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J. Biol. Chem., Vol. 277, Issue 20, 17733-17742, May 17, 2002
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andFrom the Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908-0735
Received for publication, January 16, 2002, and in revised form, March 6, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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TASK-1 and TASK-3, members of the
two-pore-domain channel family, are widely expressed leak potassium
channels responsible for maintenance of cell membrane potential and
input resistance. They are sites of action for a variety of modulatory
agents, including volatile anesthetics and neurotransmitters/hormones,
the latter acting via mechanisms that have remained elusive. To clarify
these mechanisms, we generated mutant channels and found that
alterations disrupting anesthetic (halothane) activation of these
channels also disrupted transmitter (thyrotropin-releasing hormone,
TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C
terminus virtually abolished both anesthetic activation and transmitter
inhibition. The only sequence motif identified with a classical
signaling mechanism in this region is a potential phosphorylation site;
however, mutation of this site failed to disrupt modulation. TASK-1 and
TASK-3 differed insofar as a large portion of the C terminus was
necessary for the full effects of halothane and TRH on TASK-3 but not
on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels
were fully modulated by anesthetic and transmitter, and introduction of
the identified mutations either into the TASK-1 or the TASK-3 portion
of the channel was sufficient to disrupt both effects. Thus, both
anesthetic activation and transmitter inhibition of these channels
require a region at the interface between the final transmembrane
domain and the cytoplasmic C terminus that has not been associated
previously with receptor signal transduction. Our results also indicate
a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.
TASK1 channels are
members of the two-pore domain family of potassium channels, whose
presumed structure consists of two pore-forming regions flanked by four
membrane-spanning domains (1, 2). Their name (an acronym for
"TWIK-related acid sensitive
K+ channels") is derived from their
structural similarity to the first mammalian member of the two-pore
domain family to be characterized (TWIK-1), and from the fact that the
activity of these channels is sensitive to changes in extracellular pH
in the physiological range (3). Like other two-pore domain family
members, these channels show little time or voltage dependence. Thus
they have characteristics of leak K+ channels, generating
background currents that contribute to membrane potential and the
shaping of cell excitability.
The first TASK channel to be cloned, TASK-1 (designated KCNK3 by the
Human Genome Organization), has been established as a site of
modulation by a variety of agents, including hydrogen ions, oxygen,
volatile anesthetics, and hormones/neurotransmitters (4). This
modulation appears to be important in a number of physiological
contexts, including the regulation of breathing (5, 6), control of
aldosterone secretion (7), and transmitter and anesthetic regulation of
neuronal activity (8-10).
In addition to TASK-1, four other two-pore domain channel genes have
also been given the name TASK; two of these (TASK-2 (KCNK5) and
TASK-4/TALK-2 (KCNK17)) are only distantly related to TASK-1 (11-13),
and one (TASK-5 (KCNK15)) has not produced functional channels in
heterologous expression systems (14-17). The fourth, TASK-3 (KCNK9),
is >50% identical to TASK-1 at the amino acid level, and in
whole-cell recordings the two channels have similar physiological
properties but different pH sensitivities (3, 18-21). They are
co-expressed in a number of different neuronal populations (16, 22, 23)
and can form heterodimers when expressed in Xenopus oocytes
(24).
For both TASK-1 and TASK-3, a molecular basis has been established for
modulation by extracellular protons, as pH sensitivity is essentially
abolished by replacing a histidine residue adjacent to the presumed
selectivity filter region of the ion-conducting pore (18, 21, 25).
Progress also has been made in identifying channel regions required for
the actions of volatile anesthetics on human TASK-1 (26) (see below).
However, the mechanisms underlying effects of neurotransmitters on
these channels are poorly understood. For TASK-1 (and recently TASK-3)
(24), inhibition via G cDNA encoding rat TASK-1 (GenBankTM accession
number AF031384) and rat TASK-3 (GenBankTM accession number
AF391084) were obtained from Andrew Gray (University of California, San
Francisco) (27) and Donghee Kim (the Chicago Medical School) (18). Both
cDNAs were subcloned into a mammalian expression vector
(pcDNA3, Invitrogen). Portions of mouse TREK-1 (29)
(GenBankTM accession number U73488.2, obtained by PCR from
mouse cerebellar RNA (22)) and mouse TRAAK (30) (GenBankTM
accession number AF056492, obtained from Andrew Gray) were used to make
chimeric constructs. Site-specific mutations and chimeras were
generated by PCR using the overlap-extension method (31). A
TASK-1/TASK-3 tandem dimer also was produced using PCR. The resulting
channel contains a three-residue (glycine-serine-alanine) linker
region, placed after the C-terminal amino acid of TASK-1 and fused to
the start methionine of TASK-3. Mutations were introduced into this
dimeric construct by ligation of fragments excised from cDNAs
coding for the relevant monomeric mutant channels.
For both TASK-1 and TASK-3, a hemagglutinin epitope tag was added to
the wild-type channel and to a number of different mutant constructs.
