From the Henry Hood MD Research Program, Department of Cellular and Molecular Physiology, Penn State College of Medicine, Danville, Pennsylvania 17822
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ABSTRACT |
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The membrane-delimited activation of muscarinic K+ channels by G protein subunits plays a
prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying
K+ channels. Here, we demonstrate that muscarinic K+ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel,
which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of G
subunits to the channel was tested, using recombinant G
1
5. G
1
5 was able to control the
equilibrium between the four gating modes of the channel in a manner consistent with binding of G
to four equivalent and independent sites in the protein complex. Surprisingly, however, G
1
5 lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K+ channel gating by G
provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.
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INTRODUCTION |
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The cardiac muscarinic inward rectifier potassium channels (KACh channels) are responsible for the acetylcholine- (ACh)1 and adenosine-induced deceleration
of the heart rate and atrioventricular conduction.
When either m2-muscarinic cholinergic or A1-purinergic receptors are activated in cardiac atrial myocytes,
they interact with heterotrimeric regulatory GTP-binding proteins (G proteins), promoting dissociation of
the G proteins into a G-GTP subunit and a G
complex. Once dissociated, both the G
-GTP and G
subunits proceed to regulate several effectors in the myocytes (Clapham and Neer, 1993
). It is now well recognized that the G
dimers confer the activation of the
KACh channels (Reuveny et al., 1994
; Wickman et al.,
1994
). At the same time, the G
subunits are thought to interact with additional signaling constituents, the
regulators of G protein signaling (RGS proteins), that
stimulate the intrinsic GTPase activity of the G
subunit, leading to rapid reassociation of the G protein
heterotrimer and termination of the signal (Doupnik et al., 1997
; Saitoh et al., 1997
).
Several members of an expanding family of structurally
related G protein-activated inward rectifiers (GIRKs)
have been recently identified (Dascal et al., 1993; Kubo
et al., 1993
; Lesage et al., 1994
; Doupnik et al., 1995
).
The cardiac KACh channels consist of two homologous
subunits, GIRK1 and GIRK4 (also known as CIR) (Krapivinsky et al., 1995
; Wickman et al., 1998
), that are thought to form tetrameric structures with a (GIRK1)2(GIRK4)2
stoichiometry (Silverman et al., 1996
; Tucker et al.,
1996
). Both GIRK1 and GIRK4 subunits are composed
of two putative transmembrane domains, flanked by
large hydrophilic NH2- and COOH-terminal domains
residing within the cell. These NH2- and COOH-terminal regions of GIRK1 and GIRK4 are thought to be
involved in G
interactions to mediate channel activation (Takao et al., 1994
; Dascal et al., 1995
; Huang et al., 1995
; Kunkel and Peralta, 1995
; Huang et al.,
1997
).
While the structural domains involved in the G
binding have been identified, the molecular mechanisms directing KACh channel gating upon G
binding
remain enigmatic. Previous electrophysiological studies
have shown that the native cardiac KACh channels exhibit sigmoidal stimulus-response curves, suggesting
cooperative binding of three or four G
subunits to a
single channel molecule (Ito et al., 1991
, 1992
). It has
been further postulated that the function of the KACh
channels is controlled by two independent mechanisms: a G protein-independent fast gating of the
channel and a G protein-dependent slow transition
from an unavailable to an available KACh channel state
(Hosoya et al., 1996
). Another intriguing possibility,
however, is that G
subunits may control the function
of the KACh channel by governing the equilibrium between several functional modes of the channel. In fact,
we have recently shown that multiple gating modes of
the KACh channel do exist in the atrial myocytes of the
bullfrog Rana catesbeiana (Ivanova-Nikolova and Breitwieser, 1997
). Yet it remains to be determined whether
the modal regulation of the KACh channel is evolutionarily conserved and, if so, how G
subunits control the
balance between the different functional states of the
channel in mammalian systems.
To address these questions, we studied the interactions of G with the native KACh channels in atrial myocytes of the neonatal rat heart. First, we examined the
KACh channel gating upon activation of muscarinic receptors in the atrial myocytes and identified the presence of four distinct patterns of KACh channel gating. The different gating modes were characterized by differences in both the frequency of channel opening and
the mean open time of the channel, which accounted
for a 76-fold increase in channel open probability from
mode 1 to mode 4. Further, to reveal the mechanisms
underlying modal behavior of the KACh channel, we reconstructed channel activation in excised membrane
patches using purified recombinant G
1
5. Surprisingly, in the presence of G
1
5 alone, the four gating
modes of the KACh channel differed only in the frequency of channel openings, while the mean open time
of the channel remained the same. Finally, the analysis
of the equilibrium among the functional modes of the
channel at different G
1
5 concentrations demonstrated
that the four gating modes reflected binding of a different number of G
1
5 subunits to four equivalent and
independent sites in the channel protein complex.
