From the Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France
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ABSTRACT |
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The novel structural class of mammalian channels
with four transmembrane segments and two pore regions comprise
background K+ channels (TWIK-1, TREK-1, TRAAK, TASK,
and TASK-2) with unique physiological functions (1-6). Unlike its
counterparts, TRAAK is only expressed in neuronal tissues, including
brain, spinal cord, and retina (1). This report shows that TRAAK, which
was known to be activated by arachidonic acid (3), is also opened by
membrane stretch. Mechanical activation of TRAAK is induced by a convex
curvature of the plasma membrane and can be mimicked by the amphipathic
membrane crenator trinitrophenol. Cytoskeletal elements are negative
tonic regulators of TRAAK. Membrane depolarization and membrane
crenation synergize with stretch-induced channel opening. Finally,
TRAAK is reversibly blocked by micromolar concentrations of gadolinium,
a well known blocker of stretch-activated channels. Mechanical
activation of TRAAK in the central nervous system may play an important
role during growth cone motility and neurite elongation.
A key feature of all classes of K+ channel
pore-forming subunits is a conserved signature sequence constituting
the pore segment (P)1 (7).
Mammalian K+ channels can be divided into three major
structural classes encoding channels with six transmembrane segments
(TMS), 4TMS and 2TMS (7). The 6TMS class comprises voltage-gated as
well as Ca2+-gated K+ channels. The inward
rectifier IRK, the G protein-coupled GIRK and the ATP-gated
KATP K+ channel subunits are members
of the 2TMS structural class. The most recent structural class of 4TMS
mammalian K+ channel subunits consists so far of five
members (TWIK-1, TREK-1, TRAAK, TASK, and TASK-2) (1-4, 6). Besides
the presence of 4TMS, the other major structural characteristic is the
presence of two P regions as well as an extended M1P1 extracellular
loop (8). Although these subunits display the same structural motif (4TMS/2P), they only share 25-40% sequence identity. These unusual K+ channel structures are associated with unique
physiological properties. TWIK-1 is an ubiquitous subunit, which
directs the expression of a time- and voltage-independent
K+-selective weak inward rectifier in Xenopus
oocytes (4, 8). TREK-1 is highly expressed in brain, heart, lung,
kidney, and several other tissues and encodes a mammalian
serotonin-sensitive-like K+ channel (3, 5). TASK and TASK-2
encode background K+ channels that are blocked by external
acidification near the physiological pH (2, 6). TASK is highly
expressed in heart and brain, while TASK-2 is mainly expressed in
kidney (2, 6).
TRAAK is the only 4TMS subunit to be specifically expressed in the
nervous system (1). TRAAK is opened by polyunsaturated fatty acids,
including arachidonic acid (AA) (1). The present report demonstrates
that TRAAK is also opened by membrane stretch and thus belongs to the
class of mechano-gated ion channels (9-16).
Cell Culture--
COS-7 cells were maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum. The cDNA for TRAAK was subcloned into the
pIRES-Neo expression vector (CLONTECH), and the
resulting construct was transfected in COS cells using the phosphate
calcium method. After 48 h in normal medium, the transfected cells
were dissociated and subcultured by 1:20 dilution in medium containing
500 µg/ml Geneticin (Life Technologies, Inc.). Antibiotic-resistant
clones were selected at random after 2 weeks. A TRAAK-expressing clone
was then identified by using an 86Rb+ efflux
assay as well as electrophysiology and expanded to maintain a stock culture.
Western Blots and Immunocytology--
Anti-TRAAK antibodies were
raised against a glutathione S-transferase fusion protein
containing the carboxyl-terminal 102 amino acids of TRAAK (residues
Pro296 to Val398). The antibodies were purified
by using a glutathione S-transferase fusion protein
containing the same domain of TRAAK. The preparation of fusion
proteins, rabbit immunization, and antibody purification were performed
as described previously (8).
Proteins from COS cells and from synaptic plasma membranes of mouse
spinal cord were prepared and analyzed in the presence of reducing
agents (8). For immunocytology, TRAAK immunodetection was performed as
described previously (8), except that cells were permeabilized by
adding 0.1% Triton X-100 in the blocking solution (phosphate-buffered
saline supplemented with 2% BSA and 5% goat serum).
