- Email: [email protected]
G-protein control of cardiac potassium channels
activate two disparate targets after the occupation of a single receptor.
G-Protein Control of Cardiac Potassium Channels Yoshihisa Kurachi
Two cardiac potassium (K+) channels are activated by pertussis toxin (PTX)-sensitive G proteins either directly or in a “membrane-delimited” manner. They are muscarinic K+(KAcH) and ATP-sensitive K+(KATp) channels. KAch channels are responsible for acetylcholine (ACh)- or adenosine-induced deceleration of the heartbeat and atrioventricular conduction, while KATpchannels are responsible for the ischemiainduced shortening of the cardiac action potential and possibly for the adenosine-mediated protection from ischemic damage. Distinct molecular mechanisms underlie G-protein activation of these cardiac K+ channels; the a subunit of PTX-sensitive G proteins activates the KATp channels, while &subunits activate the KAchchannel. The physiologic significance of this heterogeneous mechanism remains to be determined. (Trends Cardiovasc Med 1994;4:64-69)
Regulation of potassium (K+) channels via specific receptors is an essential component of neurohumoral control of the heart. GTP-binding (G) proteins are now known to be involved either directly or indirectly (via second messengers) in the regulation of a number of cardiac K+ channels (Table 1). This direct or at least membrane-delimited regulation of ion channel function by G proteins is a newly recognized mode of the remotesensor-type regulation of ion channels (Hihe 1992). This system was first identified for the muscarinic activation of a cardiac K+ (KAch) channel. In addition, a number of other K+ channels, Ca2+ channels, Na+ channels, and Cl- channels have been shown to be regulated by G proteins in a membrane-delimited manner in a variety of tissues, including cardiac, neuronal, and endocrine cells.
Yoshihisa Kurachi is with the Division of CardiovascularDiseases, Departmentof InternalMedicine and Departmentof Phannacology, Mayo Clinic, Rochester, MN 55905, USA;and with the Departmentof Phannacology II, Osaka UniversitySchool of Medicine, Siuta,Osaka565, Japan. 64
This is one of the most important cellsignaling mechanisms in the regulation of cellular function [for a review, see Brown and Birnbaumer (1990), Hill (1992), and Kurachi et al. (1992)]. The G protein is a heterotrimer composed of G,, Gg’ and Gy, which is functionally dissociated into the GTPbound form of G, (Ga_oTp)and G& upon receptor stimulation (Gilman 1987). Either of these subunits of the G protein may regulate effecters, such as adenylyl cyclase, phospholipase C, and ion channels. The main object of this review is to assess the current situation of the controversy concerning whether it is the or the GButhat forms the physioGa_GTP logic activator of the KAch channel of atrial muscle. The evidence is now overwhelming that G& serves to activate the K*ch channel. This does not necessarily make G, nonfunctional, however, since this subunit of the activated G protein has been found to stimulate another cardiac K+ channel, the ATP-sensitive K+(K,,) channel. This elegant system, the “remote control” of an effector by a receptor via the intermediary step of G-protein utilization, is thus able to 01994, Elsevier Science Inc., lOSO-1738/94/$7.00
l
“Membrane-Delimited” G-Protein Regulation of the Cardiac KAch Channel
Pfaffinger et al. (1985) were the first to report the involvement of pertussis toxin (PTX or islet-activating protein)-sensitive G proteins in the muscarinic cholinergic induction of a specific inwardrectifying K+ current in cardiac atria1 muscle. The G protein was designated as G, based on this function [see Breitwieser and Szabo (1985)]. In inside-out patch membranes of cardiac atrial cells, Kurachi et al. (1986 a and b) showed the KACh channel to be activated by intracellular GTP (GTP,) in the presence of suitable agonists (ACh or adenosine) or by the nonhydrolyzable GTP analogue, GTP$% Intracellular AlF,- also induced activation of KAch channel in an agonistindependent manner (Kurachi et al. 1992). This AIF,- activation was prevented when the inside-out patches were pretreated with GDP@% The agonistdependent GTPi-induced channel activation was blocked by treating the insideout patch with the A protomer of PTX and nicotinamide-adenine dinucleotide. Since PTX is known to specifically ADPribosylate a certain population of G proteins and uncouple the membrane receptors and G proteins (Gilman 1987) it was concluded that a PTX-sensitive G protein (G,) couples m2-muscariniccholinergic and A,-purinergic (adenosine) receptors and the KAChchannel in the atrial cell membrane. Involvement of soluble second messengers can be excluded from this system, since these experiments have been performed in the excised inside-out patches. These observations clearly indicate the “membrane delimited” nature of G-protein activation of K*ch channels. The questions that we now address are (a) how G, activates the KAC,,channel and (b) which G, subunit activates the channel.
l
Physiologic Mode of G-Protein Activation of the Cardiac KACh Channel
To define the physiologic interaction between activated GK(GK*) and KAch channel, we examined the concentrationdependent effect of GTPi on the KACh
TCM Vol.4,No. 2,1994
Table 1. Receptor-dependent-G-protein-mediated
regulation of cardiac K+ channels Intracellular second-
Effect mediated
Channel Membrane-delimited Muscarinic
messenger
systems
regulation Activation by m,-muscarinic, cholinergic, and A,-purinergic Rs Activation by A,-purinergic R
K+ channel
ATP-sensitive
G twoteins
bv recerDtors
K+ channel
Second-messenger-mediated
PTX-sensitive
G (G,)
No
PTX-sensitive
G (G,?)
No
regulation
Delayed outward I, channel
Transient outward K+ channel Inward-rectifier Ix, channel
Activation by l3-adrenergic Inhibition by m2-muscarinic, ergic, and A, -purinergic Activation by a-adrenergic Inhibition by a-adrenergic Inhibition by a-adrenergic
R cholinRs R R R
CAMP-A kinase
G, G, PTX-insensitive PTX-insensitive PTX-insensitive
DAG-C kinase Unidentified Unidentified
G (G,?) G (G,?) G (G,?)
R, receptor.
channel.