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MATERIALS AND METHODS |
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Isolation and Cell Culture of Neonatal Cardiac Myocytes
Viable atrial myocytes were obtained from 1-2-d-old Sprague
Dawley rats by a trypsin/chymotrypsin/elastase dissociation procedure as described previously (Foster et al., 1990). The dissociated cells were centrifuged through Percoll step gradients to obtain cell preparations consisting of >94% myocytes. The myocytes were suspended in Modified Eagle's Medium containing
5% newborn calf serum and 0.1 mM 5-bromo-2'-deoxyuridine
and were plated at a density of 105 cells/cm2 on cover slips precoated with 0.1% gelatin. After overnight incubation, the cells
were washed to remove nonadherent cells and cultured in a defined serum-free media for four additional days (Hansen et al.,
1994
).
Expression and Purification of G Protein Subunits
Procedures for construction and selection of recombinant Baculoviruses encoding various or
subunits of the G proteins have
been described previously by Iniguez-Lluhi et al. (1992)
. cDNA
containing the entire coding region for a particular subunit was
transferred to the baculovirus expression vectors pVL1392 and
pVL1393. Recombinant viruses were generated by cotransfection of Spodoptera frugiperda (Sf9) insect cells with the recombinant pVL1392 and pVL1393 transfer vectors along with mutant Autographa californica nuclear polyhedrosis virus, as described by the
supplier (PharMingen, San Diego, CA). All recombinant viruses
were plaque purified and were verified by their ability to direct
the expression of the appropriate proteins, as detected by immunoblotting. Gi
1
1
5 heterotrimers containing a hexahistidine
(H6) tag inserted into the Gi
1 were expressed in Sf9 cells as described (Kozasa and Gilman, 1995
). H6-tagged heterotrimers
were solubilized by 0.5% Lubrol and purified by adsorption to an
H6-affinity nickel column. The
1
5 dimers were then selectively
eluted from the column using a HEPES buffer (pH 8) containing
0.03 mM AlCl3, 50 mM MgCl2, and 10 mM NaF. The elution of
the G
1
5 complex was monitored by immunoblotting, using
1-
and
5-specific antibodies. The
1
5 dimers were further purified
to homogeneity using Fast Protein Liquid Chromatography
Mono Q column (Pharmacia LKB Biotechnology Inc., Piscataway, NJ). Purity of the final product was determined by sodium
dodecyl sulfate (SDS)-PAGE and silver staining before use.
Electrophysiology
Single-channel currents through KACh channels were recorded
from cell-attached and inside-out patches of atrial myocytes using standard high resolution patch-clamp method (Hamill et al.,
1981). The membrane potential was zeroed with the following
bath solution (mM): 150 KCl, 5 EGTA, 5 glucose, 1.6 MgCl2, 5 HEPES, pH 7.4. Pipette solution contained (mM): 150 KCl, 1 CaCl2, 1.6 MgCl2, 5 HEPES, pH 7.4. Patch pipettes were made
from borosilicate glass (World Precision Instruments, Inc., Sarasota, FL) on a Flaming Brown micropipette puller (Sutter Instruments, Co., Novato, CA) and firepolished on a microforge (Narishige Scientific Instrument Lab., Tokyo, Japan). The resistance
of the patch pipettes (when filled with pipette solution) was
10-20 MOhm. Either acetylcholine or adenosine was added to
the pipette solution to activate m2-muscarinic or A1-purinergic receptors, respectively. Currents were recorded with a Patch Clamp
List-Medical EPC-7 amplifier (ALA Scientific Instruments Inc.,
Westbury, NY), additionally filtered at 2 kHz with an eight-pole
Bessel filter (Frequency Devices Inc., Haverhill, MA) and the acquired data was stored on the hard disk of a computer. Data acquisition and analysis were performed using Digidata 1200 (Axon
Instruments, Foster City, CA) supported by version 6.0 of pCLAMP software (Axon Instruments). Channel opening and
closing transitions were identified with the half-amplitude threshold-crossing algorithm. Idealized records of the amplitudes, and
channel open and close times, were generated by the FETCHAN
Events List function. To determine the residence time of the
KACh channel in the different gating modes, the continuous
records were divided into consecutive segments, the frequency of
apparent openings, f, was calculated for each segment and f histograms were generated for further analysis.