These included the four TASK/TREK chimeric channels (i.e.
TASK-1 and TASK-3 containing either a portion of the TREK-1 C terminus
or six residues substituted from TREK-1, see "Results"). The
sequence encoding the epitope was excised from pKH3 (32) (obtained from
Ian Macara, University of Virginia). Each construct was modified by PCR
to allow for insertion of the epitope upstream of the start methionine,
with an intervening three-residue (glycine-serine-alanine) linker. For
all six of these epitope-tagged constructs, the responses to halothane
and TRH were not different from the responses of constructs without the
epitope, and therefore the data were pooled. All subsequent mutations,
including the point mutations for TASK-1 and C-terminal deletions for
TASK-3, were made in the context of the epitope-tagged channels.
Plasmids were prepared by column elution (Endo-Free Plasmid Maxi Kit,
Qiagen, Inc.) and further purified by phenol/chloroform extraction. All
constructs were fully verified by sequence analysis.
HEK 293 cells stably expressing the TRH-R1 receptor (E2 cells (33),
obtained from Graeme Milligan) were grown under standard conditions and
transfected by calcium phosphate precipitation. Cells were
co-transfected with green fluorescent protein (pGreenLantern, Invitrogen) at a ratio of 6 µg of channel cDNA to 1 µg of green fluorescent protein. DNA precipitant was washed from cells 1-4 h after
transfection, and cells were plated onto polylysine-coated coverslips
4-15 h later. Electrophysiological recordings were performed within
24 h of transfection. Cells were visualized by differential
interference contrast optics and by fluorescence using a standard
fluorescein isothiocyanate filter set. Care was taken to select cells
for recording that were not coupled to neighboring cells. Bath
recording solution consisted of (in mM) 130 NaCl; 3 KCl; 2 MgCl2; 2 CaCl2; 1.25 NaH2PO4; 10 HEPES; and 10 glucose. pH was
adjusted using NaOH and HCl. A continuous gravity perfusion system
(flow, 5-7 ml/min) was used to change solutions.
Patch electrodes were pulled from borosilicate glass to resistances of
2-7 megohms and coated with Sylgard 184 (World Precision Instruments).
Internal solution contained (in mM): 120 potassium methane
sulfonate; 4 NaCl; 1 MgCl2; 0.5 CaCl2; 10 HEPES; 10 EGTA; 3 MgATP; 0.3 GTP-Tris, pH 7.2. Recordings were obtained
using an Axopatch 200A amplifier and digitized with a Digidata 1200 A/D
converter (Axon Instruments). Series resistance was compensated by
60-75% and was carefully monitored throughout the recordings to
ensure accurate compensation. Voltage commands were applied and
currents recorded and analyzed using pCLAMP software (Axon). Cells were
held at For analysis, slope conductance was evaluated by linear fits to
currents from TRH (American Peptide) was used at 0.2 µM. Halothane was
bubbled into the perfusate via a vaporizer (Ohmeda, Austell, GA) using
a room air/gas mixture (21% O2/balance N2).
Aqueous concentrations were determined by gas chromatographic analysis
of solutions taken from the recording chamber (34).
TASK Channels: Regulation by Extracellular pH, Volatile
Anesthetics, and Neurotransmitters--
TASK-1 and TASK-3 are subject
to modulation by multiple agents; examples of this modulation are shown
in Fig. 1. HEK 293 cells stably
expressing the TRH-R1 receptor were transiently transfected with rat
TASK-1 or TASK-3 and tested for responsiveness to changes in pH, as
well as to applications of the volatile anesthetic halothane and the
neuropeptide TRH. Cells were subjected to voltage ramps at 5-s
intervals (see "Experimental Procedures") to assess changes in
whole-cell conductance. As expected, decreasing extracellular pH from
7.3 to 5.9 resulted in a complete inhibition of both TASK-1 (Fig.
1A) and TASK-3 (Fig. 1B). Switching to an
alkalized extracellular solution (pH 8.4) restored the currents. In the
case of TASK-1, alkalization induced an increase in conductance
relative to physiological pH (pH 7.3), whereas for TASK-3 there was
little change. These distinct responses to bath alkalization reflect a
difference in the pH sensitivities of the two channels (pK
~7.2-7.5 for TASK-1 versus pK ~6.0-6.7 for
TASK-3) (3, 10, 18-21).