We found that GTPi activates channel in a highly positive
the KACh cooperative manner (It0 et al. 1991). Figure la shows the effects of GTPi on K *ch channel openings in the absence and presence of ACh. Figure lb shows the concentration-dependent activation of the KAch channel by GTPi in the presence of various concentrations of ACh in the pipette. The relationship between GTPi and channel activity at each concentration of ACh was fitted by the following Hill equation with use of the least-squares method: y =Vw/[
l+(K,,/[GTP])H]
the maximal response and the apparent affinity of the Knch channel for GTP,, which may be due to the facilitation of functional dissociation of G, induced by agonist binding to the muscarinic cholin-
ergic receptors (Kurose et al. 1986). The positive cooperative effect of GTPi on the channel open probability may be derived from the intrinsic properties of the association of G-protein subunits and the
Figure 1. Concentration-dependent effect of intracellular GTP on the KAch channel in the absence and presence of ACh. (a) Examples of inside-out patch experiments. The concentration of ACh in the pipette is 0 or 1 p&4as indicated. The bars above each truce indicate the protocol of perfusing various concentrations of GTP and 10 ph4 GTPyS. The membrane potential was -80 mV. Note that a three- to tenfold increase in GTP concentration resulted in a dramatic increase of N.P, of K,, channels, indicating the existence of a highly cooperative process. (b) The relation between the concentration of GTP and the relative N.P,, of KAChchannels with reference to the N.P, induced by 10 @I GTPyS in each patch. Symbols and bars are mean f SD. The continuous curves are fitted by the Hill equation with the least squares method: 0 @vl ACh (open circles, n = 7), 0.0 1 @VIACh (closed circles, n = 6), 0.1 pIv4ACh (closed triangks, n = 6). and 1 pkl ACh (closed squares, n = 6). Reproduced with permission from Ito et al. (1991).
V,, = the where y = the relative N-P,, maximal N.P,,, Kd = the GTP concentration at the half-maximal activation of the channel, and H = the Hill coefficient. The channel activity was expressed as N-P,, where N is the number of the channel in the patch and P, is the open probability of each channel. The relative N.P, was
a
W ACh
defined with reference to the value induced by 10-100 FM GT@y!5 in each patch.
10 pA
ACh 1 ELM GTP 0.03 PM,
0.1
0.1
I
,-m
0.3
As the concentration of ACh in the pipette was raised from 0 to 0.01, 0.1, or 1 pM, the following points were observed: (a) the threshold concentration of GTPi necessary to induce openings of the channel decreased, (b) the concentration of GTPi for half-maximal activation of the channel (K,) decreased, and in(c) the maximal relative N-P,,(VMAX) creased; however, (d) the Hill coefficient of the fitted curve was constant around 3 and was independent of the ACh concentration. These results indicate that ACh binding to the receptor increased both
TCM Vol. 4, No. 2, 1994
concentration
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of GTP
10
I 100
65
activate the KAChchannel but also corresponds to the physiologically functional G, subunit that activates the channel (Ito et al. 1992, Kurachi et al. 1992, Yamada et al. 1993a-b, Murphy et al. 1993).
t
0
GK
Figure 2. Hypothetical model for activation of the KAch channel by G,’ in the cardiac atrial cell membrane. The K,, channel is
assumed to be composed of four identical subunits. One activated G, (Gx*) binds to each subunitto open the KACh channel.
K*c,, channel, which has multiple (>3) binding sites for G; (Ito et al. 1991, Karshin et al. 1991). Based on the models proposed for the structure of the inward-rectifier K+ channels recently cloned from rat kidney and mouse macrophage (Ho et al. 1993, Kubo et al. 1993), we assume that the KAChchannel is also a homotetramer. One G,’ may
bind to each segment in order to activate the KACh channel (Figure 2).
l
G-Protein Subunit Activation of the Cardiac KAch Channel
To identify the G-protein subunit that is
responsible for activation of the KAch channel, recombinant or purified G,s and GBrhave been applied to inside-out patches of the atria1cell membrane [for a review, see Brown and Birnbaumer (1990), Kurachi et al. (1992), and Yamade et al. (1993b)l. It has been reported that both Gi, cTWs (Codina et al. 1987, Yatani et al. 1987, Logothetis et al. 1988) and Gti (Logothetis et al. 1987 and 1988, Kurachi et al. 1989a, Kobayashi et al. 1990) could activate the KAChchannel. Considerable controversy surrounds G,+,activahas tion of the KAChchannel, since Gcl_GTp been generally recognized as the functional arm responsible for regulation of various effecters in G-protein-linked systems (Gilman 1987). By now, however, convincing evidence has been gathered to indicate that G& is not only able to
66
be made between different membrane patches that contain a different number of channels and only in -30% of patches (40 of 124)l. In contrast, GBv(10 nM) effectively activated the K,,ch channel in all patches examined (~400 patches) with comparable efficacy as 10-100 p.M The main points of the argument GTPyS. Second, after GBuwas incubated against GBuactivation of KAChchannels were (a) Gia_cTPys~activated the KAch for 2448 h (at 4°C) in Mg*+-free EDTA solution containing 2-10 @I GDP (or channel at subpico - picomolar concenGDP-BS), a treatment that would inactitrations, whereas GBuwas only effective vate any GTPyS-bound form of G,, the in nanomolar concentration; the activaG& activated the KAChchannel as effection by Gb observed by some investigatively as nontreated GW Third, boiled GBv tors was supposed to result from condid not activate the KAch channel. tamination of the GBupreparation by G, (Codina et al. 1987, Kirsch et al. 1988). Fourth, we could not activate the KAch (b) The detergent (CHAPS)used to sus- channel with CHAPS,and GBususpended in Lubrol PX activated the KAChchannel pend the hydrophobic G& itself could as effectively as that suspended in CHAPS, have activated the KAChchannel. Thus, indicating that activation was “detergent the activation of the KAch channel by independent.” Fifth, GBu preincubated detergent-suspended GBu is an artifact with excessive GDP-bound form of G, or (Yatani et al. 1990). G, failed to activate the channel. These In an exhaustive series of studies, we observations exclude the possibility that have been able to counteract the above G& activation of the KAch channel is an arguments (Kurachi et al. 1989a, Kobayashi et al. 1990, Ito et al. 1992, artifact. Furthermore, G,+,activated the Yamada et al. 1993a). First, Gi_,a_GTqS KAChchannel in a positive cooperative manner, which mimics the characterand Gi_za_Gm (100 pM to 10 nM) actiistic mode of activation of the KAch vated the KACh channel only slightly [-lo%-20% of the activation induced by channel by GTPi (Figure 3). Two lines of evidence indicating that 10-100 p.M GTPyS, which is used as a benchmark that enables comparisons to GKa_GTPphysiologically activates KAch Figure 3. Concentration-dependent activation of the KAChchannel by GTP, GTP@, brain GBy, and retinal T The relation between the KAchchannel activity and GTP (in the presence of ACh), GTPyS,%rain G&, and retinal TBuare depicted. The effect of Giacrqss is also indicated in the graph. The concentration-dependent effect of GTPi on the KAChchannel is shown in the internal solutions containing either 130 mM Cl- or 65 mM SO,*-. Intracellular Cl- disturbs the turnoff reaction of KAChchannel activity probably by inhibiting the intrinsic GTPase activity of G,, resulting in higher sensitivity of the KAChchannel to GTP, (Nakajima et al. 1992). The relative N.P, of the channel at each concentration of various substances was obtained with reference to the N.P, value induced by 10-100 fl GTPyS in each patch.
