Dipartimento di Scienze Biomediche, Sezione di Fisiologia, Università di Modena e Reggio Emilia, 41100 Modena, Italy
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ABSTRACT |
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Bigiani, Albertino.
Mouse Taste Cells With Glialike Membrane Properties.
J. Neurophysiol. 85: 1552-1560, 2001.
Taste buds
are sensory structures made up by tightly packed, specialized
epithelial cells called taste cells. Taste cells are functionally
heterogeneous, and a large proportion of them fire action potentials
during chemotransduction. In view of the narrow intercellular spaces
within the taste bud, it is expected that the ionic composition of the
extracellular fluid surrounding taste cells may be altered
significantly by activity. This consideration has led to postulate the
existence of glialike cells that could control the microenvironment in
taste buds. However, the functional identification of such cells has
been so far elusive. By using the patch-clamp technique in
voltage-clamp conditions, I identified a new type of cells in the taste
buds of the mouse vallate papilla. These cells represented about 30%
of cells patched in taste buds and were characterized by a large
leakage current. Accordingly, I named them "Leaky" cells. The
leakage current was carried by K+, and was
blocked by Ba2+ but not by tetraethylammonium
(TEA). Other taste cells, such as those possessing voltage-gated
Na+ currents and thought to be chemosensory in
function, did not express any sizeable leakage current. Consistent with
the presence of a leakage conductance, Leaky cells had a low input
resistance (~0.25 G). In addition, their zero-current
("resting") potential was close to the equilibrium potential for
potassium ions. The electrophysiological analysis of the membrane
currents remaining after pharmacological block by
Ba2+ revealed that Leaky cells also possessed a
Cl
conductance. However, in resting conditions
the membrane of these cells was about 60 times more permeable to
K+ than to Cl
. The
resting potassium conductance in Leaky cells could be involved in
dissipating rapidly the increase in extracellular
K+ during action potential discharge in
chemosensory cells. Thus Leaky cells might represent glialike elements
in taste buds. These findings support a model in which specific cells
control the chemical composition of intercellular fluid in taste buds.
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INTRODUCTION |
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Taste transduction relies on
several molecular and cellular processes (reviewed in Herness
and Gilbertson 1999; Lindemann 1996
). Among
them, firing of action potentials by taste cells appears to be one
important step in the processing of the chemosensory information at the
peripheral level. Action potentials in taste cells are generated by the
same ionic mechanisms described for other excitable cells, such as
neurons. Namely, they derived from the interplay of voltage-gated ionic
conductances that caused ionic fluxes across the plasma membrane
(Chen et al. 1996
; Kinnamon and Roper
1987
; Roper 1983
). Given the narrow
intercellular spaces between cells inside taste buds (Murray
1973
), it is expected that ion concentrations might change
during action potential discharges. In particular, potassium ions are
extruded from taste cells for membrane repolarization and can
accumulate in the extracellular space. Increased extracellular
K+ concentration,
[K+]o, can disrupt
membrane excitability. A similar situation is observed also in other
excitable tissues, such as in CNS and in the retina, where active cells
determine variations of the extracellular ion concentrations, most
notably K+ concentration (reviewed in
Somjen 1979
; Syková et al. 1998
; Walz 1989
). In these tissues, glia cells possess
membrane properties, such as a conspicuous "resting"
K+ conductance, that enable them to distribute
rapidly extracellular K+ from active to resting
areas of the tissue ("spatial buffering"), thus avoiding dangerous
accumulation of this cation in the extracellular space
(Syková et al. 1998
; Walz 1989
). It
has been suggested that also in taste buds a glialike control of the
extracellular ion concentrations should be performed by some not-yet
identified cells (Lindemann 1996
). Although several
patch-clamp studies have been carried out on single taste cells, up to
now there are no functional data on such cells. In this paper I have
addressed the issue of functional glialike cells in taste buds by
studying the membrane properties of taste cells in the mouse vallate
papilla with the patch-clamp recording technique.
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METHODS |
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Tissue preparation
Adult male C57BL/6J mice were used in this study. Vallate taste
buds were isolated with an enzymatic-mechanical procedure (e.g.,
Béhé et al. 1990; Gilbertson et al.