Efflux of 86Rb+--
COS-7-TRAAK cells
were plated at a density of 40,000 cells/well (Falcon, 24 wells) and
used 2-3 days later. Cells were preloaded for 3 h with 0.5-1
µCi/ml 86Rb+ in 0.5 ml of Dulbecco's
modified Eagle's medium, 10% fetal bovine serum, and 500 µg/ml
Geneticin at 37 °C. Release experiments were then carried out in a
standard (EXT) saline solution containing (in mM: 150 NaCl,
5 KCl, 3 MgCl2, 1 CaCl2, 10 Hepes, pH 7.4, with NaOH) during consecutive intervals of 5 min at room temperature. For
each point six independent experiments were performed at the same time.
An hour washing with EXT solution was then performed before collecting
1-ml fractions. 86Rb+ at 2-7 mCi/mg was from
NEN Life Science Products. Fractional rates of release were calculated
as 86Rb+ released during each 5-min interval
and expressed as the percentage of 86Rb+
content in the cells at the beginning of the respective intervals. 86Rb+ was counted on a Packard Tri-Carb with a
Cerenkov program.
Patch Clamp Experimental Protocols, Recordings and Data
Analysis--
The electrophysiology procedure has been described
elsewhere previously (5). For whole-cell experiments, bath solution (EXT) contained (in mM) 150 NaCl, 5 KCl, 3 MgCl2, 1 CaCl2, 10 Hepes, pH 7.4, with NaOH and
pipette solution (INT) contained (in mM) 150 KCl, 3 MgCl2, 5 EGTA, and 10 Hepes, pH 7.2, with KOH. For cell
attached experiments, the EXT solution contained 150 KCl instead of 150 NaCl, and the pipette contained the EXT solution (150 mM
NaCl). For inside-out experiments the pipette solution was EXT and the
bath solution was INT. Mechanical stimulation was applied through an
open-loop pressure generating system and monitored at the level of the
patch pipette throughout the experiment by a calibrated pressure
sensor. This system provides a stable pressure pulse (5). Colchicine
was dissolved daily in the saline solution at the concentration of 500 µM. Cytochalasin D was dissolved at the concentration of
1 mg/ml in Me2SO and kept at TRAAK was transfected in COS cells, and a stable TRAAK cell line
was characterized by immunoblot using affinity-purified antibodies directed against the carboxyl terminus of TRAAK (Fig.
1, A-C). A major band is
detected with a relative molecular mass (Mr) of 54,000-64,000 and two minor bands of Mr 49,000 and 52,000 (Fig. 1A, lane 2). No signal is
detected in nontransfected COS cells (Fig. 1A, lane
1). The difference between the observed Mr
values and the calculated molecular mass of TRAAK (43,000 Da) is
probably due to glycosylation. This hypothesis is supported by the
presence of two consensus sites for N-linked glycosylation
(residues 81 and 84) in a region of the channel that is expected to be
extracellular (1). This probable glycosylation of TRAAK is also
observed in the spinal cord (Fig. 1A, lane 3), a
tissue that is known to express a high level of TRAAK transcript (1).
Additional bands of Mr 49,000 and 52,000, which
are detected in transfected COS cells, could correspond to incompletely
glycosylated or partially degraded forms of the protein. The
heterologous expression of TRAAK was confirmed by immunocytology (Fig.
1, B and C). A strong signal is observed at the
plasma membrane of the cells expressing TRAAK (Fig. 1C),
while it is absent in control WT COS cells (Fig. 1B).
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
20 °C. AA was dissolved
in ethanol at the concentration of 100 mM, flushed with
argon, and kept at
20 °C for a week. TNP was mixed with the saline
solutions and pH adjusted. 4-Bromophenacyl bromide was dissolved daily
at 100 mM in Me2SO and AACOCF3 at
100 mM in ethanol. All chemicals were obtained from Sigma.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Characterization of a TRAAK stable cell
line. A, Western blot analysis showing TRAAK expression
in a COS stable cell line. Proteins from COS cells and mouse spinal
cord synaptic membranes were prepared and analyzed in the presence of
reducing agents (8). The purified anti-TRAAK antibody was raised
against the carboxyl terminus of TRAAK. Nontransfected WT COS cells
(lane 1), stable TRAAK cell line (lane 2), and
spinal cord synaptic membranes (lane 3) were analyzed.
B, immunocytochemistry of nontransfected COS (WT) cells.
