From the Johns Hopkins University School of Medicine, Department of Physiology, Baltimore, Maryland 21205
Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the G subunit
from G
is a highly conserved signaling strategy used by numerous extracellular stimuli. Although G
subunits
regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403-406), few tools exist for probing instantaneous G
-effector interactions,
and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the
atrial muscarinic K+ channel, which is activated by direct interactions with G
subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321-326; Wickman, K., J.A. Iniguez-Liuhi, P.A.
Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366:
654-663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133-1143), as a sensitive
reporter of the dynamics of G
-effector interactions. Muscarinic K+ channels exhibit bursting behavior upon G
protein activation, shifting between three distinct functional modes, characterized by the frequency of channel
openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated
G
) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a G
effector with allosteric regulation and an intrinsic "off" switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in G
concentration, and may be indicative of the factors regulating other G
-modulated effectors.
Heterotrimeric GTP-binding proteins (G proteins)
transduce signals from an extensive family of receptors
to a variety of cellular effectors which include plasma
membrane-localized enzymes and ion channels
(Clapham and Neer, 1993). Molecular dissection of G
protein-mediated signal transduction pathways has revealed roles for both of the products of receptor-mediated G protein activation, namely G-GTP and G
subunits (Birnbaumer et al., 1990
; Birnbaumer, 1992
;
Clapham and Neer, 1993). The distinctive functional
properties of G
and G
subunits suggest the possibility for regulatory mechanisms unique to each subunit
class.
Resolution of the kinetic features of G interactions
with cellular effectors may provide insights into the distinctive features of G
-mediated signaling. The atrial
G protein-gated inwardly rectifying K+ channel (muscarinic K+ channel), comprised of a heterotetramer
(Yang et al., 1995
) of GIRK1 and CIR (GIRK4) (Krapivinsky et al., 1995
; Hedin et al., 1996
), has been extensively studied as a direct G protein effector (Kurachi et
al., 1992
; Yamada et al., 1994a
). Both G
and G
subunits have been implicated in its activation mechanism
(Birnbaumer et al., 1990
; Kurachi et al., 1992
; Clapham
and Neer, 1993), although recent mutational analysis
and biochemical studies have clearly defined a primary
role for G
subunits (Huang et al., 1995
; Slesinger et
al., 1995
; Pessia et al., 1995
; Kunkel and Peralta, 1995
). In this study, we used the atrial muscarinic K+ channel
as a prototype G
effector to probe alterations in effector activity in response to agonist-induced alterations in G
concentrations. We find that atrial muscarinic K+ channels gate in bursts of activity, and although burst duration is not a function of G
concentration, the heterogeneous gating kinetics
within bursts are characterized by G
-mediated shifts in modal preference. These results demonstrate that
muscarinic K+ channel activity can be "titrated" by binding of variable numbers of G
, and provide evidence
for muscarinic K+ channel inactivation or desensitization.
Atrial myocytes were obtained from the bullfrog Rana catesbeiana
as described previously (Scherer and Breitwieser, 1990). Single-channel currents were recorded with standard high-resolution patch-clamp techniques (Hamill et al., 1981
) using 1 mm O.D.
