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J Biol Chem, Vol. 273, Issue 44, 28868-28872, October 30, 1998
From the Department of Molecular Physiology and Biological Physics,
University of Virginia, Charlottesville, Virginia 22906-0011
The removal of nucleotides from the solution
bathing the inner face of excised patches of frog atrial membranes was
found to activate muscarinic K+ channels in the
absence of agonists. Channel activation was also observed in
Mg2+-free solutions and blocked by low (0.1-10
µM) concentrations of GDP or GTP. After full activation
was achieved, channel openings were abolished by the application of
GDP-bound G The muscarinic K+ channel
(KACh)1 is an
inwardly rectifying, potassium-selective channel whose activation by
agonist-bound receptors is mediated by the Atrial myocytes were obtained from bullfrog hearts by digestion
with collagenase and trypsin (9). Pertussis toxin was a gift from Dr.
Erik Hewlett (University of Virginia). G protein subunits
Single channel activity in excised, inside-out atrial membrane patches
was measured, stored, and analyzed essentially as described previously
(10). The external (pipette) solution contained 0.1 M KCl,
2.5 mM CaCl2, and 20 mM HEPES, pH
7.2 with KOH; carbamylcholine (10 µM) or atropine (1 µM) was added when noted. The internal (bath) solution
contained 0.1 M KCl, 2 mM MgCl2, 1 mM EGTA-KOH, and 20 mM HEPES, pH 7.2 with KOH,
supplemented with test compounds as noted. Although the exchange of
bath solutions was usually performed with the aid of a rotary valve, to
avoid contamination of the bath with G protein subunits or nucleotides
small volumes of stock solutions of these compounds were applied
directly to the recording chamber. To ensure the access of bath
solution to the intracellular aspect of the membranes, after the
formation of a patch the tip of the pipette was briefly passed through
the air-bath solution interface. Additionally, single channel kinetics, activation of ATP-sensitive K+ channels after patch
excision, and/or sensitivity to G protein subunits or guanine
nucleotides were taken as an indication that a closed vesicle had not
formed during excision.
Channel activity in patches is reported as the number of channels
(N) multiplied by the probability of being open
(Po). To determine the relative channel
activities, the NPo during a period of 2 min at
the end of the application of control or test solutions to a patch was
averaged. The value obtained in the test solution was then normalized
to the control NPo.
Many of the patches examined during this study contained some other
types of ion channels, the most prominent being the ATP-sensitive channel of atrial myocytes, which opens when cytoplasmic ATP is reduced
or absent. These channels differed markedly from KACh in
unitary conductance (2.9 pA for KACh and 5.4 pA for
ATP-sensitive K+ channels under our conditions) and
bursting behavior. Therefore, the NPo values
reported here for KACh channels were calculated after a
selection of events with amplitudes in the range of 2.4-3.5 pA.
Although this procedure underestimates the number of KACh openings by eliminating simultaneous openings of two or more muscarinic K+ channels, it minimizes the contribution of ATP-sensitive
K+ channels to the results. NPo
diaries were constructed by plotting NPo values
calculated over consecutive periods of 12.3 s as a function of
time.
Results are reported as means ± S.E. Pertussis toxin was
activated as described previously (10) and diluted to a final toxin concentration of 1 µg/ml with bath solution containing 1 mM NAD before application to patches.
Patches of frog atrial membranes were formed without agonist in
the pipette (external) solution and subsequently excised into a bath
(internal) solution containing no nucleotides. At first, the patches
remained silent (Fig. 1A); if
ATP-sensitive K+ channels were present, these were active
for a short period and then closed, presumably due to dephosphorylation
(11). After a variable delay (0.2-5 min), openings of another channel
were detectable in 76% of the patches examined (349 of 459 patches). The activity of this channel increased gradually, reaching a steady level within 20 min of recording. The channel was characterized by a
single channel slope conductance of 41 pS, strong inward rectification,
bursting behavior (Fig. 1A), and a mean open time of 1.5 ms
(n = 3). When the total K+ concentration in
the bath was changed from 107.3 to 57.3 mM, the single
channel reversal potential shifted by +15.5 mV, in good agreement with
the theoretical value expected for a K+ selective channel,
+16.1 mV (data not shown). These properties are identical to those of
the muscarinic K+ channels present in atrial cells
(KACh; Refs. 12-14). Nevertheless, the time course, the
rate of activation, the conductance, and the gating properties of these
channels were not significantly affected by the presence or absence of
a muscarinic agonist (carbamylcholine, 10 µM) or an
antagonist (atropine, 1 µM) in the pipette (Fig. 2). Moreover, a comparison of the final
levels of channel activity attained in the absence of ligands
(NPo = 0.018 ± 0.006; n = 6), with atropine (NPo = 0.031 ± 0.015;
n = 5), and with carbamylcholine (NPo = 0.087 ± 0.037; n = 6) shows no significant differences (t test,
p > 0.05), suggesting that channel activation in
nucleotide-free solutions is a receptor-independent process. Because
stimulation of KACh channel activity by muscarinic
receptors is mediated by a heterotrimeric G protein and therefore
requires GTP, our results suggest that a prolonged exposure of atrial
membranes to nucleotide-free solutions alters the mechanism of
activation of KACh channels to the extent that the
dependence on activated receptors and GTP ceases to exist.
