 of 224 mM ± 37 mM (n = 6).
The I-V relationships plotted in Fig.
2B show the effects of reducing external Na+
over the voltage range from
90 to +10 mV. Although the size of the
betaine responses decreased as external sodium was eliminated, Erev was unaffected, indicating that although
Na+ is required it is not a major permeant ion. Identical
results were obtained by substituting two additional and structurally different monovalent cations, Tris+ (n = 13) or NMDG+ (n = 28), suggesting that the
elimination of Na+ rather than some sort of nonspecific
blocking effect by Li+ reduced the betaine-Cl
responses. These results indicate that the betaine-induced
Cl
current in GFL neurons is Na+ dependent.
Betaine-induced Cl currents are dose dependent
To determine whether betaine acts in a physiologically relevant
range, we measured the EC50 for the betaine response.
Figure 3A shows current
responses to 1-s applications of 1, 5, and 50 mM betaine. The amplitude
and kinetics of betaine-induced Cl currents increased
with increasing betaine concentration. Figure 3B shows the
concentration-response curve for betaine, obtained by normalizing the
betaine responses for a given cell to that of the response of 100 mM of
betaine in the same cell and plotting against the log of the betaine
concentration. The data were fit with the Hill equation yielding an
EC50 for the betaine response of 5.1 ± 1.0 mM
(n = 11) and a Hill coefficient of 1.5 ± 0.3 (n = 11). This EC50 is in a physiologically
relevant range assuming that Lolliguncula brevis have
similar (mM) concentrations of betaine in their blood as
Loligo (4 mM) (Deffner 1961
).
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Pharmacology of betaine-induced Cl currents
Our selectivity studies indicated that betaine activated a
Cl-selective current. We tested whether the
Cl
channel blocker niflumic acid could block betaine
current responses. In Fig. 4A
we show that application of 100 µM niflumic acid completely and
reversibly blocked the betaine-elicited Cl
current.
Figure 4B shows the averaged percent current remaining from
seven to nine cells, with current responses normalized to the peak of
the control betaine application. The amplitude of the control response
(100%) was lowered to 6 ± 6% (n = 9) with the
20-s application of niflumic acid and down to 3 ± 2%
(n = 7) with 40-s niflumic acid application. After
60 s of washing out niflumic acid, the average current response
recovered to 92 ± 10% (n = 9) of the original
response. Bath application of 500 µM SITS reversibly reduced 5 mM
betaine current responses by 29 ± 11% (n = 5).
Collectively, our results show that betaine is activating a
Na+-dependent, Cl
- selective current. To rule
out the possibility that the Cl
current was associated
with betaine activation of a verapamil-sensitive P-glycoprotein
(Tominaga et al. 1995
), we applied 25 µM verapamil to
four cells and saw no effect on the betaine-elicited current. In
addition, we found that elimination of external Ca2+ by
applying betaine in a 0-Ca2+ bath solution with 10 mM EGTA
had no effect on the betaine-induced currents (n = 7).
Finally, external application of 1 mM ATP did not reduce the
betaine-induced Cl
current (n = 8).
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Betaine responses are reversibly reduced by GABA
Because of the structural similarity of GABA and betaine, we
tested whether GABA affected betaine responses. Figure
5 shows data from a cell in which
application of 5 mM GABA alone did not activate a current; however,
coapplication of 5 mM GABA and 5 mM betaine reversibly reduced the
amplitude of the betaine response by 42%. On average, 5 mM GABA
reduced 5 mM betaine responses by 34 ± 14% (n = 8). Application of 5 mM choline activated a small current with kinetics
similar to the betaine-elicited current (n = 4),
whereas 5 mM glycine, proline, alanine, or taurine did not elicit any
current (n = 4). A subset of GFL neurons contained ionotropic GABA receptors and responded to 5 mM GABA with rapid, transient Cl currents; however, those cells were not
included in the present analyses.