C, immunocytochemistry of the stable TRAAK cell line. D,
kinetics of 86Rb+ efflux evoked by 50 µM AA in COS-TRAAK cells ( ) and in COS wild-type cells
(
). Six independent kinetics are shown per experimental condition.
50 µM AA were added as indicated by an horizontal
bar. E, kinetic of 86Rb+ efflux
evoked by 50 µM AA in COS-TRAAK cells without (
) or
with 50 µM Gd3+ (
). Gd3+ was
preincubated for 20 min before collecting fractions. F,
dose-dependent inhibition of 86Rb+
efflux activated by 50 µM AA in COS-TRAAK cells.
Activated efflux was equal to the flux at 50 min (maximal) minus the
flux at 30 min (basal). Half-maximum inhibition was IC50 = 5 ± 1 µM. Experimental data are presented as mean
associated with their S.E. (n = 6).
The expression of exogenous K+ channel activity was initially detected in the TRAAK cell line using 86Rb+ efflux (Fig. 1, D-F). Control COS cells (WT) do not show any significant increase in 86Rb+ efflux in the presence of AA (Fig. 1D). However, cells expressing TRAAK display a reversible 3-4-fold increase in 86Rb+ efflux induced by AA (Fig. 1, D and E). AA activation is reversed by the addition of micromolar concentrations of Gd3+ (IC50: 5 ± 1 µM) (Fig. 1E).
The TRAAK cell line was then used for electrophysiological
investigations. In the whole cell configuration, basal channel activity
is low (TRAAK: 12.2 ± 1.3 pA/picofarad, n = 12;
COS WT: 4.2 ± 0.5 pA/picofarad, n = 18; at 100 mV) (Fig. 2A). An outward current is slowly and reversibly induced at a holding potential of 0 mV
by AA superfusion (4.8 ± 0.4-fold, n = 12) (Fig.
2A, bottom inset). This current is absent in
mock-transfected COS cells (n = 18) (5). The I-V curve
of the current induced by AA displays a strong outward-going
rectification (Fig. 2A). The reversal potential of the
current activated by AA reverses at 83.7 ± 0.7 mV
(n = 12), which is the predicted value for
EK+. Opening of TRAAK is also
reversibly induced by the amphipathic membrane crenator TNP (6.7 ± 1.1-fold, n = 6 with 400 µM TNP) (15,
17). The kinetics for current activation is faster in the presence of
TNP compared with AA (Fig. 2A, insets). I-V
curves in the presence of TNP and AA are similar. Finally, as observed
with 86Rb+ efflux experiments, TRAAK activation
by AA is also blocked by Gd3+ (
64.2 ± 4.7%,
n = 8 with 10 µM Gd3+) (Fig.
2B).
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In the cell-attached patch configuration, no channel activity is
detected under resting conditions (NPo: 0.05 ± 0.03, n = 6). However, we observed that TRAAK opens when a
negative pressure is applied to the patch pipette (Fig.
3A). No channel activity is
observed in mock-transfected COS cells (5). In the experiment illustrated in Fig. 3, A and B, channel activity
is absent at atmospheric pressure but three TRAAK 38-picosiemens
channels readily and reversibly open during the application of a 66
mm Hg pressure. As reported previously for AA activation, TRAAK opening
was characterized by flickering kinetics (1).
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Both the number of active channels and the sensitivity to mechanical
stretch are strongly enhanced after a treatment for 20 min with
colchicine (500 µM) (Fig. 3, C-E).
The threshold for mechanical activation is lowered from 70 to
20 mm
Hg, and maximal channel activity is enhanced at all indicated pressures
in the presence of colchicine (Fig. 3, C and D).
Similarly, addition of cytochalasin D (5 µg/ml) enhances channel
activity with, however, a weaker effect (Fig. 3E). Finally,
excision of the patch in the inside-out configuration, which is known
to disrupt cytoskeletal elements, produces an almost 10-fold increase
in channel activation induced by a
70 mm Hg pressure (Fig.
3E). Moreover, the threshold for mechanical activation is
also significantly lowered after excision of the patch (Fig.
4A). In the inside-out patch
configuration, channel activity is basically absent at atmospheric
pressure, and only negative, but not positive, pressure induces channel opening (Fig. 4B). The I-V curve performed with a voltage
ramp protocol in physiological K+ conditions is
outwardly rectifying and reverses at
80 mV, the predicted value
for the equilibrium potential for K+ (Fig.