1993
; Miyamoto et al. 1996
). Briefly, mice were
deeply anesthetized by CO2, followed by
dislocation of cervical vertebrae. Tongues were rapidly removed and
placed in Tyrode solution (in mM: 120 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES,
10 glucose, 10 Na pyruvate, and 20 Na methanesulfonate, pH 7.4 with
NaOH). Two milligrams of elastase (Worthington Biochemical, Freehold,
NJ), and 2 mg of dispase (grade II; Boehringer Mannheim, Mannheim,
Germany) in 1.0 ml of Tyrode solution were injected (0.2-0.4
ml/tongue) between the lingual epithelium and muscle layer. Tongues
were incubated in Ca2+-free Tyrode solution at
30°C for ~15-20 min. After incubation, the lingual epithelium
could be peeled free from the underlying tissue with gentle dissection.
The freed epithelium was pinned serosal side up in a silicone elastomer
(Sylgard)-lined Petri dish and incubated in
Ca2+-free Tyrode solution for ~5-10 min to
loosen the attachments of taste buds to the papilla. Vallate taste buds
were removed from the epithelium by gentle suction with a fire-polished
pipette (tip diameter, about 50 µm) and plated on the bottom of a
chamber that consisted of a standard glass slide onto which a silicon ring 1-2 mm thick and 15 mm ID was pressed. The glass slide was precoated with Cell-Tak (~3 µg/cm2;
Collaborative Research, Bedford, MA) to improve adherence of isolated
taste buds to the bottom of the chamber. The chamber was placed on the
stage of an upright Olympus microscope (model BHWI), and taste buds
were viewed with Nomarski optics. During the experiments, isolated
taste buds were continuously perfused with Tyrode by means of a
gravity-driven system. Drugs were dissolved in modified Tyrode solution
to maintain osmolarity.
Whole cell recording
Membrane currents of single taste cells in isolated taste buds were studied at room temperature (20-22°C) by whole cell patch-clamp, using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Signals were recorded and analyzed using a 486-based computer equipped with Digidata 1200 data acquisition system and pCLAMP software (Axon Instruments). Signal filtering and digitization was adjusted according to the specific features of the membrane currents. For voltage-gated currents, signals were prefiltered at 5 kHz and digitally recorded at 25-µs intervals; for slow current response, signals were usually prefiltered at 100-200 Hz and digitized at 3- to 10-ms intervals.
Patch pipettes were made from soda lime glass capillaries (Baxter
Scientific Products, McGaw Park, IL) on a two-stage vertical puller
(model PB-7; Narishige, Japan). Patch pipettes were filled with
intracellular solution containing (in mM) 120 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES,
11 EGTA, 2 ATP, and 0.4 GTP, pH 7.3 with KOH. Pipette resistances
typically were 3-5 M when filled with intracellular solution. The
access resistance of the patch pipette tip was estimated by dividing
the amplitude of the voltage steps by the peak of the capacitive
transients (from which stray capacitance had been subtracted).
Values typically ranged from about 8 to 15 M
. Leakage and capacitive
currents were not subtracted from currents under voltage clamp unless
otherwise specified, and all voltages have been corrected for liquid
junction potential (LJP, ~4 mV) measured between pipette solution and
Tyrode (bath) solution (Neher 1992
).
Input resistance of taste cells was measured as the slope of the linear
current-voltage (I-V) relationship around 84 mV, unless
otherwise indicated. Cell membrane capacitance was measured by
integrating the capacitative current transient during application of a
20-mV voltage step from a holding potential of
84 mV.
Data analysis
Results are presented as means ± SE. Data were analyzed using a Student's t-test. Significance level was taken as P < 0.05, except as noted.
Concentration-inhibition curve for the effect of
Ba2+ on zero-current potential
(V0) was obtained by adding increasing
concentrations of Ba2+ into the bath solution and
by measuring the corresponding values of
V0. The data were fitted to the
logistic equation
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The Goldman-Hodgkin-Katz equation (Hille 1992) was used
to evaluate the relative membrane permeabilities to
K+ and Cl
. Values of
V0 were measured at different
[K+]o and corrected for
the shunt to ground by the seal resistance (Bigiani et al.
