I. Introduction

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Upon stimulation of vagal nerves, acetylcholine (ACh)^c is released from axonal terminals and decelerates the heart beat. This historic discovery by Otto Loewi in the 1920s established the concept of chemical synaptic transmission (Loewi, 1921; Loewi and Navaratil, 1926). Since then, many physiologists have been trying to elucidate the mechanism(s) underlying neurotransmitter (Vagusstoff)-induced bradycardia. Del Castillo and Katz (1955) first described hyperpolarization of the membrane induced by ACh in frog heart. Hutter and Trautwein (1955) measured an increase of K⁺ efflux across the cardiac cell membrane with vagal stimulation. Trautwein and Dudel (1958) showed an increase of K⁺ conductance under voltage-clamp conditions. Trautwein and colleagues analyzed the kinetics of the ACh-induced K⁺ current in the rabbit sinoatrial node and proposed that ACh induces activation of a specific population of K⁺ channels, named muscarinic K⁺ (K_ACh) channels, to decelerate pacemaker activity (Noma and Trautwein, 1978; Osterrieder et al., 1981). The single channel currents of the K_ACh channels were recorded for the first time by Sakmann et al. (1983), who showed that the channel exhibited kinetic properties that clearly differed from those of the background inwardly rectifying K⁺ (I_K1) channel in cardiac myocytes.

The next big step was the discovery that pertussis toxin (PTX)-sensitive heterotrimeric G proteins are involved in the activation of the K_ACh channel by M₂-muscarinic and A₁ adenosine receptors (Pfaffinger et al., 1985; Breitwieser and Szabo, 1985; Kurachi et al., 1986a and b). Because the K_ACh channel could be activated by intracellular guanosine 5'-triphosphate (GTP) (in the presence of agonists) and GTPS (even in the absence of agonists) in cell-free inside-out patches, the system seemed to be delimited to the cell membrane, which led to the proposal that the channel is directly activated by G proteins (Kurachi et al., 1986a,b,c). The G protein responsible for activation of the K_ACh channel was designated G_K according to its function (Breitwieser and Szabo, 1985).

It was quite a surprise that the subunit (G) but not the  subunit (G) of the G_K protein, was proposed to mediate the G_K-induced activation of K_ACh channels (Logothetis et al., 1987, 1988; Kurachi et al, 1989a), because it was strongly believed at that time that regulation of different effectors by G proteins was mediated only by G, although G merely served to bind to the GDP-form of G (G_-GDP) to anchor the trimeric G protein to the cell membrane (Gilman, 1987). Actually, Brown, Birnbaumer, and their colleagues proposed G_K and not G_K as the physiological activator of K_ACh channels (Yatani et al., 1987, 1988; Codina et al., 1987; for review see Brown and Birnbaumer, 1990). The dispute concerning the G protein subunit responsible for the physiological activation of K_ACh channels continued for nearly a decade (Ito et al., 1992; Yamada et al., 1993, 1994a,b; Nanavati et al., 1990; Kurachi, 1989, 1990, 1993, 1994, 1995; Kurachi et al., 1992; Clapham and Neer, 1993; Wickman and Clapham, 1995) until the functional interaction between the channel and G was shown at the molecular level with cloned G protein-gated K⁺ (K_G) channel and/or G protein subunits (Kubo et al., 1993b; Dascal et al., 1993; Wickman et al., 1994; Reuveny et al., 1994; Krapivinsky et al., 1995a; Inanobe et al., 1995b). Now it is established that G_K is the physiological activator of K_G channels not only in cardiac myocytes, but also in neurons and endocrine cells. Recently, it was indicated that G protein-inhibition of neuronal Ca²⁺ channels is also mediated by G and not by G (Herlitze et al., 1996; Ikeda, 1996). Efforts are now being made to elucidate the molecular mechanisms underlying G-control of K_G and N-type Ca²⁺ channels.

The importance of the G protein-activation of K_G channel system in receptor-mediated regulation of cell responses is now more widely appreciated than before because a wide variety of membrane receptors, such as M₂-muscarinic, A₁ adenosine, ₂-adrenergic, D₂ dopamine, µ-, -, and -opioid, 5-HT_1A serotonin, somatostatin, galanin, m-Glu, GABA_B, and sphingosine-1phosphate receptors, have been shown to use this system in inhibiting cell excitation in various organs (North et al., 1987; Lacey et al., 1988; Hille, 1992a; Grudt and Williams, 1993; Oh et al., 1995; Saugstad et al., 1996; Sharon et al., 1997; Bünemann et al., 1995; Koppen et al., 1996). In this review, we will first summarize ACh-activation of cardiac K_ACh channels, the prototype of this system, and then recent progress in molecular dissection of the K_G channel system.