After bath pH was raised, a clinically relevant dose of halothane (0.3 mM) activated the channels further. For TASK-1 the activation represented an ~50% increase over the pH-sensitive conductance (52.5 ± 3.6%; n = 5), with an even
larger activation of TASK-3 (134.6 ± 20.0%; n = 6). This value for rat TASK-1 is consistent with data for the human
channel (26), but the rat TASK-3 response was somewhat greater than
that reported for the corresponding human orthologue (20). For both
channels, application of TRH in the continued presence of halothane
resulted in a robust inhibition of the total (i.e.
halothane- and pH-sensitive) current (TASK-1, 94.2 ± 1.5%
inhibition, n = 6; TASK-3, 86.4 ± 1.6%,
n = 3). TASK channel inhibition by TRH is consistent
with our previous work (10) and with results showing inhibition of
these channels by a number of other G Removal of the C Terminus of TASK-1 Fails to Disrupt Modulation by
Halothane and TRH--
To identify regions of the channel necessary
for receptor-mediated inhibition of TASK-1, intracellular domains of
the channel were targeted for mutation. The presumed intracellular
domains of TASK-1 include a minimal (seven-residue) N terminus, a
30-40-residue linker between transmembrane domains two and three (the
M2-M3 linker), and a C terminus of ~160 residues. The first domain
targeted for mutation was the C terminus, which was removed by
introducing a stop codon after the fourth transmembrane domain (after
residue 248). This mutation left a core channel with currents that were indistinguishable from those of wild-type TASK-1 (Fig.
2B). As expected from previous
studies of the human orthologue (26), this core channel was fully
responsive to changes in pH and to halothane (Fig. 2B). In
addition, we found that this mutant was fully inhibited by bath
application of TRH (Fig. 2B). Thus the C terminus is not
necessary for either anesthetic activation or transmitter inhibition,
even though this region contains all of the identified consensus motifs
for phosphorylation by protein kinase C and protein kinase A, as well
as for tyrosine phosphorylation (3, 19, 27, 35).
We also tested whether mutations in the M2-M3 cytoplasmic linker region
could perturb receptor modulation. Chimeras were generated in which a
20-residue section of rat TASK-1 (amino acids 132-151) was replaced
with a corresponding section of mouse TREK-1 (residues 181-203) or of
mouse TRAAK (residues 143-165), but these mutant constructs failed to
make functional channels (data not shown). Mutations also were
introduced in the M2-M3 linker domain by substituting alanine residues
for a threonine at position 134 and for arginines at positions 137, 142, 145, and 150. For each of these point mutants, the responses to
changes in pH, halothane, and TRH were indistinguishable from wild-type
TASK-1 (data not shown).
A Six-residue Segment at the Beginning of the Cytoplasmic C
Terminus of TASK-1 Is Necessary for Both Halothane Activation and TRH
Inhibition of the Channel--
To probe further into the
transmembrane-spanning regions of the channel, we took advantage of
earlier mutagenesis of human TASK-1 (26). Results from this previous
work indicated that further deletion of the C terminus (by introducing
a stop codon after residue 242, i.e. six residues upstream
of our C-terminal deletion) resulted in a loss of channel function.
However, replacing the C terminus of human TASK-1 (starting from
residue 243) with the corresponding region from TREK-1 resulted in a
construct generating functional channels that were not activated by
volatile anesthetics (26). We made a similar construct using rat
TASK-1, but we used a truncated portion of the C terminus from TREK-1
(residues 293-327) lacking regions critical for modulation of the
channel by phosphorylation (36, 37). As shown in Fig 2C,
this TASK-1/TREK-1 chimeric construct produced currents that were
pH-sensitive but not activated by halothane. In fact, halothane caused
an inhibition of these currents (see below). The same chimeric channel
was essentially unaffected by application of TRH (Fig. 2C).
These results suggested that the six-amino acid stretch of TASK-1
(residues 243-248; VLRFMT) that was contained in the C-terminal
deletion mutant but not in the TASK-1/TREK-1 chimera is critical for
both halothane activation and transmitter inhibition of the channel. To
test this possibility, the corresponding six amino acids from TREK-1
(residues 293-298, GDWLRV, without the rest of the TREK-1 C-terminal
region) were substituted into the full-length TASK-1. The resulting
construct generated pH-sensitive currents, but once again the channels
were not activated by halothane and were only minimally inhibited by TRH (15.6 ± 2.2%).
As noted above, introduction of residues from TREK-1 into the proximal
C terminus of TASK-1 created a channel that was inhibited, rather than
activated, by halothane. For the construct containing a six-residue
TREK-1 substitution, the inhibition was substantial (30.6 ± 0.2%
of the pH-sensitive conductance). It also was
dose-dependent, being much greater at higher concentrations
of halothane (reaching 85.2 ± 1.3% inhibition at 1 mM). One possible interpretation of this result is that
there are multiple and competing sites for halothane effects on TASK-1.
Consistent with this view, we found that halothane activation of the
wild-type channel diminished with time (see Fig. 1A and Fig.
2A), particularly at higher concentrations of the anesthetic
(data not shown). An analysis of the basis for the biphasic response to
halothane will require further investigation. In any case, mean data
showing peak activation by halothane (derived from the maximal
conductance obtained during halothane treatment, Fig. 2E)
demonstrated that channel activation was eliminated in these two
TASK-1/TREK-1 chimeras. Likewise, TRH inhibition also was abolished for
the two chimeric channels (Fig. 2F). Therefore, this
six-residue segment of TASK-1 (243-248) is essential for both
halothane activation and TRH inhibition of the channel.