square bore glass (Glass Co. of America, Millville, NJ). The membrane potential was zeroed with the following bath solution (in
mM): 150 KCl, 5 EGTA, 5 glucose, 1.6 MgCl2, 5 HEPES (pH adjusted to 7.4). Pipettes contained (in mM): 150 KCl, 1 CaCl2, 1.6 MgCl2, 5 HEPES (pH adjusted to 7.4). Acetylcholine (ACh)1
was
added to the pipette solution. The excised patch configuration was used to examine channel activity in the presence of GTP
S; bath solution for these experiments contained pipette solution plus 100 µM GTP
S ± ATP or AppNHp (as described in the individual experiments). Whole cell currents were recorded as previously described (Scherer and Breitwieser, 1990
). Currents were
recorded with a LIST EPC-7 amplifier, filtered at 2 kHz (8-pole
Bessel filter; 17 µs rise time), and stored on computer. Data acquisition and analysis were performed using PCLAMP software
(versions 5.5.1 and 6.0; Axon Instruments, Foster City, CA). Channel opening and closing transitions were identified with the half-amplitude threshold-crossing algorithm. Bursts were defined as a
series of openings (single events were eliminated from the analysis) separated by closed intervals shorter than some critical interval, tc . In most records the occurrence of bursts was infrequent so
the choice of tc was not critical and the division of the record into
bursts was unambiguous. In a few cases of high activity, when the
separation was less clear, tc was calculated as described previously
(Magleby and Pallotta, 1983
) and had values between 120 and
140 ms. For each defined burst, the duration, the number of apparent open intervals and the probability of a channel being
open within a burst, po, were calculated. The po value was calculated as the ratio of the total open time in the burst to the burst
length. When indicated, the individual events from selected
bursts were compiled to generate histograms of open- and
closed-interval durations. All experiments were done at a holding
potential of
90 mV (unless otherwise noted) and at 20-22°C.
Heterogeneity of Unitary Muscarinic K+ Currents
Acetylcholine (ACh)1 released upon stimulation of the
vagus nerve causes slowing of heart rate by activation of
muscarinic receptors and the subsequent opening of
muscarinic K+ channels in the sinoatrial node and
atrium. Unitary currents through muscarinic K+ channels were recorded from cell-attached patches on frog
atrial myocytes in the presence of different concentrations of ACh in the pipette. Fig. 1 A shows an example
of a continuous record of single-channel currents activated by 1 µM ACh; the membrane potential of the
patch was held at 90 mV. Channel gating behavior is
heterogeneous, characterized by both apparent clustering of openings into bursts and spontaneous changes
in the pattern of channel activity.
Muscarinic K+ Channels Gate in Bursts
Records, such as that illustrated in Fig. 1 A, indicate
that bursts of muscarinic K+ channel openings are separated by long silent periods; individual bursts were
therefore considered to reflect the activity of a single
ion channel and analyzed further. Fig. 1 B illustrates the burst length histogram obtained from activity elicited by 1 µM ACh, plotted on a logarithmic time scale.
The histogram was best fitted by a single exponential
term with b = 193.4 ms. Burst length distributions were
also obtained at 50 nM and 0.5 µM ACh (Table I). No
significant differences between burst length time constants were evident, indicating that agonist concentration does not determine burst length. Furthermore,
the holding potential (
60 or
90 mV) did not significantly affect the burst length distribution obtained at
0.5 µM ACh (Table I).
Table I. Burst Duration Time Constants for Atrial Muscarininc K+ Channels Determined under a Variety of Experimental Conditions |
The transient nature of G interactions with effectors presents the possibility that the dynamics of G protein turnover may limit channel burst length under
physiological conditions. All heterotrimeric G proteins
exploit a highly conserved signaling strategy in which
the metastable GTP-bound state of the G
subunit is
used as a molecular clock (Bourne et al., 1990
, 1991
).
When GTP is hydrolyzed to GDP, G
is inactivated, and
its affinity for free G
is increased. The potential contributions of G protein turnover to the kinetics of G
protein-mediated signaling can be eliminated with the
hydrolysis-resistant GTP analogue, GTP
S. Binding of
GTP
S to G
persistently activates G proteins, constraining both G
and G
to their active states, reducing the signaling pathway to diffusion-limited interactions between G
and the channel. Fig. 2 A illustrates
representative unitary K+ currents recorded from an
excised, inside-out membrane patch in the presence of
100 µM GTP
S. In the presence of GTP
S, channel activity was similar to that observed in cell-attached
patches in the presence of 1 µM ACh. Indeed, comparison of the burst length distributions displayed in Fig. 1
B and 2 B reveals no significant differences in
b for the
two conditions (Table I). Thus, the combined data in
Figs. 1 and 2 suggest that muscarinic K+ channel burst
duration is not determined by agonist concentration, nor the dynamics of G protein turnover, i.e., GTP hydrolysis, presenting the possibility that burst length is
determined by a property intrinsic to the muscarinic
K+ channel itself, i.e., inactivation or desensitization in
the continued presence of G
, or may be a feature of
the G
-channel interaction.