	II. Functional Analysis of G Protein-Mediated Activation of Muscarinic K+ Channels in Cardiac Atrial Myocytes

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ACh added to the extracellular solution elicits a K_ACh channel current in cardiac atrial myocytes (fig. 1). The activation time-course is sigmoidal and takes several hundred milliseconds to reach a peak (Breitwieser and Szabo, 1988). Thereafter, the evoked current gradually decreases to a quasi-steady-state level within 1 min in the presence of high concentrations of ACh (> 0.3 µM). This reduction of cell K⁺ current in the continuous presence of ACh is called "short-term" desensitization (Kurachi et al., 1987b). After wash-out of the agonist, the current disappears within several seconds (deactivation). It is worth noting that in the inside-out patch configuration of the patch-clamp method, one measures K_ACh channel activity only in the steady-state phase. Thus, in these experiments, limited information is available regarding the desensitization of the channel.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Time-dependent response of the whole-cell muscarinic K+ channel current to acetylcholine. By using the whole-cell voltage clamp method of the patch-clamp technique, the response of the whole-cell current of a guinea-pig atrial myocyte to 11 µM acetylcholine (ACh) was measured. In the presence of normal Tyrode solution that contained 5.4 mM external K+, the cell membrane potential was clamped at 53 mV. The patch pipette contained (in mM): 150 KCl, 2 MgCl2, 5 EGTA, 5 HEPES, and 0.1 GTP (pH = 7.3). ACh was applied to the bath for the period indicated by the horizontal bar above the cell membrane current trace. An arrowhead indicates the zero current level. An upward deflection of the cell current record indicated an outwardly directed cell membrane current that would be carried by the movement of K+ ions under these circumstances.

These three phases of the response involve interactions between an agonist (i.e., ACh), an M₂-muscarinic receptor, a PTX-sensitive G protein, and the K_ACh channel. Therefore, to understand the reaction of the K_ACh channel to ACh, it is necessary to know how the receptor-generated signal is transferred to the channel through the G protein and how this signal transmission might be modulated by other factors interacting with these different reactions.

1. The G protein cyclic reaction mediating the receptor-to-channel signal transmission. Activation of K_ACh channel induced by M₂-muscarinic receptor stimulation is mediated by a heterotrimeric G protein (G_K) (fig. 2). The heterotrimeric G proteins are membrane-bound proteins which transduce signals from receptors to effectors such as adenylyl cyclase, phospholipase C, the K_ACh channel, and other ion channels (Gilman, 1987). These proteins are composed of , , and  subunits (G, G, and G, respectively). Up to now, at least 16 G, 5 G, and 11 G genes have been identified (Bourne, 1997). Heterotrimeric G proteins interact with receptors through G. It is well known that the interaction between M₂-muscarinic receptors and G_K is blocked by the toxin from Bordetella pertussis (PTX) (Ui, 1984; Kurose et al., 1986). PTX modifies covalently a cysteine residue at the carboxyl-terminal end of G subunits belonging to G_i, G_o, and G_t families by transferring an ADP-ribose group from the nicotinamide adenine dinucleotide moiety to the cysteine residue (Gilman, 1987). Because the receptor-mediated activation of K_G channels in cardiac atrial myocytes and neurons are inhibited by PTX (Pfaffinger et al., 1985; Kurachi et al., 1986a), G_K seems to belong to one of these G protein families. However, its molecular identity has not been fully elucidated, although G_K is proposed to be a member of the G_i class of G proteins in some systems (Kozasa et al., 1996; Takano et al., 1997)

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the G protein cycle involved in the activation of the muscarinic K+ channel in response to acetylcholine.