1996
; Lynch and Barry 1991
). Briefly, the
relative contribution of the cell's resting potential to
V0 was evaluated to be about 0.928. Data in Fig. 10 have been corrected for this factor to estimate the
cell's resting potential at different
[K+]o.
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RESULTS |
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Electrophysiological identification of taste cells
I studied the membrane currents elicited in mouse taste cells from
the vallate papilla by depolarizing the membrane under whole cell
voltage-clamp. I chose a holding potential of 84 mV (LJP corrected)
as standard reference potential so that membrane currents from
different taste cells could be compared. In these conditions, I patched
a total of 112 cells and was able to identify three groups of cells
according to their membrane currents.
About 53% of recorded cells (59 of 112 cells) displayed
voltage-dependent inward and outward currents (Fig.
1A). Ionic substitution and
pharmacological dissection showed that the inward current was mediated
by sodium ions, whereas the outward current was mediated by potassium
and/or chloride ions (data not shown). The relative proportion of
potassium current and chloride current to the outward currents was
highly variable. Thus for convenience I named this type of cells as
"Na/OUT" cell, where "Na" stands for the presence of sodium
currents and "OUT" for the presence of outward
(K+/Cl) currents.
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Another group of taste cells (about 15%; 17 of 112 cells) displayed
only voltage-dependent outward currents similar to those found in the
previous group (Fig. 1B). For convenience, I named this
second type of cells as "OUT" cell to indicate that they possessed
only outward (K+/Cl) currents.
Taste cells similar to the Na/OUT and OUT types have been already
described in mouse (Furue and Yoshii 1997;
Spielman et al. 1989
) as well as in other vertebrates
(Akabas et al. 1990
; Avenet and Lindemann
1987
; Béhé et al. 1990
;
Bigiani and Roper 1993
; Chen et al. 1996
;
Miyamoto et al. 1988
; Sugimoto and Teeter
1990
). These cells, or at least of part of them, may represent
sensory cells in different stages of their normal turnover (e.g.,
Mackay-Sim et al. 1996
).
In addition, I identified a new cell type (36 of 112 cells)
characterized by the presence of a strong leakage current (Fig. 1C). For this reason, I named it as "Leaky" cell. The
leakage component of the whole cell current could be readily
appreciated from the I-V plot (Fig. 1, right). In the
voltage range between approximately 85 and
20 mV, Leaky cells
displayed a higher membrane conductance (slope of the I-V
curve) than the other two groups of cells. Leaky cells were further
distinguished by the high negative value of their zero-current
potential (V0, a rough estimation of
the cell's resting potential in whole cell recordings) (Bigiani et al. 1996
),
74 ± 0.7 mV (mean ± SE,
n = 29) compared with that displayed by the other two
groups of cells:
47 ± 2.7 mV (Na/OUT cells; n = 35),
36 ± 4.4 mV (OUT cells; n = 12).
Consistent with the presence of the leakage current, Leaky cells also
displayed a low input resistance (Rin;
Fig. 2). Finally, Leaky cells had a
larger surface area than Na/OUT cells and OUT cells, as indicated by
measurements of the cell membrane capacitance
(Cm; Fig. 2).
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Membrane currents in Leaky cells
A more complete I-V characteristic for Leaky cell's
membrane was obtained by applying hyperpolarizing and depolarizing
voltage pulses from a holding potential of 84 mV. Representative
recordings from two Leaky cells are shown in Fig.
3A. Leakage currents were apparent in all the voltage range tested. In some cells,
voltage-dependent outward currents were also present (Fig.
3A, bottom records). Figure 3B shows
the I-V plot of the mean values of membrane currents recorded from 27 Leaky cells. For membrane voltages greater than about
20 mV, the I-V relationship was nonlinear, suggesting the presence of a voltage-dependent component in the membrane current in
addition to the leakage component. This outward rectification varied
considerably among Leaky cells (compare records in Fig. 3A).