i-
r-
1.0 -I
GTP(SO,s)
9
retinal Tpy
brain Gpy
GTP(CI-)
0.8
0.6
0.6
0.6
P ‘Z $ 0.4 Gia-GTPrj 0.2
:
: .. IO-8
104
IO-4
IO-2
GTP & GTPyS (:.I)
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IO-‘2
1o-10
10-s
104
lo”
G protein subunits (M)
TCM Vol. 4. No. 2, 1994
channels have also been provided: (a) A monoclonal antibody to the transducin a subunit (4A) irreversibly inhibited the agonist-dependent-GTPi-induced activation of the KAChchannel, which could be
brain G& (Ito et al. 1992)
restored
(see also Yamada et al. 1993~). Kim et al. (1989) proposed that the arachidonic acid-eicosanoid pathway mediated the exogenous G& activation of the KACh channel in inside-out patches, which is not involved in the physiologic G, activation of the KACh channel. This proposal is not valid, since the stimulatory effect of arachidonic acid metabolites on the KAch channel clearly requires intracellular GTP (Kurachi et al. 1989b and 1992) and the activation of the KACh channel by G&was not affected by the inhibitors of arachidonic acid metabolism (Ito et al. 1992). The arachidonic acid metabolites are stimulatory modulators of the G, function (Kurachi et al. 1989b) and may be involved in stimulation of the basic activity of KAch channels by a-adrenergic agonists and platelet-activating factor (Kurachi et al. 1992) and also in the short-term desensitization of ACh activation of KAch current (Scherer et al. 1993). Current data support a direct activation of the KAch channel by Gw
only by exogenously applied (Yatani et al. 1988). This result Gia-GTPfS was interpreted as indicating that the antibody inhibited the interaction between G, and the KAch channel. (b) Both transducin & dimers (Tt,J purified from bovine retina and G& from other mammalian tissues not only failed to activate but actually inhibited agonistdependent-GTP,-induced KACh channel activation (Okabe et al. 1990). Contrary to these reports, we found that the antibody 4A interferes with the interaction between muscarinic receptors and G, and not that between G, and (Nanavati et al. 1990, K *ch channels Kurachi et al. 1992). Thus, one cannot differentiate which subunit of G, is physiologically responsible for the activation of KAch channel based on the results obtained with 4A. We also found that, although Gb purified from bovine brain consistently and fully activates K *ch channels, it never inhibits agonistdependent-GTPi-induced KACh channel activation (Ito et al. 1992). Furthermore, detergent-free T& does not inhibit but in fact activates the KAch channel in a positive cooperative manner (Figure 3; Yamada et al. 1993a). Thus, we could not support the earlier findings and notion that G,, is the physiologic activator of the KAch channel. A new set of experiments in fact indicate that GxBu is the physiologic activator of the KAch channel (Yamada et al. 1993a). We found that exogenously the agonistapplied Ta_onP inhibited dependent-GTPi-induced or GTPy!!+induced activation of K,,ch channels irreversibly, probably by binding to G,,+, and forming a trimer of T,_,,,-GxBU. Since this heterotrimer may not be able to interact with mz-muscarinic or A,purinergic receptors, the KACh channel activity was inhibited irreversibly. Only exogenously applied T,,,, or GBr could restore channel activity. These results strongly suggest that it is G,& that is the physiologic functional arm of G, that activates Knch channels. The KACh channel is activated in a highly positive cooperative manner by GTP (in the presence of agonists) (Ito et al. 1991), GTP$S (Nakajima et al. 1992)
TCM Vol. 4, No. 2, 1994
and retinal T&
(Yamada et al. 1993a) (Figure 3). It would therefore appear that G,‘, brain GBv’ and T& interact with the KACh channel
l
through
a common
mechanism
G-Protein Activation of Cardiac KATp Channel
Since the initial reports that G proteins might be involved in the regulation of the channel in the pancreatic l3 cell $p unne et al. 1989) and in skeletal muscle fibers (Parent and Coronado 1989), G proteins have also been found to activate the KATp channel in cardiac myocytes (Kirsch et al. 1990, Ito et al. 1992). In inside-out patches of guinea pig ventricular cell membrane, where KAch channels are not expressed, GTP (in the presence of ACh or adenosine) and GTPyS induced an increase of KATp channel activity, which had been suppressed by intracellular ATP (ATP,) (Kurachi et al. 1992). AlF,- also induced an increase of the KATp channel activity in the presence of ATP,, which was prevented by pretreating the patches with GDP-l3S. Gia_oTFFs purified from bovine brain also increased KATpchannel activity that had been suppressed by ATPi (Ito et al. 1992). G,+, did not affect the baseline activity of the channel but inhibited the receptor-mediated-GTPi-
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induced increase of KATp channel activity. These observations suggest that the KATp channel in the ventricular cell membrane can be activated by G proteins, and in this case it is the a subunits of PTX-sensitive
G proteins
that activate
the KATpchannel.
l
Distinct Molecular Mechanisms Underlie G-Protein Activation of the Cardiac K+ Channels
Figure
4a shows an experiment where and G,, were applied to an Gi-la-GTQ5 inside-patch of atrial cell membrane, where both KACh and KATp channels are found (Ito et al. 1992). In the cellattached patch, the KAch channel was activated vigorously by ACh (1 p.M) in the pipette. In the excised inside-out patch, the openings of the KAChchannel decreased to a minimal background level (Figure 4a-1). The internal solution contained 100 @I MgsATP to suppress the KATpchannel openings and also to maintain availability of the channels. When was applied to the internal Gi-la-GTqS side of the membrane, K+ channel openings with a conductance of -90 pS were clearly induced. On the other hand, the openings of the KACh channel were not affected significantly (Figure 4a-2). Openings of the 90-pS K+ channel were inhibited by 1 @I glibenclamide, indicating that this was the KATp channel. Subsequent application of G& to the patch dramatically increased openings of the 40 to 45-pS KAChchannel (Figure 4a-3 and -4). The Gt,,-induced openings of the KAch channel were not affected by glibenclamide. Thus, distinct molecular mechanisms underlie G-protein activation of these cardiac K+ channels. Stimulation of m2muscarinic or A,-purinergic receptors results in PTX-sensitive G proteins dissociating into Gn_oTP and Gw Ga_oTP activates the KATpchannel and Gti activates the KACh channel (Figure 4b).