- GDP

-GDP

-GTP

-GTP

View this table: [in this window] [in a new window]   TABLE 1 G protein effectors regulated by G protein subunits

2. Activation phase. The time to peak of the ACh-induced response of the K_ACh channel is dependent on ACh concentration: the higher the concentration of ACh, the faster the activation. In the presence of a maximum effective concentration of ACh, the time to peak is several hundred milliseconds. If the M₂-muscarinic receptor, G_K, and the K_ACh channel encountered by simple diffusion in the membrane, the response time requires that all these signaling molecules be within less than 1.5 µm of each other (Hille, 1992a). The molecular mechanism satisfying such a topological requirement has not been clearly identified. However, it was recently suggested that K_ACh channel subunits may directly interact with not only G_K but also G_K, trimeric G_K, and the receptor and thereby might form a complex with these proteins (Huang et al., 1995; Slesinger et al., 1995) (detailed in the Sections III.F.3. and 4.).

3. The phase of short-term desensitization. Short-term desensitization becomes more prominent as the concentration of ACh is increased above 0.3 µM (Kurachi et al., 1987b). This may at least partly arise from the transition of M₂-muscarinic receptors from the high to low affinity-binding state due to dissociation of G_K from receptors after agonist application (Gilman, 1987). Recent studies demonstrated that heterologous coexpression of RGS proteins with M₂-receptors and recombinant K_G channels reestablishes the short-term desensitization, which normally cannot be seen in the absence of RGS proteins in the reconstituted system (Doupnik et al., 1997). Therefore, this protein may also be one of the molecules responsible for the short-term desensitization of the K_G channel system. Other possible candidates for the short-term desensitization include phosphorylation of M₂-muscarinic receptors by -adrenergic receptor kinase (ARK), dephosphorylation of K_ACh channels and functional modulation of G proteins.

4. Deactivation of the response of the muscarinic K⁺ channel. The ACh-induced K⁺ current disappears quickly when the agonist is washed out from the extracellular solution (fig. 1). The rate of deactivation of the whole-cell K_ACh channel current was estimated as ~30 to 200/min, which is much more rapid than either the GTP hydrolysis rate of G proteins (~1 to 5/min) or the rate of dissociation of G from K_G channel subunits (~0.01/min) estimated in vitro (Breitwieser and Szabo, 1988; Nakajima et al., 1992; Gilman, 1987; Doupnik et al., 1997; Krapivinsky et al., 1995c). This discrepancy might in part be attributed to positive cooperativity in the interaction between the channel and G_K that will be described in the Section II.B.2., where even a slight decrease in free G_K concentration in the membrane should cause a larger reduction of the channel activity.

4

1. Single-channel characteristics of the muscarinic K⁺ channel. Fig. 3A shows single-channel recording of the K_ACh channel obtained from a cell-attached membrane of a guinea-pig atrial myocyte (Kurachi et al., 1986a). In general, K⁺ ions flow through K⁺ channels depending on the electrochemical gradient for K⁺ ions across the plasma membrane. This gradient is the difference between the membrane potential (V_m) and the K⁺ equilibrium potential (E_K): V_m  E_K. The single-channel current flowing through a K⁺-selective channel can be described as follows:

(1)

where i is the single-channel current amplitude, and  is the single-channel conductance of the channel. The current is positive (outwardly flowing across the membrane) at V_m positive to E_K, although it is negative (inwardly flowing) at V_m negative to E_K.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Single-channel properties of the muscarinic K+ channel. A: Cell-attached recordings of muscarinic K+ channel currents from a single guinea pig atrial myocyte at different membrane potentials. The patch pipette contained 150 mM K+ and 5.5 µM acetylcholine, although K+ concentration in the bath was 5.4 mM. The resting membrane potential (Er) of the cell was 52 mV. Membrane potentials are indicated to the left of each trace as the difference from Er. Arrowheads indicate the zero current level. Downward reflection of the current record represent ion current passing inwards into the cell from the pipette. Upward deflections represent passing from the cell outwards into the pipette. B: The single-channel current-voltage relationship of the muscarinic K+ channel shown in A. The line was fitted to the data by eye, and the single channel conductance was 46 pS at potentials between Er 60 and Er +40 mV. C: Open time histogram of the muscarinic K+ channel at Er. The line is the fit of the data with a single exponential curve with a time constant of 1.35 msec. [Modified from Kurachi et al. (1986a)].