Heterogeneity of Muscarinic K+ Channel Gating within Bursts
Although channel burst duration is independent of the
agonist concentration, inspection of current records reveals two features of muscarinic K+ channel gating worthy of further exploration. First, gating is heterogeneous over a wide range of conditions including different agonist concentrations (Fig. 4 A), in excised patches
following GTPS-mediated activation of the channel
(Fig. 2 A), and at different membrane potentials over
the range from
110 to
40 mV (Fig. 3). Second, despite the apparent gating heterogeneity, there is a systematic increase in the open probability during individual bursts as the ACh concentration is increased, as illustrated in Fig. 4 A for individual bursts recorded in
the presence of two different ACh concentrations. To
determine whether these features of muscarinic K+
channel gating could be related to the level of activated
G
, we employed several complementary approaches,
at low (50 nM) and high (1 µM) ACh concentrations,
to separate bursts into two or more discrete and kinetically simplified gating modes. First, we examined the
distributions of the number of apparent openings for
bursts composed of two or more openings. These distributions should consist of a sum of geometric components, the number of components being equal to the
number of open states, although the contributions of
some components may be too small to resolve (Colquhoun and Hawkes, 1983
). Examples of histograms of
the apparent openings per burst are presented in Fig. 4
B for 50 nM and 1 µM ACh. Both distributions were
best fitted by a sum of two geometrics, µ1 and µ2: the
first component had a mean value of approximately three openings per burst, while the mean of the second
component was ~15. Comparison of the fits to these distributions reveals that the agonist concentration determines the proportion of bursts in the two groups: the
fraction of bursts containing a larger number of apparent openings increased from 36 ± 6% to 62 ± 5% when
comparing bursts obtained in 50 nM and 1 µM ACh, respectively, while the mean values for µ1 and µ2 were
similar for the two concentrations.
Classification of bursts into groups according to the
two geometric components in Fig. 4 B hinted at the existence of distinct gating modes, but even among bursts
with an equivalent number of openings, the gating behavior remained heterogeneous with respect to both
mean open time and open probability. Therefore, as
an alternative approach to grouping bursts and identifying distinct patterns of gating, we plotted the number
of apparent openings in the burst, N, vs. the length of
the burst, B, for each burst. Illustrated in Fig. 4 C are
plots for bursts recorded in the presence of either 50 nM ACh or 1 µM ACh. Each symbol in the figure represents the kinetic behavior of an individual burst. Histograms of frequencies of openings within individual
bursts (f = N/B) were generated from the same bursts
illustrated in Fig. 4 C to empirically determine the most
populated regions of the N-B plots. The histograms, illustrated in Fig. 4 D, define three distinct peaks centered around f values of 0.03 ms1, 0.06 ms
1, and 0.12 ms
1. These frequencies were therefore used as the criteria for separation of channel gating behavior into
low-, medium-, and high-f modes (and were used to
generate the lines in Fig. 4 C). Although the predominant frequencies of channel openings were independent of ACh concentration, comparison of the two
plots in Fig. 4 D indicates that modal preference is a
function of the ACh concentration. At 50 nM ACh, low-
and medium-f behavior dominate the f -histogram, while saturating ACh concentrations favor the high-f
mode. To determine the maximal effect on modal preference, we also analyzed the frequency of openings
within individual bursts, f, for a total of 273 bursts, recorded in excised patches exposed to 100 µM GTP
S. The frequency distribution after GTP
S activation revealed three distinct peaks, corresponding to the low-f
(0.032 ms
1; relative area 13.5%), medium-f (0.064 ms
1; relative area 36.5%), and high-f (0.116 ms
1; relative area 50%) modes characterized previously in the
cell-attached configuration in the presence of ACh
(Fig. 4 D). Thus, the heterogeneous kinetic behavior
observed in the presence of GTP
S reflects sojourns of
the channel in the same dominant gating modes found
in the presence of ACh.