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RESULTS |
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Evidence for the Presence of Different Functional States of the KACh Channels in the Neonatal Rat Cardiac Myocytes
We have previously demonstrated that upon activation
of endogenous G proteins in frog atrial myocytes, the
KACh channels exhibit bursting behavior, shifting between three predominant patterns of gating. They were
termed low, medium, and high frequency modes, according to the frequency of channel openings during
individual bursts (Ivanova-Nikolova and Breitwieser,
1997). Under basal conditions, infrequent, agonist-independent channel openings display exclusively a low frequency behavior, while a maximal activation of G proteins by GTP
S favors the high frequency behavior of
the KACh channels. To test whether such a multistep activation of the KACh channel has been evolutionarily conserved as a signal transduction mechanism, we examined
the gating behavior of KACh channels after m2-receptor stimulation in rat neonatal atrial myocytes.
The individual KACh channels, activated in cell-attached
configuration by 1 µM ACh, differed considerably in
their gating behavior. One gating pattern was characterized by infrequent channel openings separated by
long silent intervals, while a second one was dominated
by the apparent clustering of openings into long bursts
of activity. Fig. 1 provides an illustration of the differences between these patterns of gating. First, a comparison of the all-points histograms unveiled an order of
magnitude difference in channel open probability, Po.
Second, a comparison of the open time distributions revealed a correlation between the proportion of brief
and long KACh channel openings and the pattern of
gating. In 10 cell-attached patches selected for analysis
(no recordings with superimposing openings were accepted for this or the following types of analysis), a sum
of two exponentials provided an adequate fit to the
open time distributions. In addition, the time constants
of both fast and slow exponential components were
similar for different KACh channels and had values of
~1 and ~7 ms, respectively. The fractions of the histograms fitted by each of the two components, however, were different for the channels exhibiting the patterns
of activity illustrated in Fig. 1, A and B. Thus, the
changes in the kinetic behavior of the KACh channels
were associated with both a change in the open probability and a shift from a brief open state to a long open
state of the channel. These two observations provided a
basis for classification of the KACh channel gating into
functionally distinct modes. In bullfrog atrial myocytes,
such classification was entirely based on analysis of
channel gating within well defined individual bursts, assuming that each burst reflected a single event of interaction between the ion channel and G. In rat neonatal atrial myocytes, however, similar unambiguous division of the single channel records into bursts was not
always possible. Therefore, we used a different approach for the classification of heterogeneous channel
behavior into discrete gating modes. The continuous
recordings were divided into consecutive, equally
spaced time intervals and the channel behavior was assessed within these intervals. A duration of 400 ms was
selected for the time intervals in this analysis based on
the burst duration determined for the KACh channels
exhibiting well-delineated bursting behavior. For each
400-ms data segment, the frequency of openings, f, and
the probability of the channel being open, Po, were calculated from the events list files and plotted vs. time.
Fig. 2 illustrates this approach, using the two segments
of KACh channel recordings shown in Fig. 1. Visual inspection of a large number of f and Po plots verified
that the selected time interval provided an adequate
representation of the fluctuations in the KACh channel
gating. Similar plots were generated from each of the
10 cell-attached recordings selected for analysis (total
of 30 min of single-channel data). The f and Po values
within each 400-ms data segment were used to calculate the mean open time of the channel, topen. The topen values were further analyzed with regard to the frequency
of openings to determine to what extent the changes in
the frequency of gating could be correlated with changes
in the mean open time of the KACh channel. The resulting topen-f plot, shown in Fig. 3 A, revealed a multistep augmentation in the average topen from 2.28 ± 0.10 ms
at f = 2.5 Hz (n = 999 segments) to 6.24 ± 0.28 ms at
frequencies above 47.5 Hz (n = 231 segments). Each
statistically significant step in the topen augmentation is
indicated by an arrowhead in the topen-f plot and presumably reflects a rather abrupt transition from one pattern of channel gating to another. The analysis of KACh
channel gating, outlined above, identified transitions between four functional modes accessible to the channel
upon activation of muscarinic receptors. A histogram of
the frequency of openings was also generated to estimate
the relative occupancy of different modes (Fig. 3 B).