13

1

2. Positive cooperative effect of GTP on muscarinic K⁺ channel activity. Activation of K_ACh channels by intracellular GTP (GTP_i) can be reproduced in inside-out patch membranes of atrial myocytes in the presence of ACh in the pipette (Kurachi et al., 1986a, 1990; Ito et al., 1991). Fig. 4 shows the concentration-dependent effect of GTP_i in the presence of different concentrations of ACh.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effect of intracellular GTP on the muscarinic K+ channel in the absence and presence of acetylcholine. A: Examples of inside-out patch experiments obtained from guinea-pig atrial myocytes. The channel currents were recorded at 80 mV with the symmetrical 145 mM K+ solutions. The concentration of acetylcholine (ACh) in the pipette was 0 or 1 µM as indicated. The bars above each trace indicates the protocol of application of different concentrations of GTP or 10 µM GTPS to the internal side of the patch membrane. The 3- to 10-fold increase in GTP concentration resulted in a dramatic increase of N.Po of muscarinic K+ channels, indicating the existence of a highly cooperative process. B: The relation between the concentration of GTP and the N.Po of muscarinic K+ channels normalized to the maximum N.Po induced by 10 µM GTPS in each patch. Symbols and bars are mean ± SD. The continuous lines indicate the fit of the relationship between GTP and channel activity in the presence of each concentration of ACh with the following Hill equation: where f is the relative N.Po; Vmax, the maximum N.Po available in the presence of 10 µM GTPS; Kd, the apparent dissociation constant of GTP; and n, the Hill coefficient. [Reproduced with permission from Ito et al. (1991)].

(2)

K

K

K

3. Spectral analysis of the muscarinic K⁺ channel currents in the presence of different concentrations of intracellular GTP. Precise and reliable analysis of the single-channel kinetics of the K_ACh channel is difficult because multiple K_ACh channels are usually included in a single membrane patch of atrial myocytes (fig. 4A). In these cases, the spectral analysis of the channel currents (an analysis based on a frequency domain) is one of the most reliable and powerful ways to assess the channel kinetics (fig. 5). The power spectrum constructed from inside-out patch recordings of the K_ACh channel currents is always well fitted with the sum of two Lorenzian curves irrespective of GTP_i concentration (Hosoya et al., 1996). These observations indicate that the K_ACh channel possesses three distinct open/closed states. Because the channel possesses a single open state (Sakmann et al., 1983), the equilibrium of the states can be described as C2C1O, where O represents the open state although C1 and C2 are closed states. It is likely that the transition among these three states is responsible for the open and closure of K_ACh channel currents observed at the single-channel level (figs. 3A, 4A and 5A). The corner frequencies of the two Lorenzian functions (the frequencies at which the power of the each component is the half-maximum) were constant irrespective of GTP_i concentration (fig, 5B). The ratio of the powers of the two Lorenzians at 0 Hz was also unaffected by GTP_i concentration. These results indicate that the kinetics of the fast open-close transition of the channel is not a function of G_K activity. In other words, G_K activates the K_ACh channel without altering the channel's fast open-close kinetics.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Spectral analysis of the muscarinic K+ channel currents in an inside-out patch. A: The muscarinic K+ channel currents in the inside-out patch membrane of a guinea pig atrial myocyte. The channel currents were recorded at 60 mV with the symmetrical 150 mM K+ solutions. The patch pipette also contained 0.5 µM acetylcholine. Different concentrations of GTP were applied to the internal side of the patch membrane as indicated above each current trace. B: Power density spectra calculated from the data shown in A. Each spectrum could be fitted with the sum of two Lorentzian functions. F1 and F2 indicated by arrows indicate the corner frequencies of the slow and the fast Lorentzian components, respectively. [Reproduced with permission from Hosoya et al. (1996)].

U A

C2C1O

View larger version (34K): [in this window] [in a new window]   Fig. 6.   The relationship between GTP concentration and the fraction of the "available" state of the muscarinic K+ channel, and the concerted allosteric model of Monod, Wyman, and Changeux. A: The fraction of the "available" state (A/(A + U)) was calculated from inside-out membrane patch experiments such as shown in fig. 5. Symbols indicate the relationship between GTP concentration and the calculated fraction of the "available" state. Lines indicate the fit of the data with the Monod, Wyman, and Changeux allosteric model with different assumed numbers of n (see Section II.B.4.). B: Schematic representation of the Monod, Wyman, and Changeux allosteric model. In this scheme, each muscarinic K+ channel is assumed to be an oligomer composed of four identical subunits (i.e., n = 4). Each subunit is in either the tense (T) or the relaxed (R) state, which is represented by squares and circles, respectively. Each subunit in the T or R state binds with one dissociated G protein subunit (solid circles) independently of each other with the microscopic dissociation constant of KT or KR, respectively. In this model, all subunits in the same oligomer must change their conformations simultaneously. Therefore, the channel can be either T4 or R4. T4 and R4 are in the equilibrium through an allosteric constant L. [Reproduced with permission from Hosoya et al. (1996)].