Kinetics of Muscarinic K+ Channel Gating within Distinct Gating Modes
Although ACh concentrations establish modal preference for muscarinic K+ channels, all modes are represented at any given ACh concentration. To characterize
the kinetic behavior of the channel in each gating mode, we analyzed histograms of open and closed interval durations generated from low-, medium-, and
high-f burst populations recorded in 1 µM ACh (Fig.
5). For this analysis, only bursts with f values within 2 SD of the mean of a particular f population were included (eliminating bursts in which there was an obvious switch in gating mode during the burst). Each
open and closed state adds an additional exponential
component to these distributions (Colquhoun and
Hawkes, 1981). Within each mode, a sum of three exponentials provided a satisfactory fit to the open time
distributions (Fig. 5, left). The fast, intermediate, and
slow components showed little variation from one
mode to the next, and had time constants (
o1
o3) of
~0.18, 1.5, and 5.0 ms, respectively. The relative areas
under the individual exponentials, however, were quite
different for the three modes and verified the general
observation that the low-f bursts were predominantly
made of short openings while high-f bursts were dominated by longer ones.
Closed intervals were distributed over a wider time
range than open times, and fitting of closed-time histograms obtained from randomly compiled bursts required more than three exponentials. After modal classification of the bursts at 1 µM ACh, however, three exponential terms were sufficient to describe the closed times in each mode (Fig. 5, right). The fast and intermediate components had similar time constants for the
three kinetic modes (c1 = 0.23 ms and
c2 = 1.4 ms),
whereas the third time constant,
c3 , decreased as the
frequency increased, being 67.8 ms for the low-f bursts,
23.9 ms for medium-f, and 12.3 ms for the high-f bursts.
Similar analysis of mode-segregated bursts recorded
from GTPS-activated patches was performed (data not
shown). The fits to the open-time distributions yielded
fast, intermediate, and slow time constants which were
similar in all modes and comparable to those observed
in the presence of ACh (
o1 = 0.14 ms;
o2 = 1.4 ms;
o3 = 4.0 ms). The fast and intermediate closed time constants had mean values of
c1 = 0.15 ms and
c2 = 1.3 ms
in all modes, while the slow closed time constant,
c3,
was once again the primary discriminator between
modes, being 80.0 ms for the low-f mode, 23.1 ms for
the medium-f mode, and 12.4 ms for the high-f mode. These results suggest that the channel enters the same
gating modes whether the G proteins are activated by
GTP or GTP
S, with a minimum of three open (O1-O2-O3) and three closed (C1-C2-C3) states within each
mode. The values for all three open time constants (
o1-
o3) as well as the fast and intermediate closed time
constants (
c1 and
c2) are similar for the three modes.
As long as the conducting and nonconducting states of
the channel can be regarded as reporters of distinct
conformational states of the protein, our results imply
that gating in any mode arises from a common set of
five conformational states O1 . . . C2 and one additional nonconducting state C3 which is the main kinetic discriminator between the modes. The concentration of agonist (and presumably the concentration of
G
) has the unique role of establishing modal preference, i.e., determining the ease with which the channel enters the higher frequency gating modes.