Activation of KACh channels in GTP-free solutions has been
previously observed under two conditions. One condition involves the
application of high (0.25-1 mg/ml) concentrations of trypsin to the
internal aspect of excised patches, which induces a gradual increase in
muscarinic channel opening in the absence of GTP (15, 16). Activation
of KACh by trypsin presumably is a result of the release of
Receptor-independent Activation of Atrial Muscarinic Potassium
Channels in the Absence of Nucleotides*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
i2 but were not affected by exogenous G
dimers, suggesting that effector activation is a consequence of the
liberation of subunits from endogenous G proteins. The process
of channel activation in the absence of nucleotides seems to be
receptor-independent, because it is not influenced by muscarinic
receptor agonists and antagonists or by treatment with uncoupling
agents such as pertussis toxin or N-ethyl maleimide. Taken
together, these results suggest that the loss of GDP from the G protein
nucleotide binding site promotes its uncoupling from receptors and
destabilizes the G(empty) heterotrimer. Therefore,
the nucleotide-free form of G proteins has some of the characteristics
of the GTP-bound, activated form.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunits of a
pertussis toxin (PTX)-sensitive GTP-binding protein (reviewed in Ref.
1). In the heart, the primary components of this system of signal
transduction have been identified. These include the muscarinic
m2 receptor, heterotrimeric G proteins of the
Gi subfamily, and the heterotetrameric channel formed by
Kir3.1 (GIRK1) and Kir3.4 (GIRK4 or CIR)
subunits (2-5). Several neurotransmitter-regulated channels share the
basic structural and functional features of atrial KACh
channels; therefore, the latter are considered a prototype for the
study of G protein-channel interactions (6). In addition, because patch
clamp methods allow the study of signal transduction with high time
resolution and under circumstances in which the composition of the
milieu can be strictly controlled, atrial muscarinic K+
channels can also be viewed as rapid, selective, and sensitive monitors
of G protein function (7, 8). In the present work, we used excised,
inside-out patches of atrial membranes to examine how G proteins might
be affected by the total absence of intracellular nucleotides. Our
results provide new insights into the behavior of a nucleotide-free
form of G proteins that appears regardless of receptor occupancy and
suggest that the empty subunit shares some characteristics of the
activated, GTP-bound form.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
i2-GDP and were a gift from Dr. S. Graber (West
Virginia University). Protease inhibitors and nucleotides were from
Boehringer Manheim, except for Mg·ATP, which was from Sigma. All
other chemicals were of the highest purity available.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (13K):
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Fig. 1.
Time course of activation and single
channel properties of K+ channels that open in the absence
of agonists and nucleotides. A, continuous recording from an
atrial patch formed with no agonist present in the pipette, held at
85 mV. After the excision of an inside-out patch
(arrowhead) in bath solution with no additions (see
"Experimental Procedures"), KACh channels activated
slowly. An expanded trace is shown below the recording. B,
current-voltage relationship for the channel activated in the absence
of agonists and nucleotides. The slope conductance in this experiment
was 41 pS, and the mean open time was 1.4 ms. In frog atrial
membranes, KACh channels activated in the presence of 10 µM GTP and carbamylcholine have a slope conductance of 38 pS and a mean open time of 1.3 ± 0.17 ms (n = 3).
View larger version (27K):
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Fig. 2.