The region we identified as essential for transmitter inhibition of
TASK-1 was not expected, because initial descriptions did not identify
consensus motifs for receptor signaling in this part of the channel (3,
19, 27, 35). However, the sequence VLRFMT does contain a threonine
(residue 248) that could serve as a site of phosphorylation by
calcium-calmodulin-dependent protein kinase (38).
Nonetheless, substitution of the corresponding valine from TREK-1
failed to affect modulation by halothane or TRH (Fig.
3, A, C,
and D), indicating that phosphorylation of this residue does
not participate in anesthetic activation or transmitter inhibition of
TASK-1. Thus, there are no known motifs in this region that would
suggest a modulatory mechanism underlying anesthetic and transmitter
effects.
We also targeted an arginine at position 245 for mutation, because this
positively charged residue could be important for interaction with the
head groups of phospholipids implicated in receptor signaling, such as
phosphoinositides. As shown in a representative cell (Fig.
3B) and in the mean data (Fig. 3, C and
D), substitution of the corresponding residue from TREK-1
(tryptophan) resulted in a channel in which the actions of both
halothane (29.7 ± 3.7% peak activation) and TRH (29.6 ± 4.0% inhibition) were partially attenuated (to 57 and 35%,
respectively, of levels for the wild-type channel). Grouped comparisons
combining all TASK-1 constructs indicated that both effects on this
mutant were smaller than those on the wild-type channel (and on the
C-terminal deletion and T248V mutants) and greater than the effects on
the two TREK-1 substitution mutants. So, anesthetic activation and
neurotransmitter inhibition of this mutant were partial in both cases,
suggesting once again that these two forms of modulation share
structural requirements at the channel.
The Corresponding Six Residues Critical for Transmitter Inhibition
of TASK-1 Are Also Essential for Modulation of TASK-3 by Halothane and
TRH--
The protein sequences of rat TASK-1 and TASK-3 differ
substantially in the C terminus (18), but the six-residue sequence necessary for halothane activation and TRH inhibition in TASK-1 also is
contained in TASK-3 with only one minor difference: TASK-3 contains a
leucine instead of methionine at position 247, resulting in a sequence
of VLRFLT (residues 243-248). To test if this region is required for
halothane activation and TRH inhibition of TASK-3, the same mutations
used for TASK-1 were introduced into TASK-3, with slightly different
results (Fig. 4). As with TASK-1, removal of the C terminus from TASK-3 did not abolish halothane activation or
TRH inhibition (Fig. 4B), although both of these effects
were diminished (Fig. 4, E and F, see below).
Substitution with residues from TREK-1, either replacing the C terminus
or only replacing residues 243-248, resulted in a loss of halothane
activation (to less than 15% of the wild-type levels) and TRH
inhibition (to less than 10% of wild-type levels), indicating that
this six-residue stretch is essential for effects of these compounds on
both TASK-1 and TASK-3.
Both Halothane Activation and TRH Inhibition Are Increasingly
Attenuated by Removal of Successive Portions of the TASK-3 C
Terminus--
Removal of the C terminus of TASK-3 partially attenuated
halothane activation and TRH inhibition, indicating that a portion of
this domain is necessary for the full effects of these agents. We
investigated this issue in more detail using two more constructs, one
in which the final 11 residues were deleted ( Introduction of a Single Mutated Domain (Substitution of Six
Residues from TREK-1) into a Tandem-linked Channel Is Sufficient to
Disrupt the Effects of Halothane and TRH--
As noted above, TASK-1
and TASK-3 are co-expressed in a number of different cell types,
suggesting the possibility that they form heterodimeric channels (16,
22, 23). To test for neurotransmitter and anesthetic effects on such
channels, we generated a construct in which the C terminus of TASK-1
was fused to the N terminus of TASK-3 (via a short linker region, see
"Experimental Procedures"). As shown in Fig.
6, these heterodimeric channels were
fully activated by halothane (150.8 ± 12.6%) and inhibited by
TRH (82.4 ± 5.4%). As was found for TASK-3, the effect of
halothane on the tandem construct was more potent than on TASK-1 (see
Figs. 2 and 3). We also made constructs with substitutions of the six
residues from TREK-1 (identified above) into either the TASK-1 or the
TASK-3 portion of the channel. This created channels with only a single mutated domain, as opposed to un-linked channels, which (because they
assemble as dimers (25)) contain two mutated domains per channel. One
might predict that a single mutated domain would be only half as
effective in attenuating the actions of halothane and TRH. Instead, we
found that the effects of these compounds on the singly mutated
tandem-linked channels were reduced to levels very similar to those
seen for the mutated un-linked channels (Fig. 6, D and
E). Thus a single substitution of six residues from TREK-1,
into either the TASK-1 or the TASK-3 portion of the heterodimeric
channel, was sufficient to disrupt the effects of halothane and
TRH.