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Fig. 4 provides a direct comparison of several functional properties of the four KACh channel gating modes in neonatal rat atrial myocytes. Taken together, the differences in the frequency of channel openings and in the average open times account for significant changes in the open probability of the channel from one mode to the next. Thus, the mean open probability was ~20- and ~76-fold higher in modes 3 and 4, respectively, when compared with the open probability in mode 1. Such differences in the open probability would certainly have profound effects on the contributions of individual modes to the total current. Accordingly, modes 3 and 4 together contributed ~80% of the total current, although the KACh channels spent only 23% of their active time in these two modes.
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These data indicate that the modal behavior of the
KACh channels in neonatal rat atrial myocytes is qualitatively similar to the one described by us in bullfrog
atrial myocytes (Ivanova-Nikolova and Breitwieser, 1997).
The different number of gating modes identified in the
rat myocytes (four, vs. three in the bullfrog myocytes) can be attributed to the elimination of mode 1 from the
data selected for analysis in the bullfrog myocytes. In
that case, individual bursts were defined as series of
openings separated by closed intervals shorter than
some critical interval, usually between 120 and 140 ms.
As a result, the singular events (separated by closed intervals longer than 140 ms) that delineate gating mode 1 were completely excluded from the analysis.
Regulation of Modal Behavior of the KACh Channels by G
Protein Subunits
To understand the mechanisms underlying the modal
behavior of the KACh channel, we reconstructed channel activation in excised membrane patches using recombinant G. A major advantage of this approach
over receptor-mediated activation of the KACh channel is that it provides a strictly controlled environment,
abolishing potential contributions of intermediate signaling molecules to the KACh channel-G
interactions. Since the composition of the G
involved in the
physiological regulation of the KACh channel is unknown, we selected the G
1
5 isoform for our experiments, based on its relative abundance in the heart
(Hansen et al., 1995
). G
1
5 dimers, expressed and purified from Sf9 cells, as previously described by Kozasa
and Gilman (1995)
, were applied to activate directly
the KACh channels in inside-out membrane patches excised from the rat atrial myocytes. Fig. 5 illustrates the
activity recorded from the same KACh channel in a cell-attached patch in the presence of 1 µM adenosine (A),
upon patch excision in GTP-free solution (B), and after
application of 0.6 and 1.5 nM G
1
5 (C and D, respectively). Nanomolar concentrations of G
1
5 activated the KACh channels in a concentration-dependent manner similar to that reported by Wickman et al. (1994)
.
The activation of the KACh channels developed over the
course of 2-11 min after G
1
5 application and was sustained during continued presence of G
1
5. The gating
behavior of the individual KACh channels remained heterogeneous even under such strictly controlled conditions, suggesting that the heterogeneity in the channel
gating is an intrinsic property of KACh channel-G
interactions.
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To reveal the source of this heterogeneity, the KACh
channel gating in the presence of G1
5 was subjected
to the same analysis routine described for analysis of
the data recorded upon muscarinic receptor stimulation. The unitary currents through KACh channels activated by different concentrations of G
1
5 were recorded for 20-30 min after the initial time interval required for incorporation of G
1
5 in the membrane.
The continuous single-channel recordings were divided into consecutive 400-ms segments, and three parameters, f, Po, and topen, were calculated for each data
segment. The results from the analysis were then combined to generate a topen-f plot and a frequency histogram for each individual experiment. Fig. 6 illustrates
the topen-f plots and the f histograms obtained from the
experiment with the two different G
1
5 concentrations, shown in Fig. 5, C and D.
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The comparison of the topen-f plots obtained at different G1
5 concentrations (ranging from 0.15 to 12 nM)
with the topen-f plot in Fig. 3 A revealed a key difference
in the function of the KACh channels activated by G
1
5
alone. While the mean open time of the receptor-activated channels underwent synchronized changes with
the increase in the frequency of gating, the mean open
time of G
1
5-activated channels (topen = 1.86 ± 0.09 ms, n = 10) was unaffected by the frequency of gating
(Fig. 6 A). Such a difference in the gating of receptor-activated and G
1
5-activated KACh channels suggests
that a combination of molecular interactions might
contribute to the phenomenon of modal behavior.