Physiological Relevance of Modal Shifts in Gating
Activation of whole cell muscarinic K+ current (IK[ACh])
by application of ACh incorporates both the kinetic
changes within bursts which have been described in
previous sections, as well as potential alterations in interburst intervals and the number of functional channels. To determine the physiological relevance of the
modal shifts in gating within bursts, three measures of muscarinic K+ channel activity were compared at a
range of ACh concentrations, Fig. 6. As a standard for
comparison, the steady state whole cell dose response
for ACh was plotted (data normalized to the current at
1 µM ACh). To assess the dose response relation for
ACh-mediated shifts in po within bursts, the average po
within 150 random (i.e., non-mode-segregated) bursts
was tabulated, at three ACh concentrations (50 nM, 0.5 µM, and 1 µM). The average pos were normalized to the value obtained at 1 µM ACh (po = 0.3). Finally, to
determine the overall dose response relation for ACh
at the single channel level (bursts plus interburst intervals), the po of representative recordings in the cell-attached configuration was determined at 50 nM, 0.5, and 1 µM ACh (normalized to the po obtained at 1 µM ACh 0.047). All three measures of the dose response
relation are similar, suggesting that ACh does not differentially affect the interburst intervals, and therefore
that the ACh-induced shifts in modal gating within
bursts are the primary determinant of the overall,
whole cell current response.
This study reveals two novel aspects of muscarinic K+
channel gating. First, muscarinic K+ channels gate in
discrete bursts of activity. Burst duration is monoexponentially distributed and insensitive to a variety of factors which modulate muscarinic K+ channel activity, including agonist concentration, membrane potential, and G protein turnover (i.e., GTP hydrolysis). Second,
the gating of muscarinic K+ channels within bursts is
heterogeneous, but discrete modes of gating can be defined based on the frequency of channel opening within a burst. Agonist concentration determines
modal preference, although all modes are represented
at all agonist concentrations. Agonist-induced shifts in
modal preference are the main determinant of whole
cell IK[ACh]. We will consider these results in light of extensive studies which suggest G to be an integral activator of muscarinic K+ channels (Logothetis et al.,
1987
; Kurachi et al., 1992
; Wickman et al., 1994
; Yamada et al., 1994a
; Krapivinsky et al., 1995
), with direct
binding to the channel recently demonstrated in vitro (Huang et al., 1995
; Slesinger et al., 1995
; Pessia et al.,
1995
; Kunkel and Peralta, 1995
). It is also possible that
additional, muscarinic receptor- or G protein-activated
signaling pathways modulate muscarinic K+ channel
activation (e.g., Scherer and Breitwieser, 1990
; Yamada et al., 1994b
). Since these additional pathways are presumably also activated in a dose-dependent manner by
ACh, our results at present cannot distinguish between
the primary G
interaction with the muscarinic K+
channel and these modulatory influences. We thus
limit our discussion to the minimal model which requires only direct G
interaction with the channel
heteromultimer.
Burst Duration Is Independent of G Concentrations
The simplest model for determination of muscarinic
K+ channel burst duration consistent with the data is
one in which the channel enters a discrete inactivated
or desensitized state (independent of G), as indicated in the following scheme.
![]() |
The channel undergoes repeated transitions between
closed and open states (as determined by G interactions) and, with a rate constant k1 (reflected in the time
constant for burst duration), enters the inactivated
state. k
1 represents the time constant for exit from the
inactivated state, and can in principal be obtained from
interburst intervals. In practice, however, this is not
possible since it is difficult to unequivocally establish
the number of channels in the patch (maximal patch
open probability is 0.08-0.1). Nevertheless, the existence of an inactivated, nonresponsive state limits the
activity of the channel, and, if G
subunits interact directly with muscarinic K+ channels, has implications for
G
-mediated signaling.
G subunits lack an intrinsic enzymatic activity
which serves to periodically alter the conformational
state of the complex (Sondek et al., 1996
; Neer and
Smith, 1996
) and hence the affinity for either effectors
or G
subunits. Dissociation of G
from effector binding sites may therefore be the rate-limiting step in termination of G
-mediated signaling. Inactivation of
muscarinic K+ channels presents the possibility that effectors themselves may contribute to termination of
G
-mediated responses. Channel inactivation may be
accompanied by either a transient decrease in channel
affinity for G
(i.e., a conformational change may occur in the G
binding site), or it may occur despite
the continued presence of bound G
, with subsequent dissociation of G
while the channel is inactivated.