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The membrane conductance responsible for the leakage current in Leaky
cells was selective for potassium ions: elevation of external potassium
concentration resulted in a marked increase in current amplitudes
associated with a positive shift of the I-V curve (Fig.
4). Current reversal potentials were
close to the theoretical equilibrium potentials
(EK) for each of the imposed ionic
gradients for K+, indicating that currents were
almost exclusively carried by K+ ions. The mean
membrane potentials were 75 ± 2 mV (n = 8),
60 ± 1 mV (n = 4),
47 ± 1 mV
(n = 7), and
26 ± 1 mV (n = 6)
in 5, 10, 20, and 50 mM K+-containing solutions,
respectively. These shifts in membrane potential suggest an
approximately
52-mV change in potential per 10-fold change in
[K+]o, supporting the
relative selectivity of the leakage conductance for
K+ ions. This value is similar to that previously
observed for currents through the inwardly rectifying
K+ channels in astrocytes (Ransom and
Sontheimer 1995
), ventricular cells (Sakmann and Trube
1984
), and rat taste cells (Sun and Herness 1996
).
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To establish the pharmacological properties of the leakage currents, I
used known blockers of K+ channels such as
tetraethylammonium (TEA) and barium (reviewed in Castle et al.
1989; Rudy 1988
). Figure
5 illustrates data that are
representative of all Leaky cells that were tested with TEA (n = 5). TEA (20 mM) was unable to affect the linear
(leakage) component of the whole cell currents. However, TEA did block
the voltage-dependent component of the outward currents (Fig. 5). On
the contrary, the leakage current was sensitive to
Ba2+ in all cells tested (n = 14;
Fig. 6). This result supported the view
that the leakage current was K+ dependent and
indicated that it exhibited a specific sensitivity to
Ba2+, similarly to the inward rectifying
K+ currents
(KIR) described in glia cells (e.g.,
Linn et al. 1998
; Newman 1989
;
Ransom and Sontheimer 1995
). It is interesting to note
that in the presence of Ba2+, the reversal
potential in the I-V plot shifted toward zero (Fig. 6B), whereas no change could be detected when TEA was
applied (Fig. 5B).
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Steady inward currents and resting conductances in Leaky cells
Leaky cells held at approximately 80 mV, that is, below their
zero-current potential, showed a steady inward current (Fig. 7A).
Ba2+ caused an increase in this holding current
recorded when the cell was kept at approximately
80 mV (Fig.
7A). Concomitant with the increase in holding current, an
increase in the membrane resistance could be detected, as indicated by
measurement of cell input resistance (Rin; Fig. 7A). The average
increase in Rin was approximately sixfold (Fig. 7B; P < 0.001). The change in
Rin was consistent with channels being
closed by Ba2+.
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Ba2+ strongly affected also the membrane potential of Leaky cells: 10 mM Ba2+ caused about 50 mV depolarization (Fig. 8A; P < 0.001). This shift in membrane potential suggested that Ba2+ blocked a membrane conductance directly involved in maintaining the resting membrane potential. As indicated by the concentration-inhibition curve shown in Fig. 8B, Ba2+ induced half-maximal reduction of V0 at a concentration of ~0.6 mM. This result was consistent with the sensitivity of the K+ leakage current to Ba2+ described above (see Fig. 6).
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The increase in the steady inward current in the presence of
Ba2+ could be due to unmasking of an outward flux
of Cl or of an inward flux of
Na+, which were the main ions, in addition to
K+, in my experimental conditions. At a membrane
potential of
84 mV and in regular Tyrode, both ions were not at the
Nernst equilibrium, as indicated by their equilibrium potentials
(ECl
1 mV;
ENa
+130 mV). The involvement of
Cl
conductance in the steady inward current was
tested by applying 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
(DIDS), a known Cl
conductance blocker in taste
cells (e.g., Herness and Sun 1999
; Taylor and
Roper 1994
). Figure 9A
shows the steady inward current recorded from a Leaky cell during bath
application of 10 mM Ba2+. In this condition, 500 µM DIDS markedly reduced the inward currents (Fig. 9A),
indicating that it was mediated by a Cl
flux.