Given the ability of GIRK1 subunits of the KACh channel
to bind G
i to a binding site different from that for
G
(Huang et al., 1995
), one intriguing scenario is
that binding of both G
i and G
to the KACh channel
is necessary to achieve the augmentation of topen found
after muscarinic receptor activation. Alternatively, different subclasses of G
might exert different effects
on the KACh channel regulation and the method outlined in the present work is sensitive enough to capture
the distinctions between channel gating in the presence of G
1
5 and in the presence of the unidentified,
but hypothetically distinct, G
released upon muscarinic receptor stimulation. Further studies will be required to distinguish between these two possibilities.
At the same time, the comparison of the frequency
distributions obtained at different G1
5 concentrations (Fig. 6 B) revealed that the increases in G
concentrations are translated into increases in the frequency of KACh channel openings. These distributions should consist of a sum of geometric components, and
the number of components should reflect the number
of conducting conformations of the channel (Colquhoun and Hawkes, 1981
, 1983
). We therefore used the
geometric components in the f histograms to classify the gating behavior of G
1
5-activated channels into
distinct modes. In the presence of 0.15 nM G
1
5, a single geometric component was sufficient to fit the f histograms, while a sum of up to four geometric components was required for the adequate fit of the data generated in the presence of higher G
1
5 concentrations. The mean frequencies of the first through fourth components showed little variations from one KACh channel
to another and averaged 2.2 ± 0.2 Hz (n = 24), 12.7 ± 0.6 Hz (n = 20), 33.6 ± 1.9 Hz (n = 18), and 65.0 ± 4.2 Hz (n = 8), respectively. These values are similar to the
frequencies found for modes 1-4 of the receptor-activated KACh channels in the atrial myocytes (see Fig. 4
for comparison). Accordingly, with regard to the frequency of openings, the G
1
5-activated KACh channels
behaved in a manner approaching that of the receptor-activated channels, converting between four functional
modes.
On the basis of these results, we proposed that binding of a different number of G subunits to four G
-binding sites in the tetrameric KACh channel structure
gives rise to its four functional modes. For simplicity,
we further assumed that the four G
-binding sites are
functionally equivalent and independent. These assumptions imply that the four sites have the same binding affinity for G
, and that G
binding to one site is
not affected by the G
occupancy of the remaining
sites. Such a simple model predicts that the probability
of observing each gating mode is given by the binomial distribution [N!/k!(N
k)!]Pk(1
P)N
k, where N is
the total number of G
-binding sites, k is the number of the occupied binding sites, and P is the probability
that one of the four G
-binding sites is occupied.
To test this prediction, we quantified the equilibrium
among the four gating modes at different G
concentrations. The equilibrium probability (or relative occupancy) of different modes was estimated from the fraction of the total f histogram fit by the corresponding
geometric component. In experiments using the same
concentrations of G
1
5, some variations in the relative
occupancy of different modes were observed. These
variations can be explained by differences in the probability of G
binding, P, and can be attributed either to
a different amount of G
1
5 incorporated in the membrane or to a different binding affinity of the channel
for G
1
5. Therefore, in each experiment, the probability of G
binding was calculated from the fraction
of the f histogram fit by the first geometric component
and the relative occupancy of different gating modes
was examined as a function of G
binding to the
channel, rather than as a function of G
concentration. The corresponding plot from this analysis is
shown in Fig. 7 and verifies that the equilibrium probability of each mode is binomially distributed as predicted by the model. In this way, the analysis of the
equilibrium among the four KACh channel functional
states in the presence of G
1
5 predicts the existence of
four equivalent and independent G
binding sites in
the channel structure.
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It is interesting to note that in a small subset of G1
5
experiments (3 of 27), the mean frequencies of gating
for each mode deviated by a factor of ~2 from the values estimated for m2 receptor-activated KACh channels.
In that case, the mean frequencies of the four modes
were f1 = 3.7 ± 0.3, f2 = 21.4 ± 1.1, f3 = 54.7 ± 1.9, and
f4 = 104.5 ± 5.6 Hz, while the conductance and the mean open time of these channels were similar to those
determined for the rest of the G
1
5-activated channels. Such differences in the channel gating, although
infrequent, point to certain structural variability among
native KACh channels in the heart. Evidently, a combination of K+ channels susceptible to G
regulation
shapes the responsiveness of the atrial myocytes to G
protein activation and the basic approach for classification of the KACh channel gating outlined in the present
work creates a sensitive tool for capturing the functional variety among these channels.