We generated mutations in TASK-1 and TASK-3 in
order to identify regions critical for neurotransmitter inhibition of
these channels, and we found that alterations disrupting anesthetic activation also interfered with neurotransmitter inhibition. For TASK-1, removal of the cytoplasmic C terminus failed to affect either
form of regulation, even though this domain contains in its primary
sequence a number of motifs associated with receptor signal
transduction (3, 19, 27, 35). Also, mutations in the M2-M3 cytoplasmic
linker region failed to disrupt modulation. Instead, a region not
associated with known signal transduction-related motifs, at the
interface between the presumed fourth transmembrane domain and the
cytoplasmic C terminus, was critical for both halothane activation and
TRH inhibition of TASK-1 currents. Substitution of six residues from
TREK-1 into this region (in the context of full-length TASK-1 or in the
replacement of the C terminus with a section from TREK-1) essentially
abolished both effects. Mutagenesis of TASK-3 produced similar although
not identical results. We used the same strategy (substitution of
residues from TREK-1) and found that the corresponding region in TASK-3
was critical for modulation by halothane and TRH. However, a large
portion of the C terminus of TASK-3 was necessary for the full effects of these agents.
Identification of a Domain in TASK-1 and TASK-3 Critical for
Transmitter Inhibition--
We identified a region in both TASK-1 and
TASK-3 at the cytoplasmic face of the fourth transmembrane domain that
is critical for channel inhibition by transmitters. Similar or
identical sequence is present in the corresponding regions of presumed
channels from Caenorhabditis elegans (GenBankTM
accession number AF083652) and Drosophila melanogaster
(GenBankTM accession numbers AAF54970 and AAF54374),
indicating that this sequence is ancient and may have the same
importance in regulating channel activity in those species as well.
Also, it has been noted that an analogous region (immediately following the S6 transmembrane domain) is likely to be important for transmission of intracellular gating signals in voltage- and cyclic nucleotide-gated channels (39).
The importance of this region was unexpected, because the relevant
sequence is not identified with any known signal transduction motifs.
However, this fact is consistent with previous negative findings
regarding second messenger pathways responsible for TASK channel
inhibition by neurotransmitters and hormones (3, 19, 27, 28).
Inhibition of TASK-1 and TASK-3, or native counterparts, has been
demonstrated for a number of different G protein-coupled receptors
besides the TRH-R1 receptor, all of which specifically couple to G
proteins of the
Although most of the evidence has been negative, two proposed signaling
mechanisms have received some positive support. One proposal suggests
that the mechanism does not involve pathways downstream of PLC but
instead results from PLC-mediated depletion of PI(4,5)P2
(28), as has been suggested for receptor-mediated inhibition of members
of the inwardly rectifying K+ channel family (41-43).
Supporting a role for PLC in receptor-mediated TASK-1 inhibition, the
G
A second signaling pathway proposed for receptor actions on TASK-1
involves the endocannabinoid anandamide, which inhibits human TASK-1
currents at sub-micromolar levels, although it is less potent at
inhibiting TASK-3 (40). This compound can be released in neurons by
activation of G Convergence of Anesthetic and Transmitter Effects--
Our results
suggest an unexpected convergence of effects of anesthetics and
neurotransmitters at the molecular level, because the same region of
the channel was critical for both anesthetic activation and transmitter
inhibition. Moreover, mutations resulting in partial blockade of one
effect also produced partial obstruction of the other. These mutations
included an arginine-to-tryptophan substitution in TASK-1, and TASK-3
constructs in which increasing portions of the C terminus were removed.
The TASK-3 C-terminal deletion mutants were especially interesting in
this regard, because halothane and TRH shared the same rank order of
potency in their effects on these channels, with increasing lengths of
the C terminus resulting in incrementally greater effects for both
anesthetic and transmitter. The data clearly indicate that these agents
share structural requirements at the channel and may act via a common mechanism, albeit with opposite effects.
It is not clear if the identified convergence occurs directly at the
channel or involves interaction with some upstream intermediary signaling molecule(s). A direct anesthetic effect has been suggested, because halothane activated TASK-1 channels in excised patches (26).
Therefore the effects of these compounds, if not direct, are at least
likely to be closely associated with the channel. If the receptor
mechanism involves depletion of an endogenous channel-activating
compound (such as PI(4,5)P2) (28), then it might be
proposed that volatile anesthetics substitute for this compound, acting
as agonists at the same site on the channels. If this were true, then
it would be expected that depletion of the endogenous compound would be
compensated by anesthetics and that receptor-mediated channel
inhibition therefore would be offset in their presence. However, our
data indicate that TRH inhibition is fully effective during the
continued application of halothane (see Fig. 1) and therefore do not
support this model.