Modal Preference Determination by G Concentrations
The native muscarinic K+ channel in atrial myocytes
contains multiple structurally heterogeneous, G
binding sites within what is most likely a tetrameric
channel structure (Krapivinsky et al., 1995
; Yang et al.,
1995
). In this context, the simplest model of channel
gating would require the occupancy of all G
binding
sites before channel activation. The ability of ACh (i.e., G
) to titrate modal preference, however, favors more
complex models, in which distinct functional consequences, i.e., modal shifts, accompany binding of successive G
. While our data at present cannot be constrained to a model which imposes a one-for-one correlation between the number of bound G
and a
particular gating mode, a reaction scheme similar to
the Monod-Wyman-Changeux allosteric model (Monod
et al., 1965
) in which all modes are interconnected, and binding of increasing numbers of G
subunits facilitates transitions to the high-f mode is entirely consistent with our observations. A similar mechanism has
been proposed for G protein-mediated inhibition of
neuronal N-type Ca2+ channels (Boland and Bean,
1993
; Delcour et al., 1993
; Delcour and Tsien, 1993
),
which has recently been shown to be mediated by G
(Ikeda, 1996
; Herlitze et al., 1996
).
Modal Shifts as a Means of Increasing G
Signaling Specificity
Some effectors are activated in a highly specific manner by particular subtypes of G (Inguez-Liuhi et al.,
1992
; Schmidt et al., 1992
; Kleuss et al., 1993
; Wu et al.,
1993
) while other effectors are activated equally well by
all G
subtypes (Clapham and Neer, 1993; Wickman
et al., 1994
; Ueda et al., 1994
). Muscarinic K+ channels
fall into the second class, i.e., all G
subtype combinations are equally effective at the activation of the channel (Wickman et al., 1994
), with the exception of transducin
(Yamada et al., 1994a
). This presents a conceptual difficulty, since it implies that activation of any
G protein-coupled receptor within the cell should contribute to the population of activated G
and thus muscarinic K+ channel activation. Signaling specificity
must therefore arise on the basis of other mechanisms.
Our results suggest a possible means for increasing signaling specificity and/or sensitivity in spite of a lack of
G
subtype specificity, namely, the existence of multiple functional states of an effector, which are dependent upon the number of bound G
. The efficiency
of signal transduction via a population of G
would
thus depend not only on the amount of free G
and
the relative abundance of effectors, but also on the
number of functional modes accessible to the various
effectors and on the ability of G
to control the equilibrium between these modes. In the case of the muscarinic K+ channel, the combination of an intrinsic inactivation mechanism which may serve to limit the duration of the G
interaction plus allosteric regulation
via multiple G
subunits (which is poised to provide
significant whole cell K+ current only at high G
concentrations) produces a system which is rapidly regulated and highly sensitive to dynamic changes in G
concentration.
Original version received 14 May 1996 and accepted version received 8 October 1996.
Address correspondence to G.E. Breitwieser, Johns Hopkins University School of Medicine, Department of Physiology, 725 N. Wolfe Street, Baltimore, MD 21205. Fax: 410-955-0461; E-mail: gbreitwi{at}welchlink.welch.jhu.edu
We thank E. Nikolova, F. Sigworth, M. Li, and W. Agnew for helpful comments at different stages of the work. We thank C. Frederick Lo for providing the whole cell acetylcholine dose response data.
This work was supported by the National Institutes of Health (HL41972), a National Science Foundation Career Advancement Award (9407251) and an E.I. from the American Heart Association (AHA) National Center (900126) to G.E. Breitwieser. T.T. Ivanova-Nikolova was partially supported by a Fellowship from the AHA Maryland Affiliate.
ACh, acetylcholine.