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DISCUSSION |
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The results outlined in the present work characterize
the mechanism of membrane-delimited activation of
muscarinic K+ channels by G subunits in neonatal
rat atrial myocytes and render an important clue to understanding the principles underlying the specificity of
G
-mediated signaling in general. These two aspects
of our findings will be discussed below.
Mechanism of G Regulation of KACh Channels
Activation of KACh channels by acetylcholine and adenosine in sinoatrial nodal cells and atrial myocytes modulates both heart rate and cardiac contractility. The
channel activation is brought about by a pertussis
toxin-sensitive G protein (Breitwieser and Szabo, 1985;
Pfaffinger et al., 1985
) in a membrane-delimited manner (Soejima and Noma, 1984
), using the G protein
subunits as direct information carriers between the
membrane receptors and the KACh channels (Reuveny
et al., 1994
; Wickman et al., 1994
). Because of their significance in the regulation of the heart function, KACh
channels have been extensively studied at both whole-cell and single-channel levels (for review see Kurachi,
1995
), yet the molecular mechanism underlying G
-
KACh channel interactions remains obscure. The only
available functional model for G protein activation of
the KACh channel was based on data from spectral analysis of current fluctuations at different GTP concentrations (Hosoya et al., 1996
). In that case, the spectral
analysis identified transitions only between two closed
(C1 and C2) and one open (O) state of the KACh channel. Accordingly, the proposed model was based on two
independent processes: a GTP-independent fast gating (C2
C1
O) and a slow, GTP-dependent transition
(not detected in the power spectra) between unavailable and available channel states. Our recent single-channel analysis of the KACh channel regulation, however, clearly indicated that the channel activity is much
more complex than previously thought and this activity is distributed between several gating modes (Ivanova-Nikolova and Breitwieser, 1997
). To understand the
mechanisms underlying such modal behavior of the
KACh channel, in the present study, we performed an
extensive single-channel analysis of the activation of the
native KACh channel by recombinant G
1
5 subunits.
On the basis of this analysis, we formulated an alternative model of G
-KACh channel interactions in which
binding of a different number of G
subunits to four
G
-binding domains in the KACh channel structure
gives rise to four conducting conformations of the protein complex, linked in a dynamic equilibrium by the G
concentration.
The heterogeneity of the KACh channel gating is a major obstacle in the interpretation of the single-channel
recordings from both receptor- and G1
5-activated
channels. Therefore, in the present study, we initially
developed a basic approach for classification of the
KACh channel gating into functionally distinct modes.
The method is sensitive enough to capture subtle changes
in the KACh channel regulation and greatly simplifies
the kinetic analysis of the data. The analysis routine is
based on fragmentation of continuous single-channel
recordings into consecutive, equally spaced segments
and subsequent characterization of the channel gating
within individual segments. The characterization of channel gating combines the analysis of three different parameters: the frequency of openings, the probability of
the channel being open, and the mean open time of
the channel. The results from this analysis were then
compiled to generate the frequency histograms and
topen-f plots as final readouts that convey the information about the dynamics of the KACh channel-G
interactions.
In the present work, this method was successfully applied to classify the KACh channel gating into functionally distinct modes either by the appearance of several
kinetic components in the frequency distributions, or
by the orchestrated augmentations of KACh channel
open time within the topen-f plots. Two different series
of experiments, one of which conserved the integrity of transduction cascade from the activation of muscarinic
receptors to the activation of the KACh channels, and
another in which KACh channel activation was brought
about solely by purified recombinant G1
5, indicated
the presence of four functional modes of the channel.
Remarkably, the two series of experiments yielded similar values for the predominant frequencies of channel
openings within different gating modes. The values for
the m2 receptor-activated channels were: f1ACh = 2.5, f2ACh = 10.3 ± 0.1, f3ACh = 31.7 ± 0.3, and f4ACh = 69.1 ± 1.2 Hz, while the values for the G
1
5-activated channels were: f1G
= 2.2 ± 0.2, f2G
= 12.7 ± 0.6, f3G
= 33.6 ± 1.9, and f4G
= 65.0 ± 4.2 Hz. Thus, our analysis identified the frequency of channel openings as a
principal parameter in the classification of the KACh
channel activity into distinct gating modes.
Intriguingly, G1
5 alone was not able to reproduce
the augmentation in the mean open time of the KACh
channel in modes 3 and 4 associated with the channel
activation through muscarinic receptors (Fig. 3 A).