Muscarinic ligands have no effect on
muscarinic K+ channel activation in nucleotide-free
solutions. Conditions were as described in the Fig. 1 legend, but
the pipette solution contained 10 µM carbamylcholine
(top panels) or 1 µM atropine (bottom
panels). The left panels (A) show continuous
recordings of channel activity. The recording obtained in the presence
of atropine (bottom left panel) contains two channels aside
from KACh. The larger channel that is very active in the
early portion of the experiment and eventually runs down is the
ATP-sensitive K+ channel. A smaller (unitary
conductance < 2 pA), short-lived channel with poorly defined
openings remains active throughout this experiment. The two right
panels (B) show the respective plots of
NPo (N = number of
KACh channels in the patch; Po = open probability) versus time for events corresponding to
muscarinic K+ channels (see "Experimental
Procedures").
dimers after cleavage of the N terminus of G, which is
essential for binding to (17). Therefore, we initially considered the possibility that a protease associated with atrial membrane patches was responsible for KACh activation in
nucleotide-free solution. However, the excision of patches in a bath
solution containing a mixture of protease inhibitors (1 µg/ml each of
aprotinin, leupeptin, and pepstatin) had no effect on KACh
activation in the absence of nucleotides (Fig.
3; n = 5). Furthermore,
KACh channels arising through exposure to nucleotide-free
solutions remain open for extended periods of time and are blocked by
GDP (see below); in contrast, the open probability of trypsin-activated channels decays markedly with time (15), and channel activity is not
affected by the GDP analog GDPS after trypsin treatment (16). These
results suggest that membrane-bound proteases are not involved in the
activation of KACh in nucleotide-free solutions.
View larger version (13K):
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Fig. 3.
Activation of muscarinic K+
channels in nucleotide-free solutions is not affected by protease
inhibitors. Conditions were as described in the Fig. 1 legend,
except that the bath solution was supplemented with aprotinin,
leupeptin, and pepstatin (all at 1 µg/ml) for the period indicated by
the horizontal bar. The arrowhead indicates the
time at which the patch was excised.
Another condition that results in the activation of KACh in
the absence of GTP (and agonist) is the perfusion of patches with purified G subunits (13, 18), which bind directly to the channel
protein (reviewed in Ref. 6) and increase the probability of opening,
bypassing the GTP-dependent steps of channel activation. By
analogy, channel activation in the absence of GTP or agonist might be
achieved through the spontaneous liberation of subunits from
endogenous G proteins. This hypothesis allows us to make several
predictions regarding the behavior of the system.
First, if the release of endogenous subunits is the cause of
channel activation, the application of exogenous subunits should
have different effects on the system, depending on the extent of
channel activation. Namely, subunits should increase NPo when applied during the early stages of each
experiment, when channel activation is still underway. However, when
the system is allowed to attain maximal activation levels so that
endogenous subunits occupy most of the channel sites available,
the presence of additional dimers should have little or no
effect on channel activity. Fig.
4A shows that when
subunits were applied 8-10 min after patch excision, that is, before
full activation was reached, the channel activity
(NPo) increases rapidly, by an average of
5.92-fold (±1.61; n = 5). In contrast, the application
of subunits to patches 20 min after the patch has been excised
(Fig. 4B) had little or no effect on channel activity, and
the ratio of NPo after application to
NPo in bath solution was 1.1 ± 0.24 (n = 4). These results indicate that the sites involved
in channel activation through exposure to nucleotide-free solutions are
identical to the activation sites.
|
Second, G-GDP subunits bind dimers with high affinity (19)
and inhibit their actions through formation of the inactive G protein
heterotrimer (reviewed in Ref. 20). If endogenous subunits are
responsible for KACh activation in nucleotide-free solutions, then this process should be blocked by exogenous G-GDP. Therefore, we examined the effect of Gi2-GDP on channels
that had reached maximal activation levels in the absence of
nucleotides (Fig. 4C). At 25 nM,
Gi2-GDP decreased NPo to 4.4%
(±0.02; n = 4) of the control value, and similar
results were obtained at 50 nM (NPo
in Gi2-GDP/NPo control = 0.053 ± 0.09; n = 3), supporting the idea that
the opening of muscarinic K+ channels in nucleotide-free
solutions involves the liberation of endogenous subunits.
Third, the activation of channels through the release of
subunits should occur equally well in the presence and absence of
Mg2+, because the latter is only required for channel
opening when the mechanism involves GTP hydrolysis (5, 18). Indeed, as seen in Fig. 4D, activation of KACh channels in
the absence of nucleotides also took place when Mg2+ was
omitted from the bath solution and replaced by 1 mM EDTA (14 of 14 patches). On the other hand, EDTA markedly changed the behavior of ATP-sensitive K+ channels, which remained open
for the duration of these experiments (Fig. 4D). Note that
the lack of effect of EDTA on muscarinic K+ channels
eliminates a possible contamination of our solutions with
AlF4
as a cause for channel activation, because
AlF4 does not activate G proteins in the absence of
Mg2+ (21).