Modulation of TASK-1/TASK-3 Heterodimers--
As noted in the
Introduction, mRNAs encoding TASK-1 and TASK-3 are co-expressed in
a number of different neuronal populations (16, 22), including cells
known to express functional TASK-like currents (8-10, 23). Recently,
heterodimerization of these two channel subunits was demonstrated in
Xenopus oocytes (24). Whether these two genes form
heterodimers in vivo remains to be established, but it is
possible that such heterodimeric channels have properties that are
emergent, as opposed to having properties common to those of TASK-1 and
TASK-3. Here we show that heterodimers composed of TASK-1 and TASK-3
(in a forced tandem-linked conformation) are regulated by TRH and
halothane. This regulation apparently occurs via the same mechanism as
in un-linked homodimeric channels, because substitution of individual
domains into either the TASK-1 or the TASK-3 portion of the channel
resulted in nearly complete disruption of the effects of halothane and
TRH. The magnitude of the TRH effect was not different between the
un-linked channels and the tandem heterodimer. However, halothane
activated TASK-3 and the tandem construct much more potently than
TASK-1, with a nearly 3-fold difference in the degree of activation.
The difference in magnitude of the halothane effect raises the question
of how these values compare with those from native cells in which both
TASK-1 and TASK-3 are expressed, such as motoneurons (10, 15, 16, 22).
In previous recordings of hypoglossal motoneurons (HMs) from our
laboratory, a pH- and neurotransmitter-sensitive leak K+
current was identified as resulting from expression of TASK-1 (10),
because the pH sensitivity (pK ~7.4) of the current was inconsistent with any other known leak K+ channel
(including TASK-3). This motoneuronal leak current also was activated
by volatile anesthetics (9), once again consistent with the expression
of TASK-1. However, in the present study we found that TASK-1 was only
activated by ~50% using 0.3 mM halothane; in HMs, the
same concentration of halothane produced an activation that was
~100% relative to the pH-sensitive current (9). Thus, although the
motoneuronal experiments were performed somewhat differently from those
of the present report, the potency of the anesthetic effect appears to
have been larger than would be expected solely from the expression of
TASK-1. Therefore it is possible that the magnitude of the anesthetic
effect in HMs results from the activation of some combination of
TASK-1, TASK-3, and/or TASK-1/TASK-3 heterodimers. The existence of
heterodimers in these neurons would appear to be even more likely if
the heterodimeric channels had a pH sensitivity close to that of TASK-1
(and not TASK-3), given the TASK-1-like pH sensitivity of the currents
observed in HMs.
In any case, here we demonstrate that a domain contained in both TASK-1
and TASK-3 is necessary for the effects of transmitters and
anesthetics. This domain is not associated with any previously identified signaling motifs, consistent with the fact that the mechanism for transmitter regulation of these channels has not been
elucidated. Because the same domain is required for transmitter and
anesthetic modulation of TASK-1/TASK-3 heterodimers, the present results are equally relevant to potential heterodimeric channels existing in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11-coupled receptors has been
demonstrated for the cloned channels and for channels with similar
properties in native systems (7, 8, 10). However, experiments have
failed to implicate downstream effectors commonly associated with these
receptors (3, 19, 27, 28). Here, we report the results of experiments
in which we used mutagenesis of TASK-1 and its homologue TASK-3 to
investigate the molecular bases for neurotransmitter inhibition of
these channels in HEK 293 cells stably expressing the TRH-R1 receptor.
We found that mutations disrupting anesthetic activation of these
channels also disrupt transmitter inhibition; both of these effects
require a region of the channel that does not contain motifs
suggestive of a traditional signaling pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
60 mV, and depolarizing ramps (0.2 V/s, from 130 to +20 mV)
were applied at 5-s intervals.
130 to 60 mV. For each cell, effects of halothane and
TRH were normalized to the pH-sensitive conductance (i.e. the conductance at pH 8.4, less the conductance obtained when the
channels were fully inhibited, pH 5.9). Data are presented as
means ± S.E. Statistical analyses of anesthetic and transmitter effects on wild-type and mutant channels were performed by one-way analysis of variance (ANOVA) using a statistics software package (SigmaStat, Jandel Scientific). Pairwise comparisons were made using
the Student-Newman-Keuls method, with p < 0.05 as
criteria for acceptance as statistically significant. Linear regression analysis was performed using the same software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Opposing regulation of pH-sensitive
K+ currents by halothane and TRH in cells expressing TASK
channels. HEK 293 cells stably expressing the TRH-R1 receptor were
transfected with rat TASK-1 (A) or TASK-3 (B) and
recorded by whole-cell voltage clamp. Cells were held at 60 mV and
subjected to voltage ramps (0.2 V/s) at 5-s intervals. Conductance
(calculated using linear fits to currents obtained between 130 and
60 mV) was plotted over time to reveal changes in response to
variations in pH (7.3, 5.9, and 8.4), as well as to applications of
halothane (0.3 mM) and TRH (0.2 µM).
Insets show current traces from the indicated time points.
TASK-1 and TASK-3 channels were inhibited at acidified pH (pH 5.9) and
activated at alkalized pH (pH 8.4; trace a). Application of
halothane caused further channel activation beyond that seen in the
alkalized bath, resulting in an increase in the whole-cell conductance
(trace b). In the case of each channel, application of TRH
in the continued presence of halothane induced a potent inhibition
(trace c) of both the basal (pH-sensitive) and
halothane-activated current.
q/11-coupled
receptors (8, 24, 28).