l
Conclusions
There is now overwhelming evidence that GI<& is the physiologic functional arm of G, activating KAch channels in the heart. A growing body of evidence also shows that GBu regulates various other effecters, such as type-2 and type-4 adenylyl cyclases (Tang and Gilman 1991), phospholipase Cl32 in mammals
67
The major unsolved questions regarding G, activation of KAch channels are (1) Which pertussis-toxin-sensitive G protein serves as G,, (b) of which subtypes of G,, GP and G,,is G, composed, and (c) how does G,& interact with K,,ch channels? In other words, we have not yet fully answered the question of how information specifically passes from a membrane receptor to the effector, the K Ach channel. Similar questions are unanswered in the case of the KArp channel. The physiologic significance of this heterogeneous molecular mechanism of Gprotein activation of different cardiac K+ channels also awaits an answer.
l
Acknowledgments
The author thanks Dr. Ian Findlay (Univer-
b
ACh Adenosi ne
M R
Breitwleser GE, Szabo G: 1985. Uncoupling of cardiac muscarinic and g-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature 317:538-540.
GTP
Figure
4. (a) Effects of Gi_ta_orPis and G& on the KATpand KAch channels in the atrial cell
membrane. The pipette solution contained 1 @l ACh. The inside-out patch was formed at the arrow (I/O) above the current trace in the internal solution containing 100 pM ATP and 0.5 mM MgClz (A-1). Gt.1, oq (300 PM) was applied first to the internal side of the patch and clearly induced openings of the KATpchannel (-90 pS) (A-2) without affecting background activity of *e KACh channel. Subsequently, GBu(10 nM) was applied to the patch and caused a dramatic increase of 45pS KAChchannel openings (A-3 and -4). Numbers above the current trace indicate the location of each expanded current trace shown below. In the expanded current traces, the dotted line is the first open level of the KAChchannel and the continuous line is that of the KArp channel. The arrowhead in each trace is the zero current level. (b) Proposed mechanisms of the pertussis toxin (PTX)-sensitive G-protein-subunit activation of the KArpand KAChchannels in cardiac cell membrane. Upon stimulation of the receptors by adenosine or ACh, PTX-sensitive G proteins will be functionally dissociated into GuoTP and GBv Ga_oTPwill activate the KArr channel, while G&activates the KAchchannel. This scheme does not represent any quantitative relationship between the components of the model and does not take into account possible intermediate steps. The KATpchannel mechanism exists in both atrial and ventricular cells, whereas the KAchchannel mechanism exists in atrial cells but not in ventricular cells. Since cardiac myocytes contain millimolar concentrations of ATP,, the G-protein activation of the KArr,channel may not be operative under physiologic conditions. However, the system might play a significant role in the adenosine-mediated protection of cardiac tissues from ischemic damage (Gross and Auchampach 1992). Reproduced with permission from Ito et al. ( 1992). (Camps et al. 1992, Katz et al. 1992) and the unidentified effecters in the mating pheromone-signaling system of yeast and in the maturation of the starfish oocyte (Whiteway et al. 1989, Jaffe et al. 1993).
68
site de Tours, France) for critical reading of this review. This work has been supported by National Institutes of Health ROl HL47360 and an American Heart Association grant in aid (no. 91013540) to Y.K. This work was done during the tenure of an established investigator-ship of the American Heart Association to Y.K.
Thus, it is now well established that not only G,s but also Gp are responsible for G-protein-mediated regulation of various effector proteins, including ion channels. 01994, ElsevierScienceInc., lOSO-1738/94/$7.00
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Murphy JF, Graber SG, Garrison JC, Szabo G: 1993. Recombinant G protein by subunits activate the muscarinic K’ channel in bullfrogatrialmyocytes[abst]. FASEB J 7:All38. Nakajima T, Sugimoto T, Kurachi Y: 1992. Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane: intracellular chloride inhibition of the GTPase activity of G,. J Gen Physiol99:665-682. Nanavati C, Clapham DE, Ito H, Kurachi Y: 1990. A comparison of the roles of purified G protein subunits in the activation of the cardiac muscarinic K+ channel. In Nathanson NM, Harden TK, eds. G Proteins and Signal Transduction. New York, Rockefeller University Press, pp 29-41. Okabe K, Yatani A, Evans T, et al.: 1990. @y Dimers of G proteins inhibit atrial muscarinic K+ channels. J Biol Chem 265:12,85412,858. Parent L, Coronado R: 1989. Reconstitution of the ATP-sensitive potassium channel of
Kurachi Y, Nakajima T, Sugimoto T: 1986a. Acetylcholine activation of K* channels in cell-free membrane of atria1 cells. Am J Physio125 1:H68 1-H684.
Pfaffinger PJ, Martin JM, Hunter DD, Nathanson NM, Hille B: 1985. GTP-biding proteins couple cardiac muscarinic receptors to a K channel. Nature 3 17536-538. Scherer RW, Lo CF, Breitwieser GE: 1993. Leukotriene C4 modulation of muscarinic K+ current activation in bullfrog atria1 myocytes. J Gen Physiol 102:125-141. Tang W-J, Gilman AG: 1991. Type-specific regulation of adenylyl cyclase by G protein By subunits. Science 254:1500-1503. Whiteway M, Hougan L, Dignard D, et al.: 1989. The STE4 and STE18 genes of yeast encode potential g and y subunits of the mating factor receptor-coupled G protein. Cell 564671177. Yamada M, Ho Y-K, Katada T, Kurachi Y: 1993a. Activation of cardiac muscarinic K+ channel by transducin by subunits [abst]. Biophys J 64:A388. Yamada M, Tetzic A, Kurachi Y: 1993b. Regulation of K’ channels by G protein subunits and arachidonic acid metabolites. Methods Enzymol (in press). Yamada M, Jahangir A, Hosoya Y, Inanobe A, Katada T, Kurachi Y: 1993c. Gx* and brain G&activate muscarinic K+ channel through the same mechanism. J Biol Chem 268:24,551-24,554. Yatani A, Codina J, Brown AM, Bimbaumer L: 1987. Direct activation of mammalian atria1 muscarinic potassium channels by GTP regulatory protein Gk. Science 235:207211. Yatani A, Hamm H, Codina J, Mazzoni MR, Bimbaumer L, Brown AM: 1988. A monoclonal antibody to the a subunits of Gk blocks muscarinic activation of atrial K’ channels. Science 241:828-83 1. Yatani A, Okabe K, Bimbaumer L, Brown AM: 1990. Detergents, dimeric Gt,.,, and eicosanoid pathways to muscarinic atria1 K+ channels. Am J Physiol 258:H1507TCM Hl514.