One possible reason for this could be that the unidentified G
subunits released upon muscarinic receptor stimulation are structurally different from G
1
5 and
the active conformations of the channel vary depending on different G
combinations. Alternatively, the
augmentation in the mean open time could be regulated by different structural domains of the channel
molecule and might require the binding of the G
subunit to the channel. Validation of these possibilities
requires direct experiments, which are currently under way.
Nevertheless, G1
5 was able to govern the equilibrium
between the four functional modes of the channel.
Multiple biochemical mechanisms might underline the
modal behavior of the KACh channels. Any hypothetical
mechanism, however, should take into consideration two important structural characteristics of the channel:
its tetrameric structure (Silverman et al., 1996
; Tucker
et al., 1996
) and the presence of numerous G
-binding domains in the channel complex. Because of its
simplicity, we considered a paradigm in which binding of increasing numbers of G
subunits to four equivalent and independent positions in the channel protein
complex would give rise to four distinct conformational
states of the KACh channel. As long as the four gating
modes of the channel can be regarded as reporters of
such different conformational states, this paradigm
predicts the probability of observing each gating mode
as a function solely of the probability that one of the
four G
-binding sites is occupied, P. In this case, the
probability function of each gating mode should follow
the binomial distribution,
![]() |
(1) |
for binding of one, two, three, or four G subunits to
four G
-binding sites on the tetrameric channel. To
test this prediction, the equilibrium among the four
gating modes was quantified from the f histograms generated at different G
concentrations, and the relative
occupancy of each mode,
k, was examined as a function of the parameter P. In each instance, this parameter was computed from the relative occupancy of mode
1,
1, according to the equation
1 = 4P(1
P)3/[1
(1
P)4]. This equilibrium analysis clearly indicated
that the probability of observing each gating mode is
binomially distributed as one would predict if the four
G
-binding sites are identical and independent. The
structural basis for this functional equivalence of the
G
-binding sites in the heterotetrameric KACh channel remains to be determined. The NH2- and COOH-terminal domains of both GIRK1 and GIRK4 bind G
; however, the affinity of the COOH-terminal regions is different for the two subunits (Huang et al., 1997
). In view
of this fact, our data suggest that the GIRK1 and GIRK4 subunits must be arranged in a precise pattern to form
the four equivalent G
-binding sites, perhaps using
G
-binding blocks from both GIRK1 and GIRK4
polypeptide chains.
The probability of one of the four G-binding sites
to be occupied, P, was a hyperbolic function of the G
concentration (Fig. 8 A), and was well approximated by
the equation:
![]() |
(2) |
|
where Kd is the microscopic dissociation constant for
G binding to the channel. The least-squares fit to the
data provided a value of 0.63 for the parameter Pmax
and a Kd value of 1.29 nM. These values were used in
Eqs. 1 and 2 to calculate the probability of observing
each gating mode, Pk, as a function of the G
1
5 concentration. Then from the Pk values and the experimentally determined open probability of the KACh channel in each gating mode (Po1G
= 0.0055 ± 0.0006, Po2G
= 0.0242 ± 0.0015, Po3G
= 0.0642 ± 0.0048, and Po4G
= 0.1409 ± 0.0127), we generated the theoretical stimulus-response curve for a KACh channel
with four gating modes arising from the binding of a
different number of G
subunits to four equivalent and independent binding sites. The resulting curve (Fig.
8 B, dotted line) is sigmoidal, and its fit with the Hill
equation,
![]() |
(3) |
yields a Hill coefficient, N, of 1.2 and an apparent dissociation constant, kd, of 2.71 nM. The theoretical and
the experimental (Fig. 8 B, solid line) G1
5 concentration-response curves are identical in the concentration
range 0.15-2.5 nM; however, at higher G
1
5 concentrations, the theoretical curve approaches the saturation limit more gradually than the experimental curve (kd of 1.09 nM and Hill coefficient of 1.73). This discrepancy between the theoretical and the experimental
curves can be explained by the desensitization of the
KACh channel. We found that as G
1
5 concentration
was raised, the proportion of the blank data segments exceeded the probability of observing gating mode 0 (no G
bound to the channel), Po, calculated from
Eq. 1. This behavior is expected if the KACh channel enters a desensitized state that depends on the G
concentration. An additional possibility is that as the G
concentration increases, the G
1
5 dimers aggregate in
the membrane and, consequently, the membrane concentration of G
1
5 deviates from the one in the experimental chamber. Consistent with this possibility, the
probability of G
1
5 binding, P, saturated at Pmax of
0.63 instead of reaching a value of 1. Perhaps a combination of these two factors suppresses the KACh channel
responses at high G
concentrations, and thus creates
the steeper, switch-like stimulus-response curve observed in our experiments.