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Betaine-induced Cl currents are sensitive to changes
in osmolality
On the basis of our hypothesis that betaine activates a
osmoregulatory Na+-dependent transporter containing a
Cl channel, we predicted that the Cl
current should be sensitive to the same osmotic changes that would
affect betaine transport. To test this prediction, we applied betaine
under three osmotic conditions that span the range of salinities that
these animals can tolerate (580, 780, and 980 mOsm ASW) (Hendrix
et al. 1981
). Figure
6A shows the effect of increasing and decreasing osmolality on 5-mM betaine responses with
whole cell voltage-clamp recordings. All of the recordings were made at
70 mV, and D-mannitol was used to increase osmolality so
that Cl
and Na+ concentrations of the three
solutions were the same. Our normal recording bath and pipette
solutions were set at 780 mOsm. When the external solution bathing a
GFL cell was changed from hypertonic 980 mOsm ASW (Fig. 6,
) to
either 780 (
) or 580 mOsm ASW (
), the betaine-induced
Cl
currents were eliminated. In contrast, when the bath
solution was changed from a hypotonic 580 mOsm ASW to either 780 or 980 mOsm ASW, the betaine-induced Cl
currents returned within
the 20-s application interval. Similar results were obtained in 21 cells. These data indicate that the betaine-induced current acted
exactly as an osmotically sensitive betaine transporter would act; in
the presence of betaine, it turned on in hypertonic conditions (betaine
uptake to prevent shrinkage) and it shut off in hypotonic conditions.
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To further investigate osmotic effects on the betaine-activated
Cl current, we tested whether substitution of glucose or
urea for mannitol affected the betaine-current responses. Because
glucose is transported and therefore more permeant than mannitol, we
predicted that in glucose solutions, the osmotic effects may be
compensated for as glucose is transported into or out of the cell.
Similarly, urea, which is freely permeant, should show even faster
compensation for osmotic changes. In Fig. 6B we normalized
and superimposed peak current responses to betaine application for a
single transition from 980 to 580 mOsm ASW. When mannitol was used to
increase osmolality to 980 mOsm, the betaine-induced Cl
current was eliminated in the 580-mOsm ASW. When glucose was used, the
majority of the betaine-induce Cl
current recovered
within 60 s of hypotonic solution exposure. When urea was used,
the cells responded as if the osmolality of the solution was unchanged,
as would be expected for a freely permeant substance. The effects of
glucose and urea were repeatable and observed in all five cells tested.
These data strongly indicate that the betaine-elicited Cl
current shows the same dependency on osmotic changes as expected for a
betaine transporter. Furthermore, short-term changes in tonicity in the
absence of betaine (2-3 min) had no effect on the cell, indicating
that the Cl
current activated by betaine is not directly
shrink nor swell activated. Prolonged incubations in hypotonic seawater
(>5 min) appeared to activate volume-sensitive ion channels because
the cells became very leaky. These slow, betaine-independent
conductances were not investigated further.
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DISCUSSION |
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The work in this study showed that betaine, an organic osmolyte,
activates a Cl current in GFL motor neurons of the squid
Lolliguncula brevis. Our data showed that the
betaine-induced current is highly selective for Cl
and
that it can be completely blocked by the Cl
-channel
blocker niflumic acid and partially blocked by SITS. The strict
dependence on betaine, Na+, and hypertonicity suggests that
the Cl
current is carried through the betaine
transporter. However, the large amplitude of the Cl
current rules out the possibility that it is a stoichiometrically coupled transporter current. In contrast to a coupled transport process, we believe that as in many of the neurotransmitter
transporters, the betaine transporter is capable of acting like an ion
channel and generating a nonstoichiometric Cl
flux.
We designed a model similar to that proposed by Lester et al. (1994)
for neurotransmitter transporters, and Wadiche et al. (1995)
and
Larsson et al. (1996)
for glutamate transporters, which incorporates
the findings of Moeckel et al. (1997)
for betaine transporters. In our
model, the transporter and the ion channel are inactive in the absence
of sodium and betaine (Fig.
7A). When present, sodium and
betaine act as coligands, binding to the outside of the transporter
(Fig. 7B). The Hill coefficient of 1.5 may reflect the
requirement for the binding of two Na+ ions as the
rate-limiting step in betaine binding, as was described for the GABA
transporter (Mager et al. 1996
). Once Na+
and betaine bind, a conformational change occurs that opens a Cl
ion channel, allowing Cl
flux in and out
of the cell, depending on the membrane potential and
ECl (Fig. 7C). As Na+ and
betaine are transported across the membrane and leave their binding
sites, the Cl
channel closes (Fig. 7D). Our
model is supported by our recent betaine flux studies on whole GFL
tissue from L. brevis that show Na+-dependent
betaine uptake. As in the current work, the [3H]betaine
uptake is blocked by 100 µM niflumic acid (n = 5) and is dependent on external Cl
(J. Poulsen, D. Steel, and M. Lucero, unpublished observations). An alternative model that we cannot
rule out is that the betaine transporter and the Cl
channel are two separate proteins that are blocked by niflumic acid,
and the gating of the Cl
channel is controlled by
betaine, Na+, and salinity in an identical manner as the
betaine transporter.