The mean percent reduction in the inward current induced by DIDS was
56 ± 12% (n = 4).
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The involvement of a Na+ conductance in setting up the steady inward current in the presence of Ba2+ was tested by replacing Na+ with N-methyl-D-glucamine or choline in the bath. In these conditions, an upward deflection in the current trace should be expected if a Na+ conductance was operating in the cell. However, as shown in Fig. 9B, replacement of extracellular Na+ did not cause any detectable upward deflection in the current trace (n = 3).
In summary, the data are consistent with the steady inward current in
the presence of Ba2+ being mediated by a
Cl conductance. The contribution of this
conductance in the generation of the resting potential in Leaky cells
seems to be, however, smaller compared with the
K+ conductance.
V0 in Leaky cells was close to the
potassium equilibrium potential (about
88 mV) both when the pipette
solution contained KCl (V0 =
74 ± 0.7 mV; n = 29) or K gluconate (
75 ± 0.8 mV; n = 7). To estimate the relative membrane
permeabilities of Leaky cells to K+ and
Cl
in resting conditions,
V0 was measured at different
[K+]o and then corrected
for the shunt to ground by the seal resistance (see
METHODS). This correction allowed to evaluate the resting potential (Vr) in Leaky cells. Figure
10 shows that
Vr values at different
[K+]o could be well
fitted by the Goldman-Hodgkin-Katz equation for a permeability ratio
PCl/PK
of about 0.016. This finding indicated that in resting conditions the
membrane of Leaky cells was, on average, about 60 times more permeable
to K+ than to Cl
.
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Voltage-dependent conductances in Leaky cells
In addition to the leakage K+ conductance,
some Leaky cells also possessed voltage-dependent currents. In Fig.
3A, bottom traces, for example, an early,
downward deflection can be noted in a few records, suggesting that the
depolarizing steps activated a voltage-dependent inward current.
Moreover, the outward rectification described above (see, for example,
Fig. 3A, and the currents in Fig. 6A during
Ba2+ treatment) was suggestive of the occurrence
of voltage-dependent outward current. A direct demonstration of the
presence of voltage-dependent currents was obtained by subtracting the
leakage component from the whole cell currents. Figure
11 shows representative recordings from
two Leaky cells in control conditions (left traces) and
after subtracting electronically leakage currents (middle
traces). In one cell (Fig. 11A), leak subtraction did
not unmask any voltage-dependent currents (Fig. 11A, records
in the middle, and corresponding I-V plot on the
right). On the contrary, in the other cell (Fig.
11B), leak subtraction revealed the presence of two types of
voltage-dependent currents, a transient inward current (Fig.
11B, ) and a sustained outward current (Fig.
11B,
). The corresponding I-V curves are reported in Fig. 11B on the right. The inward
current was blocked by 1 µM TTX (n = 3; data not
shown), indicating that it was a Na+ current,
whereas the outward current was blocked by 20 mM TEA (n = 5; data not shown), indicating that it was a K+
current. The occurrence of these voltage-dependent currents as well as
their magnitude varied considerable among Leaky cells. After leak
subtraction, voltage-dependent Na+ currents
(INa) were detected in about 45% of
tested cells (10 of 22 cells), with an average peak value of
106 ± 29 pA (range
22 to
311 pA; n = 10). In the same
conditions, voltage-dependent K+ currents
(IK) were present in about 86% of
tested cells (19 of 22 cells). IK
amplitude, evaluated at the end of a 30-ms depolarizing step to +46 mV,
averaged 258 ± 38 pA (range, 60-584 pA; n = 19). Just for the purpose of comparison, in Na/OUT cells the peak value of
INa averaged
705 ± 87 pA
(range, 60-3,035 pA; n = 44), whereas voltage-dependent outward currents (mediated by
K+ and/or Cl
) at +46 mV
averaged 2,067 ± 186 (range, 235-4,954 pA; n = 44). A detailed biophysical characterization of voltage-dependent
currents in Leaky cells was beyond the scope of this study.