4B).
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The absence of channel activation by positive pressure in the inside-out patch configuration suggests that channel opening is mediated by a specific membrane deformation. Indeed, in the outside-out patch configuration, TRAAK opening is only induced by positive pressure (Fig. 5A). The activation of TRAAK is purely pressure-dependent, and moreover, as observed in both the cell-attached and inside-out configurations, channel activity is absent at atmospheric pressure. Opening of TRAAK by positive pressure in outside-out patches is reversibly blocked by the addition of 30 µM Gd3+ (n = 3) (Fig. 5B).
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We then investigated the effect of voltage on TRAAK activation in the
inside-out patch configuration. Fig. 6
shows that opening of TRAAK by membrane stretch is enhanced by membrane
depolarization. The threshold for mechanical activation by negative
pressure is lowered at depolarized voltage, and moreover maximal NPo is
significantly enhanced (Fig. 6C). The basal NPo measured at
atmospheric pressure is also significantly increased at depolarized
potential (Fig. 6, A-C). The steep modulation of
TRAAK activation occurs at voltages between 25 and 50 mV.
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We investigated the possible interaction between the membrane crenator TNP and membrane stretch. Fig. 7, A and B, show that in the inside-out configuration, internal application of TNP has no effect at atmospheric pressure. However, TNP produces a significant reversible stimulation of the opening of TRAAK by negative pressure (Fig. 7, A and B). Interestingly, in the outside-out configuration, external TNP produces a significant activation of TRAAK at both atmospheric and positive pressures (Fig. 7, C and D).
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Finally, to assay for the possible involvement of AA in the
mechano-activation of TRAAK, we investigated the effect of 30 µM lipid-free BSA, which is known to bind fatty acids and
remove them from membranes (18). Application of BSA on both sides of the membrane did not alter the activation of TRAAK by a 50 mm Hg
pressure in the inside-out patch configuration (n = 6;
data not shown). Moreover, the addition of the phospholipase
A2 blockers 4-bromophenacyl bromide (30 µM)
and arachidonyltrifluoromethyl ketone (AACOCF3) (30 µM) to the cytosolic face of inside-out patches for 5 min
did not alter activation of TRAAK by a
50 mm Hg pressure (n = 5; data not shown).
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DISCUSSION |
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TRAAK is a member of the novel K+ channel family with two P domains and four transmembrane segments (1). Despite a similar membrane topology, the 2P/4TMS channels display little sequence identity (about 25%). When compared with TWIK-1 and TREK-1, TRAAK has a shorter amino-terminal region but an extended C terminus (3, 4). TRAAK has a large extracellular loop between M1 and P1, with a cysteine residue at position Cys52 analogous to the cysteine residue Cys69 involved in the disulfide-bridged homodimerization of TWIK-1 (8). TRAAK is only expressed in brain, retina, and spinal cord (1). The most intense levels of expression are present in the olfactory system, cerebral cortex, hippocampal formation, habenula, basal ganglia, and cerebellum. This laboratory demonstrated that TRAAK is opened by AA (1). The activation of TRAAK by AA is reversible, concentration-dependent, and direct (i.e. not via protein kinase C). Other polyunsaturated fatty acids also open TRAAK, while saturated fatty acids are without effect (1). TRAAK current is instantaneous and outwardly rectifying in a physiological K+ gradient. Finally, TRAAK is only inhibited by high concentrations of Ba2+ and is insensitive to the other classical K+ channel blockers, including tetraethylammonium, 4-aminopyridine, and Cs+ (1).
The anionic amphipath TNP has been shown previously to expand the exterior half of the lipid bilayer and thereby induce human erythrocytes to crenate (17). Moreover, TNP was shown to activate bacterial mechano-gated cationic channels, and this effect was attributed to membrane crenation (15). In the present study, we demonstrate that TNP is a strong activator of TRAAK. TRAAK opening by TNP is observed in the whole cell as well as in the excised outside-out patch configurations, suggesting a rather direct mechanism (i.e. without second messenger). Interestingly, no significant effect of TNP (at the concentration used) is observed under resting conditions in the inside-out patch configuration. This observation suggests that TNP may act preferentially via the external side of the membrane. It has been hypothesized previously that because of the negative charges (mainly phosphatidylserine) of the inner leaflet, anionic amphipaths may preferentially insert in the external leaflet of the plasma membrane (15, 17). The differential insertion of these amphipaths in the external lipid monolayer induces a curvature of the membrane which generates transversal forces that may alter channel activity. This model implies that TRAAK may be sensitive to mechanical forces transmitted via the membrane.