R,IEZPRlNTS Reprints
Kurachi Y, Nakajima T, Sugimoto T: 1986b. On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atria1 cells: involvement of GTP-binding proteins. Pflugers Arch 407:264-274.
of articles in TCM are available (minimum order: 100). Please contact: Crystal Howard, Advertising Sales Elsevier Science Inc. 655 Avenue of the Americas New York, NY 10010
Kurachi Y, Ito H, Sugimoto T, Katada T, Ui M: 1989a. Activation of atria1 muscarinic K+ channels by low concent~tions of &y subunits of rat brain G protein. Pflugers Arch 413:325-327.
TCM Vol. 4,No.2,1994
skeletal muscle activation by a G proteindependent process. J Gen Physiol 94:445453.
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69
G-Protein Control of Cardiac Potassium Channels Yoshihisa Kurachi
Two cardiac potassium (K+) channels are activated by pertussis toxin (PTX)-sensitive G proteins either directly or in a “membrane-delimited” manner. They are muscarinic K+(KAcH) and ATP-sensitive K+(KATp) channels. KAch channels are responsible for acetylcholine (ACh)- or adenosine-induced deceleration of the heartbeat and atrioventricular conduction, while KATpchannels are responsible for the ischemiainduced shortening of the cardiac action potential and possibly for the adenosine-mediated protection from ischemic damage. Distinct molecular mechanisms underlie G-protein activation of these cardiac K+ channels; the a subunit of PTX-sensitive G proteins activates the KATp channels, while &subunits activate the KAchchannel. The physiologic significance of this heterogeneous mechanism remains to be determined. (Trends Cardiovasc Med 1994;4:64-69)
Regulation of potassium (K+) channels via specific receptors is an essential component of neurohumoral control of the heart. GTP-binding (G) proteins are now known to be involved either directly or indirectly (via second messengers) in the regulation of a number of cardiac K+ channels (Table 1). This direct or at least membrane-delimited regulation of ion channel function by G proteins is a newly recognized mode of the remotesensor-type regulation of ion channels (Hihe 1992). This system was first identified for the muscarinic activation of a cardiac K+ (KAch) channel. In addition, a number of other K+ channels, Ca2+ channels, Na+ channels, and Cl- channels have been shown to be regulated by G proteins in a membrane-delimited manner in a variety of tissues, including cardiac, neuronal, and endocrine cells.
Yoshihisa Kurachi is with the Division of CardiovascularDiseases, Departmentof InternalMedicine and Departmentof Phannacology, Mayo Clinic, Rochester, MN 55905, USA;and with the Departmentof Phannacology II, Osaka UniversitySchool of Medicine, Siuta,Osaka565, Japan. 64
This is one of the most important cellsignaling mechanisms in the regulation of cellular function [for a review, see Brown and Birnbaumer (1990), Hill (1992), and Kurachi et al. (1992)]. The G protein is a heterotrimer composed of G,, Gg’ and Gy, which is functionally dissociated into the GTPbound form of G, (Ga_oTp)and G& upon receptor stimulation (Gilman 1987). Either of these subunits of the G protein may regulate effecters, such as adenylyl cyclase, phospholipase C, and ion channels. The main object of this review is to assess the current situation of the controversy concerning whether it is the or the GButhat forms the physioGa_GTP logic activator of the KAch channel of atrial muscle. The evidence is now overwhelming that G& serves to activate the K*ch channel. This does not necessarily make G, nonfunctional, however, since this subunit of the activated G protein has been found to stimulate another cardiac K+ channel, the ATP-sensitive K+(K,,) channel. This elegant system, the “remote control” of an effector by a receptor via the intermediary step of G-protein utilization, is thus able to 01994, Elsevier Science Inc., lOSO-1738/94/$7.00
l
“Membrane-Delimited” G-Protein Regulation of the Cardiac KAch Channel
Pfaffinger et al. (1985) were the first to report the involvement of pertussis toxin (PTX or islet-activating protein)-sensitive G proteins in the muscarinic cholinergic induction of a specific inwardrectifying K+ current in cardiac atria1 muscle. The G protein was designated as G, based on this function [see Breitwieser and Szabo (1985)]. In inside-out patch membranes of cardiac atrial cells, Kurachi et al. (1986 a and b) showed the KACh channel to be activated by intracellular GTP (GTP,) in the presence of suitable agonists (ACh or adenosine) or by the nonhydrolyzable GTP analogue, GTP$% Intracellular AlF,- also induced activation of KAch channel in an agonistindependent manner (Kurachi et al. 1992). This AIF,- activation was prevented when the inside-out patches were pretreated with GDP@% The agonistdependent GTPi-induced channel activation was blocked by treating the insideout patch with the A protomer of PTX and nicotinamide-adenine dinucleotide. Since PTX is known to specifically ADPribosylate a certain population of G proteins and uncouple the membrane receptors and G proteins (Gilman 1987) it was concluded that a PTX-sensitive G protein (G,) couples m2-muscariniccholinergic and A,-purinergic (adenosine) receptors and the KAChchannel in the atrial cell membrane. Involvement of soluble second messengers can be excluded from this system, since these experiments have been performed in the excised inside-out patches. These observations clearly indicate the “membrane delimited” nature of G-protein activation of K*ch channels. The questions that we now address are (a) how G, activates the KAC,,channel and (b) which G, subunit activates the channel.
l
Physiologic Mode of G-Protein Activation of the Cardiac KACh Channel
To define the physiologic interaction between activated GK(GK*) and KAch channel, we examined the concentrationdependent effect of GTPi on the KACh
TCM Vol.4,No. 2,1994
Table 1. Receptor-dependent-G-protein-mediated
regulation of cardiac K+ channels Intracellular second-
Effect mediated
Channel Membrane-delimited Muscarinic
messenger
systems
regulation Activation by m,-muscarinic, cholinergic, and A,-purinergic Rs Activation by A,-purinergic R
K+ channel
ATP-sensitive
G twoteins
bv recerDtors
K+ channel
Second-messenger-mediated
PTX-sensitive
G (G,)
No
PTX-sensitive
G (G,?)