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The mammalian betaine transporter is part of the
Na+/Cl-dependent carrier superfamily, with
close homology to the GABA transporter (Lester et al.
1994
; Yamauchi et al. 1992
). Physiologically,
our transporter is similar to that of GABA transporters in that it requires Na+ for activation but differs in that the ionic
component of the GABA transporter can be activated in the absence of
GABA and appears to be a cationic channel (Cammack et al.
1994
; Mager et al. 1996
). The serotonin
transporter requires Na+, Cl
, and
K+ (Nelson and Rudnick 1979
) and transports
Na+ (Galli et al. 1997
; Mager et al.
1994
). The dopamine transporter is also
Na+/Cl
dependent and conducts a proton/alkali
cationic leak that is Na+/Cl
independent
(Sonders et al. 1997
). Of the various transporter-ion channels described, the betaine-induced Cl
current is
most similar to the glutamate transporter Cl
current
(Picaud et al. 1995
), which is in a different gene
family from the monoamine transporters (Kanner 1993
).
The betaine-induced Cl
current in squid motor neurons
differs from the glutamate transporter in that niflumic acid does not
block the Cl
current associated with the glutamate
transporter (Wadiche et al. 1995
) and internal
K+ is necessary for glutamate-induced Cl
currents (Picaud et al. 1995
), whereas the presence and
absence of K+ do not affect betaine-induced
Cl
currents.
Our study represents the first time that the electrical
properties of the betaine transporter were studied in native cells. Our
results show that betaine-Cl currents are Na+
dependent and increase when placed in hypertonic media, thereby giving
us an ionic marker for betaine transport. These findings corroborate
previous studies showing hypertonicity induced increases in betaine
uptake (Ferraris et al. 1996
; Moeckel et al.
1997
; Takenaka et al. 1994
; Wright et al.
1992
) and indicate that betaine may have a prominent role in
rapid adjustments of cellular osmolarity in squid motor neurons in
response to osmotic stress. What role might Cl
currents
play in the rapid osmotic adjustments mediated by betaine? Activation
of the betaine-induced Cl
conductance could modulate
neuronal excitability by holding the resting potential close to the
Cl
reversal potential (
46 mV). In hypertonic
conditions, a negative membrane potential would support betaine uptake.
If the cell were to depolarize or hyperpolarize, Cl
current through the transporter would tend to restore the potential and
help maintain a stable, negative resting potential in the face of
changing ionic conditions.
Recent work was done on volume-sensitive Cl
channels (ICl.swell) (see reviews by
Strange et al. 1996
, 1998
) and on volume-sensitive organic osmolyte/anion channels (VSOACs) that mediate efflux of organic
osmolytes (Kirk and Strange 1998
). These ion channels are swell activated, i.e., activated by hypotonic mediums, whereas hypertonic media have no effect and in many cell types are blocked by
extracellular ATP. VSOACs show little selectivity among osmolytes, suggesting a common anion channel pathway. In our experiments, short-term changes (2-3 min) in the tonicity alone had no effect on
ionic currents recorded from GFL neurons; 1 mM ATP did not reduce the
betaine-induced Cl
current. Taurine, proline, and alanine
did not activate the Cl
current, and hypotonic media
rapidly eliminated the betaine response. Our results indicate that the
betaine-induced conductance is not associated with swell-activated ion channels.
Our findings indirectly show that the betaine transporter in L. brevis plays a role in adapting to hypertonic conditions that may include modulation of neuronal excitability and support the evolving view of transporters acting as ion channels. It will be interesting to perform similar studies on GFL neurons from squid species that do not tolerate osmotic changes.
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ACKNOWLEDGMENTS |
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We thank Drs. L. DeFelice, W. Michel, D. Steel, and B. Olivera for helpful suggestions, and J. Lucero, D. Piper, J. Danaceau, J. Poulsen, and J. Lew for technical support.
This work was supported by National Institutes of Health Grant DC-02587-03 to M. T. Lucero and by a short-term research training grant, 5T35HL-07744, to C. N. Petty.
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FOOTNOTES |
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Address for reprint requests: M. T. Lucero, Dept. of Physiology, 410 Chipeta Way, Salt Lake City, UT 84108.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 August 1998; accepted in final form 4 January 1999.
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REFERENCES |
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