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DISCUSSION |
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The results presented in this study indicate that certain cells
(Leaky cells) in mouse taste buds of the vallate papilla possess membrane properties resembling those described for glia cells in the
CNS (e.g., Ransom and Sontheimer 1995; Sontheimer
and Waxman 1993
; Syková et al. 1998
;
Walz 1989
), and in sensory organs, such as the retina
(e.g., Linn et al. 1998
; Newman 1985
,
1989
) and the inner ear (e.g., Mammano et al.
1996
; Sugihara and Furukawa 1996
). In
particular, Leaky cells in taste buds have a conspicuous leakage
conductance highly permeable to K+. This
conductance, described here for the first time, could be involved in
potassium buffering mechanism, as proposed for glial cells in other
systems (reviewed in Syková et al. 1998
;
Walz 1989
). Taste buds are typical epithelial structures
with very narrow extracellular space between cells (Murray
1973
). Therefore variations in
[K+]o are expected during
activity of chemosensory cells, which are able to fire action
potentials in response to chemical stimulation (reviewed in
Herness and Gilbertson 1999
; Lindemann
1996
). Leaky cells, by virtue of their resting high
K+ conductance, could help in rapidly removing
the extracellular K+ released by active
chemosensory cells. Thus the resting high K+
conductance present in Leaky cells may cooperate with the
Na+/K+ pump in taste cells
(Okada et al. 1986
; Simon et al. 1993
) to reduce [K+]o and maintain
the potassium homeostasis inside the taste buds.
To implement K+ re-distribution in the
extracellular space, K+ conductance in Leaky
cells should be present all over the cell membrane. Vertebrate taste
cells are functionally polarized: tight junctions physically separate
an apical, chemosensitive membrane from a basolateral, synaptic
membrane. Whole cell recording does not allow one to establish the
membrane region where ionic currents are generated. Thus at the moment
I cannot make any statement about the membrane localization of the
leakage current in Leaky cells. However, it is unlikely that the
restricted area of the apical membrane (a few
µm2) can explain the high membrane conductance
of these cells. In Necturus taste cells, which are much
larger than mammalian taste cells and possess a resting, apically
restricted K+ conductance, the membrane
resistance evaluated with the patch-clamp methods is in the order of
gigaohms (Bigiani and Roper 1993; Kinnamon and
Roper 1988
). On the contrary, Leaky cells display a very low membrane resistance in similar recording conditions (Fig. 2). Membrane
surface area was larger in Leaky cells than in other taste cells, as
indicated by the membrane capacitance measurements (Fig. 2).
Normalization of input resistance
(Rin) to membrane capacitance
(Cm) for the different cell types
revealed that the low membrane resistance of Leaky cells was not due to
their larger membrane surface area
(Rin/Cm:
0.18 G
/pF in Na/OUT cells; 0.19 G
/pF in OUT cells; 0.03 G
/pF
in Leaky cells). Thus there is something fundamentally different about
the membrane construction of Leaky cells.
Although the membrane properties of taste cells have been extensively studied with the patch-clamp technique, no papers in my knowledge have reported the findings described in this study. This is surprising, and at the moment I am unable to explain this difference. A possibility is that patched cells with low input resistance may have been rejected in those studies. In patch-clamp recordings, poor seal resistances are associated with "leakage" current shunted across the leakage conductance around the pipette-membrane seal. In Leaky cells of the mouse taste buds, the highly negative values of V0 and the sensitivity of the leakage current to barium were inconsistent with damaged cells.
Glia cells in the nervous system as well as in sensory organs,
such as the retina, are endowed with several kinds of potassium conductances. Among them, the inwardly rectifying potassium
(KIR) current is thought to be involved in the
control of [K+]o
(Abbott 1998; Barres et al. 1990
;
Walz 1989
). As the name indicates, typical
KIR conductance exhibits strong rectification,
with little current conducted outwardly. However, several types of this
conductance are known, and some of them display weak or no
rectification (reviewed in Nichols and Lopatin 1997
). It
is then tempting to speculate that the resting K+
conductance in Leaky cells may be a KIR-like
conductance that exhibits weak or no rectification. Further
characterization of the leakage current is required to establish
whether it derived from the activity of a single type of channel or not.