Physiological Relevance of Modal Regulation of the KACh
Channel by G
The G-driven control of the modal prevalence is not
only evolutionarily conserved in KACh channel regulation from frog to mammalian atrial myocytes, as demonstrated in the present study, but is also widespread
and biologically versatile in other signaling systems.
The same mechanism is encountered in the neurotransmitter-mediated downmodulation of neuronal N-type
Ca2+ channels (Delcour and Tsien, 1993
) and in the
persistent activation of Na+ channels in mammalian
central neurons (Alzheimer et al., 1993
; Ma et al.,
1997
). In both systems, the G protein
subunits were implicated as the signal-relaying units that interact with
these channels (Herlitze et al., 1996
; Ikeda, 1996
; Ma et
al., 1997
). The identity of the G
subunits and the
mechanism through which they accomplish the modal
control may vary from one signaling cascade to another; still, in each case the existence of multiple functional states of the effector molecule helps to ensure
the sensitivity and fidelity of G
-mediated signaling.
In the case of the KACh channel, the type of regulation
described here can be exploited in at least two different
ways. The cell could downregulate the microscopic affinity of the KACh channel for G
, in which case the
system would filter out small changes in the G
concentration and yet allow the KACh channel to respond to
sufficient changes in G
concentration that occur
upon stimulation of either m2-muscarinic or A1-purinergic receptors. Alternatively, the KACh channel affinity for G
could be upregulated so that a small change
in the G
concentration could translate into a large
response. The variability in the apparent affinity of the
channel for G
1
5 encountered in our experiments implies that the cell, indeed, regulates the sensitivity of
the KACh channel to receptor stimulation. Recently, phosphatidylinositol 4,5-bisphosphate (PIP2) was identified as one of the potential factors contributing to
such regulation (Huang et al., 1998
; Sui et al., 1998
).
Sensitivity is only one of several important aspects of
the behavior of a signaling system. Another is its specificity. In this aspect, a signaling molecule with multiple
functional states like the KACh channel has the potential
to filter out the "membrane noise" associated with the
G protein-mediated signaling. Activation of G proteins
is a highly conserved signaling strategy used by a very
large number of different G protein-coupled receptors
colocalized in the cell membrane. Once the G proteins
are activated and the G subunits are released, a heterogeneous population of G
subunits is potentially
available to act directly on the KACh channels. Under
such circumstances, the specificity might arise from a
differential ability of different G
subunit combinations to activate the KACh channels. Studies with a limited number of purified recombinant G
subunits,
however, indicate that different G
combinations activate the channel with comparable efficacies (Wickman
et al., 1994
). Thus, there might be additional mechanisms for ensuring the specificity of interactions between the expanding number of signaling partners.
One attractive possibility is that the receptor, G protein, and the KACh channel exist as a precoupled complex (Huang et al., 1995
). Based on present study, an
additional mechanism, the presence of functional
states of the KACh channel with very low open probabilities, is suggested. Such states would absorb some fraction of the G
released in the membrane without producing substantial current through the channel, thus
filtering small inputs from multiple membrane receptors.
![]() |
FOOTNOTES |
---|
Address correspondence to Janet D. Robishaw, Henry Hood MD Research Program, Dept. of Cellular and Molecular Physiology, Penn State College of Medicine, Danville, PA 17822. Fax: 717-271-6701; E-mail: jdr{at}psghs.edu
Original version received 29 December 1997 and accepted version received 11 June 1998.
We are grateful to Drs. Howard Morgan, Olaf Andersen, and the two reviewers for critical comments on the manuscript. We
thank Dr. Mark Richardson for help with purification of G1
5 subunits, Tom Smink and Vivian Kalman for the expert technical assistance, and Holly Benscoter for artwork.
This work was supported by National Institutes of Health grant GM-39867 (J.D. Robishaw) and a Grant-In-Aid by the American Heart Association, Pennsylvania Affiliate (T.T. Ivanova-Nikolova).
![]() |
Abbreviations used in this paper |
---|
ACh, acetylcholine; GIRK, G protein-activated inward rectifiers.
![]() |
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