No
regulation
Delayed outward I, channel
Transient outward K+ channel Inward-rectifier Ix, channel
Activation by l3-adrenergic Inhibition by m2-muscarinic, ergic, and A, -purinergic Activation by a-adrenergic Inhibition by a-adrenergic Inhibition by a-adrenergic
R cholinRs R R R
CAMP-A kinase
G, G, PTX-insensitive PTX-insensitive PTX-insensitive
DAG-C kinase Unidentified Unidentified
G (G,?) G (G,?) G (G,?)
R, receptor.
channel.
We found that GTPi activates channel in a highly positive
the KACh cooperative manner (It0 et al. 1991). Figure la shows the effects of GTPi on K *ch channel openings in the absence and presence of ACh. Figure lb shows the concentration-dependent activation of the KAch channel by GTPi in the presence of various concentrations of ACh in the pipette. The relationship between GTPi and channel activity at each concentration of ACh was fitted by the following Hill equation with use of the least-squares method: y =Vw/[
l+(K,,/[GTP])H]
the maximal response and the apparent affinity of the Knch channel for GTP,, which may be due to the facilitation of functional dissociation of G, induced by agonist binding to the muscarinic cholin-
ergic receptors (Kurose et al. 1986). The positive cooperative effect of GTPi on the channel open probability may be derived from the intrinsic properties of the association of G-protein subunits and the
Figure 1. Concentration-dependent effect of intracellular GTP on the KAch channel in the absence and presence of ACh. (a) Examples of inside-out patch experiments. The concentration of ACh in the pipette is 0 or 1 p&4as indicated. The bars above each truce indicate the protocol of perfusing various concentrations of GTP and 10 ph4 GTPyS. The membrane potential was -80 mV. Note that a three- to tenfold increase in GTP concentration resulted in a dramatic increase of N.P, of K,, channels, indicating the existence of a highly cooperative process. (b) The relation between the concentration of GTP and the relative N.P,, of KAChchannels with reference to the N.P, induced by 10 @I GTPyS in each patch. Symbols and bars are mean f SD. The continuous curves are fitted by the Hill equation with the least squares method: 0 @vl ACh (open circles, n = 7), 0.0 1 @VIACh (closed circles, n = 6), 0.1 pIv4ACh (closed triangks, n = 6). and 1 pkl ACh (closed squares, n = 6). Reproduced with permission from Ito et al. (1991).
V,, = the where y = the relative N-P,, maximal N.P,,, Kd = the GTP concentration at the half-maximal activation of the channel, and H = the Hill coefficient. The channel activity was expressed as N-P,, where N is the number of the channel in the patch and P, is the open probability of each channel. The relative N.P, was
a
W ACh
defined with reference to the value induced by 10-100 FM GT@y!5 in each patch.
10 pA
ACh 1 ELM GTP 0.03 PM,
0.1
0.1
I
,-m
0.3
As the concentration of ACh in the pipette was raised from 0 to 0.01, 0.1, or 1 pM, the following points were observed: (a) the threshold concentration of GTPi necessary to induce openings of the channel decreased, (b) the concentration of GTPi for half-maximal activation of the channel (K,) decreased, and in(c) the maximal relative N-P,,(VMAX) creased; however, (d) the Hill coefficient of the fitted curve was constant around 3 and was independent of the ACh concentration. These results indicate that ACh binding to the receptor increased both
TCM Vol. 4, No. 2, 1994
concentration
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of GTP
10
I 100
65
activate the KAChchannel but also corresponds to the physiologically functional G, subunit that activates the channel (Ito et al. 1992, Kurachi et al. 1992, Yamada et al. 1993a-b, Murphy et al. 1993).
t
0
GK
Figure 2. Hypothetical model for activation of the KAch channel by G,’ in the cardiac atrial cell membrane. The K,, channel is
assumed to be composed of four identical subunits. One activated G, (Gx*) binds to each subunitto open the KACh channel.
K*c,, channel, which has multiple (>3) binding sites for G; (Ito et al. 1991, Karshin et al. 1991). Based on the models proposed for the structure of the inward-rectifier K+ channels recently cloned from rat kidney and mouse macrophage (Ho et al. 1993, Kubo et al. 1993), we assume that the KAChchannel is also a homotetramer. One G,’ may
bind to each segment in order to activate the KACh channel (Figure 2).