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Fig. 2.
C-terminal mutations abolish halothane
activation and TRH inhibition of TASK-1. A, responses
of a cell expressing wild-type TASK-1 to changes in pH, as well as to
applications of halothane and TRH. Whole-cell conductance was inhibited
following bath acidification (from pH 7.3 to pH 5.9) and activated
following alkalization (to pH 8.4). Halothane caused a further increase
in conductance, whereas TRH (applied after washing halothane) induced a
robust channel inhibition. B, deletion of 163 residues
(249-411) from the C terminus failed to affect the response to
halothane or TRH. C and D, substitution of a
section from the C terminus of TREK-1 (C), including a
replacement of an additional six TASK-1 residues (243-248) closer to
the presumed transmembrane domain, resulted in a channel that was
neither activated by halothane nor inhibited by TRH. Furthermore,
replacing these six residues in the full-length TASK-1 (D)
also resulted in a channel that was not activated by halothane and had
very little response to TRH. Both of these TASK-1/TREK-1 chimeric
channels actually were inhibited by halothane; this inhibition may
occur via a separate mechanism (see "Results"). E and
F, mean data demonstrate that both halothane activation
and TRH inhibition require residues 243-248 of TASK-1. Peak halothane
activation (derived from the maximal conductance obtained during
halothane treatment (E)) and TRH inhibition (F)
are plotted as a percentage of the pH-sensitive conductance
(n 
5 for each data point). Statistical significance
was established using one-way ANOVA; asterisks indicate
difference from wild-type (WT) TASK-1 (p < 0.05, pairwise comparison using the Student-Newman-Keuls test).
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Fig. 3.
Point mutations within the proximal C
terminus of TASK-1 have similar effects on the responses to halothane
and TRH. A, mutation of threonine 248 to valine failed
to diminish the effects of either halothane or TRH. B,
similar substitution of arginine 245 to the corresponding tryptophan
from TREK-1 resulted in a partial attenuation of both halothane
activation and TRH inhibition. C and D, mean
data show statistically significant effects of R245W mutation (as
determined by one-way ANOVA between the three groups; n 6 for each group); asterisks indicate difference from
wild-type (WT) TASK-1 (p < 0.05).
View larger version (25K):
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Fig. 4.
C-terminal mutations also attenuate halothane
activation and TRH inhibition of TASK-3. A, like
TASK-1, wild-type TASK-3 was activated by halothane and inhibited by
TRH. B, deletion of 148 residues from the C terminus of
TASK-3 (249-396) resulted in a construct with pH-sensitive currents
that were still activated by halothane and inhibited by TRH, although
both effects were partially attenuated. C and
D, replacement of the TASK-3 C terminus with residues
from TREK-1 (C), or replacement of six TASK-3 amino acids
(243-248) proximal to the transmembrane region (D),
resulted in channels with little response to halothane or TRH.
E and F, averaged responses of wild-type TASK-3
and the three different mutant channels to halothane (peak activation,
E) and TRH (percent inhibition, F) are plotted as
a percentage of the pH-sensitive conductance (n 6 for each data point). Statistical significance was established using
one-way ANOVA; asterisks indicate difference from wild-type
TASK-3 (p < 0.05).
386-396) and another
in which 92 residues were removed (305-396). These were compared
with the initial C-terminal deletion construct (see Fig. 4), in which
148 residues had been eliminated (249-396). As shown in Fig.
5, deletion of 11 residues (386-396)
failed to attenuate halothane activation and TRH inhibition; the
effects on this mutant were not different from those on the wild-type
channel. However, removal of a greater portion of the C terminus
(305-396) resulted in partial but significant decreases in both
effects (to 68 and 76% of the wild-type channel). The actions of both
compounds were nevertheless significantly larger than those on the
249-396 construct, which were diminished to 43% (halothane
activation) and 59% (TRH inhibition) of wild-type levels. Thus there
were incremental decreases in the effects of halothane and TRH upon
removal of 92 and 148 residues of the C terminus of TASK-3. Once again,
the results for halothane were correlated with those for TRH. This is
represented graphically in Fig. 5C, in which halothane
activation is plotted against TRH inhibition for individual cells.
Linear regression analysis of these values indicates that halothane
activation and TRH inhibition were highly correlated
(R2 = 0.669, p < 0.0005).
View larger version (12K):
[in a new window]
Fig. 5.