In taste cells possessing voltage-gated TTX-sensitive
Na+ channels (that is, Na/OUT cells in my
nomenclature), appropriate ionic manipulations have allowed to document
the presence of the KIR conductance
(Kinnamon and Roper 1988; Sun and Herness
1996
). These cells, however, present low membrane conductance
in normal ionic conditions, suggesting that the magnitude of
KIR conductance is low unless enhanced with
appropriate experimental maneuvers. Leaky cells, on the contrary,
possess a very large membrane conductance in resting conditions (Figs.
1 and 2). In addition, KIR conductance in Na/OUT
taste cells present a strong inward rectification and a partial
sensitivity to 20 mM TEA (Kinnamon and Roper 1988
;
Sun and Herness 1996
). Thus it is conceivable that the
K+ conductance expressed by Leaky cells is quite
different from the KIR described in other taste
cells. A similar situation has been described in the goldfish sacculus,
where supporting cells have a unique inwardly rectifying
K+ conductance, characterized by weak inward
rectification and insensitivity to TEA, which is different from the
typical inwardly rectifying current of hair cells (Sugihara and
Furukawa 1996
). It has been suggested that this unique
KIR conductance in supporting cells of the inner
ear likely give them a certain K+-buffering
function in both influx and efflux (Sugihara and Furukawa 1996
).
In addition to leakage K+ currents, some
Leaky cells possessed voltage-dependent Na+
currents and K+ currents. The physiological
significance of these currents in Leaky cells remains to be fully
elucidated. It is unlikely that these currents are involved in the
generation of action potentials: Leaky cells, by virtue of their large
resting K+ conductance, exhibit a very low
membrane input resistance that would shortcut any depolarizing currents
such as those associated with the activation of voltage-dependent
Na+ channels. It is interesting to note that glia
cells, such as astrocytes and Schwann cells, are endowed with a vast
array of voltage-dependent ion channels, including
Na+ channels (for review, see Barres et
al. 1990; Ritchie 1992
; Sontheimer et al.
1996
). However, they are unable to fire action potentials, at
least in physiological conditions (Sontheimer et al.
1996
).
Vertebrate taste cells are structurally heterogeneous, and
different cell morphotypes have been identified, such as elongated cells (subdivided in type I, type II, and type III cells), and round
basal cells (reviewed in Lindemann 1996; Roper
1989
). At the moment, I do not have data on the structural
features of Leaky cells. However, in a few cases (n = 4), I was able to "extract" the cells from the taste bud after
patch-clamp recording. These Leaky cells were elongated and possessed
an apical process. In one cell, this process split in two branches
(data not shown). Cm measurements
indicated that Leaky cells have a larger membrane surface area than the
other taste cells (Fig. 2). Thus Leaky cells might have lateral
cytoplasmic projections, not visible at the light microscopic level,
similar to those characterizing type I (dark) cells (Pumplin et
al. 1997
). Until recordings are correlated with cell
identification at the electron microscopic level, this interpretation
will remain speculation. Interestingly, a recent study by Lawton
et al. (2000)
indicates that the glial glutamate transporter
GLAST occurs in the plasma membrane of dark cells, suggesting that
these cells may play a glialike role in glutamate handling inside taste buds.
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ACKNOWLEDGMENTS |
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I thank Dr. Stephen D. Roper, University of Miami School of Medicine, for helpful comments and discussions. I also thank F. Vaccari and G. Nespoli for excellent technical assistance.
This work was supported by Ministero dell' Università e della Ricerca Scientifica e Tecnologica (Cofin 1998).
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FOOTNOTES |
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Address for reprint requests: Dipartimento di Scienze Biomediche, Sezione di Fisiologia, Università di Modena e Reggio Emilia, via Campi 287, 41100 Modena, Italy (E-mail: bigiani{at}unimo.it).
Received 19 September 2000; accepted in final form 8 December 2000.
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REFERENCES |
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