l
G-Protein Subunit Activation of the Cardiac KAch Channel
To identify the G-protein subunit that is
responsible for activation of the KAch channel, recombinant or purified G,s and GBrhave been applied to inside-out patches of the atria1cell membrane [for a review, see Brown and Birnbaumer (1990), Kurachi et al. (1992), and Yamade et al. (1993b)l. It has been reported that both Gi, cTWs (Codina et al. 1987, Yatani et al. 1987, Logothetis et al. 1988) and Gti (Logothetis et al. 1987 and 1988, Kurachi et al. 1989a, Kobayashi et al. 1990) could activate the KAChchannel. Considerable controversy surrounds G,+,activahas tion of the KAChchannel, since Gcl_GTp been generally recognized as the functional arm responsible for regulation of various effecters in G-protein-linked systems (Gilman 1987). By now, however, convincing evidence has been gathered to indicate that G& is not only able to
66
be made between different membrane patches that contain a different number of channels and only in -30% of patches (40 of 124)l. In contrast, GBv(10 nM) effectively activated the K,,ch channel in all patches examined (~400 patches) with comparable efficacy as 10-100 p.M The main points of the argument GTPyS. Second, after GBuwas incubated against GBuactivation of KAChchannels were (a) Gia_cTPys~activated the KAch for 2448 h (at 4°C) in Mg*+-free EDTA solution containing 2-10 @I GDP (or channel at subpico - picomolar concenGDP-BS), a treatment that would inactitrations, whereas GBuwas only effective vate any GTPyS-bound form of G,, the in nanomolar concentration; the activaG& activated the KAChchannel as effection by Gb observed by some investigatively as nontreated GW Third, boiled GBv tors was supposed to result from condid not activate the KAch channel. tamination of the GBupreparation by G, (Codina et al. 1987, Kirsch et al. 1988). Fourth, we could not activate the KAch (b) The detergent (CHAPS)used to sus- channel with CHAPS,and GBususpended in Lubrol PX activated the KAChchannel pend the hydrophobic G& itself could as effectively as that suspended in CHAPS, have activated the KAChchannel. Thus, indicating that activation was “detergent the activation of the KAch channel by independent.” Fifth, GBu preincubated detergent-suspended GBu is an artifact with excessive GDP-bound form of G, or (Yatani et al. 1990). G, failed to activate the channel. These In an exhaustive series of studies, we observations exclude the possibility that have been able to counteract the above G& activation of the KAch channel is an arguments (Kurachi et al. 1989a, Kobayashi et al. 1990, Ito et al. 1992, artifact. Furthermore, G,+,activated the Yamada et al. 1993a). First, Gi_,a_GTqS KAChchannel in a positive cooperative manner, which mimics the characterand Gi_za_Gm (100 pM to 10 nM) actiistic mode of activation of the KAch vated the KACh channel only slightly [-lo%-20% of the activation induced by channel by GTPi (Figure 3). Two lines of evidence indicating that 10-100 p.M GTPyS, which is used as a benchmark that enables comparisons to GKa_GTPphysiologically activates KAch Figure 3. Concentration-dependent activation of the KAChchannel by GTP, GTP@, brain GBy, and retinal T The relation between the KAchchannel activity and GTP (in the presence of ACh), GTPyS,%rain G&, and retinal TBuare depicted. The effect of Giacrqss is also indicated in the graph. The concentration-dependent effect of GTPi on the KAChchannel is shown in the internal solutions containing either 130 mM Cl- or 65 mM SO,*-. Intracellular Cl- disturbs the turnoff reaction of KAChchannel activity probably by inhibiting the intrinsic GTPase activity of G,, resulting in higher sensitivity of the KAChchannel to GTP, (Nakajima et al. 1992). The relative N.P, of the channel at each concentration of various substances was obtained with reference to the N.P, value induced by 10-100 fl GTPyS in each patch.
i-
r-
1.0 -I
GTP(SO,s)
9
retinal Tpy
brain Gpy
GTP(CI-)
0.8
0.6
0.6
0.6
P ‘Z $ 0.4 Gia-GTPrj 0.2
:
: .. IO-8
104
IO-4
IO-2
GTP & GTPyS (:.I)
01994. Elsevier Science Inc., lOSO-1738l94/$7.00
IO-‘2
1o-10
10-s
104
lo”
G protein subunits (M)
TCM Vol. 4. No. 2, 1994
channels have also been provided: (a) A monoclonal antibody to the transducin a subunit (4A) irreversibly inhibited the agonist-dependent-GTPi-induced activation of the KAChchannel, which could be
brain G& (Ito et al. 1992)
restored
(see also Yamada et al. 1993~). Kim et al. (1989) proposed that the arachidonic acid-eicosanoid pathway mediated the exogenous G& activation of the KACh channel in inside-out patches, which is not involved in the physiologic G, activation of the KACh channel. This proposal is not valid, since the stimulatory effect of arachidonic acid metabolites on the KAch channel clearly requires intracellular GTP (Kurachi et al. 1989b and 1992) and the activation of the KACh channel by G&was not affected by the inhibitors of arachidonic acid metabolism (Ito et al. 1992). The arachidonic acid metabolites are stimulatory modulators of the G, function (Kurachi et al. 1989b) and may be involved in stimulation of the basic activity of KAch channels by a-adrenergic agonists and platelet-activating factor (Kurachi et al. 1992) and also in the short-term desensitization of ACh activation of KAch current (Scherer et al. 1993). Current data support a direct activation of the KAch channel by Gw
only by exogenously applied (Yatani et al. 1988). This result Gia-GTPfS was interpreted as indicating that the antibody inhibited the interaction between G, and the KAch channel. (b) Both transducin & dimers (Tt,J purified from bovine retina and G& from other mammalian tissues not only failed to activate but actually inhibited agonistdependent-GTP,-induced KACh channel activation (Okabe et al. 1990). Contrary to these reports, we found that the antibody 4A interferes with the interaction between muscarinic receptors and G, and not that between G, and (Nanavati et al. 1990, K *ch channels Kurachi et al. 1992). Thus, one cannot differentiate which subunit of G, is physiologically responsible for the activation of KAch channel based on the results obtained with 4A. We also found that, although Gb purified from bovine brain consistently and fully activates K *ch channels, it never inhibits agonistdependent-GTPi-induced KACh channel activation (Ito et al. 1992). Furthermore, detergent-free T& does not inhibit but in fact activates the KAch channel in a positive cooperative manner (Figure 3; Yamada et al. 1993a). Thus, we could not support the earlier findings and notion that G,, is the physiologic activator of the KAch channel. A new set of experiments in fact indicate that GxBu is the physiologic activator of the KAch channel (Yamada et al. 1993a). We found that exogenously the agonistapplied Ta_onP inhibited dependent-GTPi-induced or GTPy!!+induced activation of K,,ch channels irreversibly, probably by binding to G,,+, and forming a trimer of T,_,,,-GxBU. Since this heterotrimer may not be able to interact with mz-muscarinic or A,purinergic receptors, the KACh channel activity was inhibited irreversibly. Only exogenously applied T,,,, or GBr could restore channel activity. These results strongly suggest that it is G,& that is the physiologic functional arm of G, that activates Knch channels. The KACh channel is activated in a highly positive cooperative manner by GTP (in the presence of agonists) (Ito et al. 1991), GTP$S (Nakajima et al. 1992)
TCM Vol. 4, No. 2, 1994
and retinal T&
(Yamada et al. 1993a) (Figure 3). It would therefore appear that G,‘, brain GBv’ and T& interact with the KACh channel
l
through
a common
mechanism
G-Protein Activation of Cardiac KATp Channel
Since the initial reports that G proteins might be involved in the regulation of the channel in the pancreatic l3 cell $p unne et al. 1989) and in skeletal muscle fibers (Parent and Coronado 1989), G proteins have also been found to activate the KATp channel in cardiac myocytes (Kirsch et al. 1990, Ito et al. 1992). In inside-out patches of guinea pig ventricular cell membrane, where KAch channels are not expressed, GTP (in the presence of ACh or adenosine) and GTPyS induced an increase of KATp channel activity, which had been suppressed by intracellular ATP (ATP,) (Kurachi et al. 1992). AlF,- also induced an increase of the KATp channel activity in the presence of ATP,, which was prevented by pretreating the patches with GDP-l3S. Gia_oTFFs purified from bovine brain also increased KATpchannel activity that had been suppressed by ATPi (Ito et al. 1992). G,+, did not affect the baseline activity of the channel but inhibited the receptor-mediated-GTPi-
01994,
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induced increase of KATp channel activity. These observations suggest that the KATp channel in the ventricular cell membrane can be activated by G proteins, and in this case it is the a subunits of PTX-sensitive
G proteins
that activate
the KATpchannel.