Both halothane activation and TRH inhibition
are increasingly attenuated by removal of successive portions of the TASK-3 C terminus. A and B,
constructs were generated by removal of different lengths of the TASK-3
cytoplasmic C terminus. Deletion of this entire region ( 249-396,
see Fig. 4) resulted in partial loss of the effects of halothane and
TRH. Removal of a smaller portion of the C terminus (92 residues,
305-396) resulted in a partial recovery of these effects, whereas
removal of only 11 residues (386-396) resulted in effects of
halothane and TRH that were not different from those of the wild-type
channel. For both halothane and TRH, there were significant differences
in effects between each of the groups (one-way ANOVA, n 5 for each group). *, different from 386-396; #, different from
249-396. C, correlation of the magnitudes of
halothane and TRH effects. Individual cells (open symbols)
transfected with the three different TASK-3 C-terminal deletion mutants
(squares, 249-396; circles, 305-396; and
triangles, 386-396) are plotted with respect to
halothane activation and TRH inhibition. Mean data (± S.E.) for each
construct are represented as filled symbols.
Linear regression analysis of the individual data points (solid
line) indicated a significant correlation between the two effects
(p < 0.0005).
View larger version (21K):
[in a new window]
Fig. 6.
Introduction of a single mutated domain
(substitution of six residues from TREK-1) into a tandem linked channel
is sufficient to disrupt the effects of halothane and TRH.
A, a tandem heterodimeric construct was generated by linking
the C terminus of TASK-1 to the N terminus of TASK-3. This construct
was fully activated by halothane and inhibited by TRH. B and
C, the six residues identified as critical for
halothane activation and TRH inhibition in the context of wild-type
TASK-1 and TASK-3 were introduced into the tandem construct, either in
the TASK-1 (B) or the TASK-3 (C) portion of the
channel. D and E, mean data (± S.E.) show
that either mutation was sufficient to reduce the effects of halothane
and TRH to an extent similar to that seen for un-linked channels.
Asterisks indicate significantly different from the
non-mutated tandem construct.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11 family (7, 8, 10, 24). The
downstream signaling pathway most commonly associated with activation
of these receptors entails hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC),
resulting in production of inositol 3,4,5-trisphosphate and
diacylglycerol. However, initial reports (3, 19, 27) and a more
complete study (28) failed to implicate the products of this lipid
hydrolysis, nor did they implicate downstream signaling events,
including release of calcium from intracellular stores and activation
of protein kinase C. Other signaling pathways initiated by
Gq/11-coupled receptors also do not appear to be
involved, including tyrosine kinase activation (28), production of
arachidonic acid via phospholipase A (36, 40), and activation of Rho
kinase.2
i/o-coupled M2 muscarinic receptor was able
to inhibit TASK-1 currents only upon overexpression of
PLC
2, an isoform that can be activated by
Gi/o-coupled receptors. In addition, the PLC inhibitor
U73122 reduced receptor effects on TASK-1 in Xenopus
oocytes, and the phospholipid kinase inhibitor wortmannin (used at
concentrations high enough to block phosphatidylinositol 4-kinases)
slowed recovery from receptor inhibition, presumably as a result of a
reduced ability of the cells to replenish PI(4,5)P2 levels
(28). However, the pharmacological evidence has not been universally
positive. In HEK 293 cells, U73122 (but not its inactive analogue
U73343) partially blocked TRH inhibition, but another PLC blocker,
D609, was ineffective.2 Also, treatment of cerebellar
granule neurons with U73122 failed to prevent receptor-mediated
inhibition of I(K)SO, a native correlate of TASK channels
(44). In any case, the possibility that PI(4,5)P2 depletion
contributes to receptor inhibition of TASK-1 and TASK-3 awaits more
experimentation, such as testing for channel activation by the compound itself.
q/11-coupled receptors (45), making it an
appealing candidate for mediation of receptor inhibition of TASK-1.
However, in their examination of the effects of this lipid on human
TASK-1, Maingret et al. (40) found that substitution of the
C terminus of TREK-1 into this channel did not prevent anandamide
inhibition. In the present study, we show that a similar substitution
did prevent receptor-mediated inhibition of rat TASK-1 and TASK-3.
Therefore, these two sets of data dissociate receptor effects from
those of anandamide and do not support a role for anandamide in
receptor inhibition of TASK channels.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Andrew T. Gray and Donghee Kim for gifts of cDNAs and Graeme Milligan for the TRH receptor-expressing cell line. We also thank Guillermo Solórzano, Jeremy F. Reed, and Qiubo Lei for assistance with the generation and preparation of mutant constructs, and Jay E. Sirois and Allison P. Berg for helpful comments on the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant NS33583 (to D. A. B.).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: Dept. of Pharmacology,
University of Virginia Health System, P. O. Box 800735, Charlottesville, VA 22908-0735. Tel.: 804-982-4466; Fax: 804-982-3878; E-mail: emt3m@virginia.edu.
Published, JBC Papers in Press, March 8, 2002, DOI 10.1074/jbc.M200502200
2 E. M. Talley and D. A. Bayliss, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: TASK, TWIK-related acid-sensitive K+ channel; TRH, thyrotropin-releasing hormone; TREK, TWIK-related K+ channel; HEK, human embryonic kidney; PI(4, 5)P2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; ANOVA, analysis of variance; HMs, hypoglossal motoneurons.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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