In every configuration tested, basal channel activity measured at atmospheric pressure was basically absent. In the inside-out patch configuration, TRAAK is opened by negative pressure, while it is opened by positive pressure in the outside-out patch configuration. These results suggest that TRAAK is selectively activated by a convex curvature of the membrane. This result fits well with the bilayer couple model of the membrane described above (15, 17). Channel activity was graded with applied pressure and was maintained throughout stimulation, demonstrating the absence of an adaptation process.
TRAAK can be opened by both arachidonic acid as well as membrane stretch. A mechanically sensitive phospholipase A2 (19) could therefore couple membrane stretch to TRAAK opening as suggested for stretch-activated K+ channels in gastric smooth muscle (18). Although the addition of lipid-free BSA as well as the phospholipase A2 (PLA2) inhibitors do not alter the activation of TRAAK by mechanical stimulation, we cannot entirely rule out such a mechanism.
Disruption of the cytoskeleton by either chemical or mechanical means (colchicine, cytochalasin D, or membrane excision) potentiates TRAAK opening by membrane stretch. These results suggest that mechano-gating does not require the integrity of the cytoskeleton. Moreover, these results imply that the activating force is coming directly from the bilayer membrane. Finally, the up-regulation of channel activity suggests that TRAAK is tonically repressed by the cytoskeleton. Dynamic regulation of cytoskeletal components through, for instance, phosphorylation/dephosphorylation is thus expected to have an important effect on the indirect regulation of TRAAK channel activity.
Opening of TRAAK is clearly facilitated at depolarized potentials
(between 25 and 50 mV). Depolarization lowers the threshold for
mechanical activation and increases the maximal channel activity (NPo).
The 2P/4TMS channels lack the positively charged S4 voltage sensor
found in Shaker-type K+ channels (7). Thus the region
responsible for the voltage-dependent modulation of TRAAK
remains to be determined. An important physiological implication of
these findings is that activation of TRAAK (induced mechanically or
chemically) will be mostly efficient at positive potentials
(i.e. during an action potential). The inside- and outside-out experiments indicate that TNP synergize with pressure activation. This result suggests that physiologically, mechanical activation of TRAAK may be most effective when endogenous amphipathic crenators are produced.
In conclusion, this report demonstrates that the mammalian neuronal
2P/4TMS K+ channel TRAAK belongs to the mechano-gated ion
channel family (9-16, 18, 20-23). Stretch-activated K+
channels have been described in several neuronal cell types, including
snail and Aplysia neurons as well as in rat hippocampal neurons
(20-24). The presence of stretch-activated K+ channels in
the growth cones of Lymnaea stagnalis neurons suggested that
these channels may be involved in the modulation of axonal pathfinding
and guidance (22). Indeed, growth cone motility is a mechanical process
where neurite membranes experience important tension changes.
Activation of TRAAK during cell locomotion may participate via membrane
hyperpolarization to the fine regulation of intracellular calcium,
actin-myosin contractions and thus neurite elongation. Finally, the
expression of TRAAK in dorsal root
ganglia2 may suggest its
possible role in more specialized sensory functions such as touch
detection and/or pain sensation. We previously identified a charged
region in the COOH terminus of TREK-1
(Arg297-Glu306:
RVISKKTKEE), which is critically involved in
channel activation by AA as well as mechanical stimulation. This region is conserved in TRAAK (Arg259-Glu268:
RAVSRRTRAE) and may also fulfill an
important function for TRAAK activation.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. A. Patel for critical and careful reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the CNRS.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.
To whom correspondence should be addressed: Institut de
Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-4-93-95-77-02 (or 03); Fax: 33-4-93-95-77-04; E-mail:
ipmc{at}ipmc.cnrs.fr.
The abbreviations used are: P, pore segment; TMS, transmembrane segment(s); AA, arachidonic acid; BSA, bovine serum albumin; TNP, trinitrophenol; WT, wild type; Npo, number of channels × open channel probability.
2 C. Heurteaux, personal communication.
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REFERENCES |
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