l
Distinct Molecular Mechanisms Underlie G-Protein Activation of the Cardiac K+ Channels
Figure
4a shows an experiment where and G,, were applied to an Gi-la-GTQ5 inside-patch of atrial cell membrane, where both KACh and KATp channels are found (Ito et al. 1992). In the cellattached patch, the KAch channel was activated vigorously by ACh (1 p.M) in the pipette. In the excised inside-out patch, the openings of the KAChchannel decreased to a minimal background level (Figure 4a-1). The internal solution contained 100 @I MgsATP to suppress the KATpchannel openings and also to maintain availability of the channels. When was applied to the internal Gi-la-GTqS side of the membrane, K+ channel openings with a conductance of -90 pS were clearly induced. On the other hand, the openings of the KACh channel were not affected significantly (Figure 4a-2). Openings of the 90-pS K+ channel were inhibited by 1 @I glibenclamide, indicating that this was the KATp channel. Subsequent application of G& to the patch dramatically increased openings of the 40 to 45-pS KAChchannel (Figure 4a-3 and -4). The Gt,,-induced openings of the KAch channel were not affected by glibenclamide. Thus, distinct molecular mechanisms underlie G-protein activation of these cardiac K+ channels. Stimulation of m2muscarinic or A,-purinergic receptors results in PTX-sensitive G proteins dissociating into Gn_oTP and Gw Ga_oTP activates the KATpchannel and Gti activates the KACh channel (Figure 4b).
l
Conclusions
There is now overwhelming evidence that GI<& is the physiologic functional arm of G, activating KAch channels in the heart. A growing body of evidence also shows that GBu regulates various other effecters, such as type-2 and type-4 adenylyl cyclases (Tang and Gilman 1991), phospholipase Cl32 in mammals
67
The major unsolved questions regarding G, activation of KAch channels are (1) Which pertussis-toxin-sensitive G protein serves as G,, (b) of which subtypes of G,, GP and G,,is G, composed, and (c) how does G,& interact with K,,ch channels? In other words, we have not yet fully answered the question of how information specifically passes from a membrane receptor to the effector, the K Ach channel. Similar questions are unanswered in the case of the KArp channel. The physiologic significance of this heterogeneous molecular mechanism of Gprotein activation of different cardiac K+ channels also awaits an answer.
l
Acknowledgments
The author thanks Dr. Ian Findlay (Univer-
b
ACh Adenosi ne
M R
Breitwleser GE, Szabo G: 1985. Uncoupling of cardiac muscarinic and g-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature 317:538-540.
GTP
Figure
4. (a) Effects of Gi_ta_orPis and G& on the KATpand KAch channels in the atrial cell
membrane. The pipette solution contained 1 @l ACh. The inside-out patch was formed at the arrow (I/O) above the current trace in the internal solution containing 100 pM ATP and 0.5 mM MgClz (A-1). Gt.1, oq (300 PM) was applied first to the internal side of the patch and clearly induced openings of the KATpchannel (-90 pS) (A-2) without affecting background activity of *e KACh channel. Subsequently, GBu(10 nM) was applied to the patch and caused a dramatic increase of 45pS KAChchannel openings (A-3 and -4). Numbers above the current trace indicate the location of each expanded current trace shown below. In the expanded current traces, the dotted line is the first open level of the KAChchannel and the continuous line is that of the KArp channel. The arrowhead in each trace is the zero current level. (b) Proposed mechanisms of the pertussis toxin (PTX)-sensitive G-protein-subunit activation of the KArpand KAChchannels in cardiac cell membrane. Upon stimulation of the receptors by adenosine or ACh, PTX-sensitive G proteins will be functionally dissociated into GuoTP and GBv Ga_oTPwill activate the KArr channel, while G&activates the KAchchannel. This scheme does not represent any quantitative relationship between the components of the model and does not take into account possible intermediate steps. The KATpchannel mechanism exists in both atrial and ventricular cells, whereas the KAchchannel mechanism exists in atrial cells but not in ventricular cells. Since cardiac myocytes contain millimolar concentrations of ATP,, the G-protein activation of the KArr,channel may not be operative under physiologic conditions. However, the system might play a significant role in the adenosine-mediated protection of cardiac tissues from ischemic damage (Gross and Auchampach 1992). Reproduced with permission from Ito et al. ( 1992). (Camps et al. 1992, Katz et al. 1992) and the unidentified effecters in the mating pheromone-signaling system of yeast and in the maturation of the starfish oocyte (Whiteway et al. 1989, Jaffe et al. 1993).
68
site de Tours, France) for critical reading of this review. This work has been supported by National Institutes of Health ROl HL47360 and an American Heart Association grant in aid (no. 91013540) to Y.K. This work was done during the tenure of an established investigator-ship of the American Heart Association to Y.K.
Thus, it is now well established that not only G,s but also Gp are responsible for G-protein-mediated regulation of various effector proteins, including ion channels. 01994, ElsevierScienceInc., lOSO-1738/94/$7.00
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Kurachi Y, Ito H, Sugimoto T, Schimizu T, Miki I, Ui M: 1989b. Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel. Nature 337:555-557. Kurachi Y, Tung RT, Ito H, Nakajima T: 1992. G protein activation of cardiac muscarinic K+ channels. Prog Neurobiol 39:229-246.
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Kurose H, Katada T, Haga T, Haga K, Ichiyama A, Ui M: 1986. Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J Biol Chem 261:6423-6428.
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R,IEZPRlNTS Reprints
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Kurachi Y, Ito H, Sugimoto T, Katada T, Ui M: 1989a. Activation of atria1 muscarinic K+ channels by low concent~tions of &y subunits of rat brain G protein. Pflugers Arch 413:325-327.
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