Finally, in the absence of agonists, GTP induces a basal stimulation of muscarinic K+ channels that results from interactions between unoccupied receptors and Gk (16, 22). In contrast, we find no evidence that muscarinic receptors affect the activation of KACh channels in nucleotide-free solutions (Fig. 2), suggesting that under these conditions, receptors cannot couple to G proteins. To verify this conclusion, we examined the effect of ADP ribosylation by PTX on channel activation in the absence of guanine nucleotides. PTX blocks basal- and agonist-stimulated GTP-induced KACh channel opening due to the uncoupling of Gk from muscarinic receptors (16, 22) but should not influence events that result from receptor-independent GDP release and dissociation of heterotrimers. As illustrated in Fig. 5A, exposure of the inner surface of patches to solutions containing 1 µg/ml PTX and no nucleotides other than 1 mM NAD did not affect spontaneous activation of muscarinic K+ channels (n = 4). This was also true when PTX and NAD were applied 5-10 min after patch excision (data not shown). Likewise, 50 µM N-ethyl maleimide, which affects muscarinic K+ channels in a manner similar to that of PTX (14, 16), had no effect on channel activation in nucleotide-free solutions (Fig. 5B; n = 7). Note that in control experiments performed in the presence of 100 µM GTP, both PTX and N-ethyl maleimide abolished channel activity (data not shown). These results are consistent with the hypothesis that activation of muscarinic K+ channels in the absence of GTP happens independently from receptor-G protein coupling.
|
Taken together, the results above support the idea that
KACh channels become active in nucleotide-free solutions as
a consequence of unstimulated release of subunits from
endogenous G proteins. Under normal conditions, the high affinity of
subunits for GDP-bound G subunits prevents GDP release and
dissociation of the G protein heterotrimer. In our experiments,
however, nucleotide-free solutions create an environment where the GDP
that is gradually released from the G protein cannot be replaced by
another nucleotide. This might promote the accumulation of a
nucleotide-free form of the G protein akin to the empty form that is a
transient intermediate in the GDP-GTP exchange process that induces
subunit dissociation and effector activation. In the present case,
complete removal of the guanine nucleotide from the medium seems to
promote the uncoupling of receptors and G proteins, generating the form
G(empty). If this receptor-free form of the empty
heterotrimer is unstable, its spontaneous dissociation into
G(empty) and dimers could fully account for our
results.
This hypothesis can be tested by examining the effects of nucleotides
on channel activity after the system has attained a steady level of
activation, because occupation of the empty nucleotide binding site
should induce reassociation of the heterotrimer and thus turn off the
system. Moreover, if the activation process observed here is indeed G
protein mediated, it should display the expected specificity for
guanine nucleotides. We applied GDP or GTP to patches where
KACh channel openings had reached a steady level after a
prolonged perfusion with nucleotide-free solutions. At 2 µM, GDP had a strong inhibitory effect on channel
activity (Fig. 6A). The
application of 10 µM GDP reduced the open probability by
93.1% (± 0.04%; n = 6). This is the outcome expected
if GDP were to bind specifically to an empty form of G, thus
promoting its reassociation with . The block by GDP (as well as
GTP, Fig. 6B) took place gradually, indicating that the
binding/reassociation process has at least one slow step. In contrast,
upon washout of GDP, KACh channels resumed activity at once
(Fig. 6A), indicating that GDP dissociates rapidly from this
state of the G protein.
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The treatment of patches with GTP had dose-dependent
effects that were similar to those of GDP (Fig. 6B),
reducing the channel opening probability by 75.4% (±14.1%;
n = 4) at 10 µM. In contrast, ATP (100 µM; Fig. 6C) and UDP (data not shown) did not
affect channel activation, attesting to the specificity of the block by
guanine nucleotides. The negative effect of GTP is reminiscent of the
results obtained when purified G proteins are incubated with GTP:
because G
hydrolyzes GTP at a rate that vastly exceeds
the rate of GDP release (19), the inactive GDP-bound form of G
accumulates, despite the presence of the activating trinucleotide. This
observation lends further support to the conclusion that
nucleotide-free conditions cause the uncoupling of endogenous receptors
and G proteins, because otherwise the basal activity of unliganded
receptors would have allowed GTP to activate the system (16, 22).
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DISCUSSION |
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Procedures
Results
Discussion
References
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The sequence of events that follow the initial binding of agonists
to muscarinic receptors and eventually lead to muscarinic K+ channel opening has been elucidated using patch clamp
methods. Thus, early experiments demonstrated that channel activation
by muscarinic agonists requires GTP or its analogs (23, 24) as well as
Mg2+ (25), implicating a heterotrimeric G protein,
Gk, in channel opening. Additionally, the demonstration
that pertussis toxin blocks channel activity indicated that
Gk belongs to the Gi/o family (13, 24). More
recently, knockout of both Go isoforms has shown that
Gk is a member of the Gi group (26). Therefore, muscarinic K+ channels are customarily described as being
activated by muscarinic agonists via a PTX-sensitive,
Gi-type G protein through a process that shows an absolute
requirement for GTP and Mg2+ (1). Nonetheless, the data
described here show that KACh channels can be activated in
the absence of agonist, GTP, and Mg2+, and that under this
particular set of conditions, the opening of channels is not affected
by PTX. At first, this might be taken as evidence for the existence of
a novel, G protein-independent pathway that also leads to muscarinic
K+ channel activation. However, the observation that GDP,
GTP, and Gi2-GDP terminate channel activation, as well
as the time-dependent effect of exogenous G dimers,
suggests that this agonist and GTP-independent pathway still relies on
the dissociation of endogenous G proteins and the release of
dimers to induce channel opening. The notable difference between the
pathway unveiled here and conventional channel activation is that in
nucleotide-free conditions the G protein heterotrimer seems to be
destabilized not through the binding of GTP, but by the continued
absence of bound nucleotide.
One important feature of the activation of KACh channels in
nucleotide-free solutions is that this condition apparently uncouples the receptor from the G protein. This is reflected in the lack of
effect of agonists and antagonists on the activation process and in the
lack of effect of PTX and N-ethyl maleimide. Thus, the G
protein form generated in our experiments is presumably distinct from
the empty state of transducin (Gt) generated in the
presence of activated rhodopsin (R*) and in nucleotide-free solutions,
which reflects the formation of a tight
R*Gt(empty) complex that does not
release dimers (27). Formation of the latter complex shows an
absolute requirement for R*, because, unlike other heterotrimeric G
proteins, Gt does not release GDP spontaneously (28). Note
that in vitro studies of the empty form of purified subunits of Gi and Gs are hindered by the
marked instability of the unliganded proteins (29, 30). Therefore, to
our knowledge, this is the first report on the functional properties of
the empty pocket conformation of a G protein generated in the absence
of activated receptors. Note that although we assume that the loss of
GDP leads to the dissociation of G into free
(empty) and dimers, there is an alternative
explanation for our observations; namely, in the absence of GTP,
G-GDP might associate into multimers such as those found in crystals
of Gi1-GDP (31). The formation of these structures is
expected to prevent interactions between G and receptors, pertussis
toxin, or dimers, and it has been hypothesized that
polymerization of G-GDP might induce nucleotide exchange
independently of activated receptors or G (32).
Finally, our results suggest that the empty state of G protein subunits bears some resemblance to the GTP-bound conformation, because
it seems to have low affinity for the dimer and for receptors.
However, this does not imply that this form of G is active and
could, for example, inhibit adenylyl cyclase as does Gi-GDP; rather, the activation of channels takes place
because G(empty) cannot act as a negative regulator of
.
In summary, our results show that KACh channels can be
activated in the absence of agonist, GTP, and Mg2+. We
propose that prolonged perfusion of atrial membrane patches with
nucleotide-free solution leads to the slow release of GDP from the
-subunit of Gk and the appearance of a form of
k with an unoccupied nucleotide binding site, the empty
state. The empty G protein heterotrimer is unstable and undergoes
spontaneous dissociation, gradually releasing subunits that can
then activate effectors such as the muscarinic K+ channel.
Our data imply that this particular empty form of the G protein can
arise independent of interactions with receptors. Furthermore, this
empty state of G protein subunits resembles the GTP-bound state,
being characterized by low-affinity interactions with the dimer
and the receptor.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Graber for the generous gift of G protein subunits and Dr. Erik L. Hewlett for pertussis toxin.
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FOOTNOTES |
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* This work was supported by Department of Health and Human Services Grants HL48726 and HL37127.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: Department of
Molecular Physiology and Biological Physics, University of Virginia, P. O. Box 10011, Charlottesville VA 22906-0011. Tel.: 804-982-1896; Fax: 804-982-1616; E-mail: ado2t{at}virginia.edu.
§ Present address: Friedrich Miescher Institute, Maulbeerstrass 66, R-1066 4.52, 4058 Basel, Switzerland.
The abbreviations used are: KACh, muscarinic K+ channels; PTX, pertussis toxin.
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