4. A possible mechanism for the G protein-mediated increase in the functional numbers of muscarinic K⁺ channels. Recent studies have revealed that Kir channels, including K_ACh channel, have an oligomeric structure (Yang et al., 1995b; Krapivinsky et al., 1995a) that may underlie the positive cooperativity of the G_K protein/K_ACh channel interaction (Monod et al., 1965).

K

K

K

K

K

K

K

K

Although the G_K/K_ACh channel interaction is the essential step of G protein-mediated activation of the K_ACh channel, this reaction is modulated by many factors such as intracellular ATP, Na⁺ ions, and arachidonic acid metabolites. Intracellular ATP has been shown to activate native and recombinant K_G channels in an Mg²⁺_i-dependent manner (Otero et al., 1988; Heidbüchel et al., 1990; Kaibara et al., 1991; Kim, 1991; Lesage et al., 1995; Sui et al., 1996). Although the molecular mechanism underlying this phenomenon has not been unequivocally identified, PIP₂ may be involved in this phenomenon (Huang et al., 1998).

The activity of K_G channels pretreated with intracellular MgATP could be further enhanced by intracellular Na⁺ (Lesage et al., 1995; Sui et al., 1996). The site of action of Na⁺ is unknown. Sui et al. (1996) showed that intracellular Na⁺ increased the activity of the K_ACh channel (and also the corresponding recombinant K_G channel) with an EC₅₀ of ~40 mM mainly by increasing the frequency of the channel's opening. They found that priming of channels with MgATP was a prerequisite for the action of Na⁺. Lesage et al. (1995), however, found that 20 mM intracellular Na⁺ activated recombinant K_G channel whether or not they had been pretreated with MgATP. This discrepancy might have occurred due to the different subunit composition of the K_G channels used in these two studies. Interestingly, Sui et al. (1996) showed that a cardiac glycoside ouabain, an inhibitor of the Na⁺/K⁺ pump, induced the opening of the K_ACh channel. They found that the N*P_o value of the channel increased although the mean open time was unchanged, indicating that the activating effect of ouabain was probably mediated by accumulation of intracellular Na⁺ but not a possible local increase in ATP concentration. However, they did not directly measure intracellular Na⁺ concentration nor reported the apparent change in the reversal potential of the K_ACh channel that might be expected when intracellular K⁺ concentration decreased due to blockade of Na⁺/K⁺ pump. Therefore, further studies may be necessary to conclude that cardiac glycosides activate the K_ACh channel through accumulation of intracellular Na⁺. This phenomenon might, at least in part, underlie the "direct" negative chrono- and dromo-tropic effects of the agent on the heart.

Arachidonic acid (AA) metabolites are known to modulate K_ACh channels (Kurachi et al., 1989c; Kim et al., 1989; Yamada et al., 1994b). The effect of AA is mimicked by leukotriene C₄ (LTC₄) and specifically blocked by AA861, a 5-lipoxygenase inhibitor (Kurachi et al., 1989c). Therefore, the effect of AA may be mediated by LTC₄ or its metabolites. Although the site of action of LTC₄ has not been clearly identified, the complete dependency of the LTC₄ effect on the presence of GTP_i indicates that LTC₄ does not directly act on the K_ACh channel (Kurachi et al., 1989c). In the absence of receptor agonists, GTP_i usually induces only 20% of the maximum K_ACh channel activity in the inside-out patch membranes even when Cl is used as an intracellular anion. However, GTP_i fully activated the channel in an agonist-independent manner when the patches were pretreated with AA before patch excision (Kurachi et al., 1989c). Thus, AA metabolites may stimulate the basal turn-on reaction of G_K. Stimulation of K_ACh channels by platelet-activating factor or ₁-adrenergic receptors may be mediated by this second-messenger pathway (Nakajima et al., 1991, Kurachi et al., 1989b).

	III. Molecular Analysis of G Protein-Gated K+ Channels

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In 1993, the molecular structure of inwardly rectifying K⁺ channels (Kir) was disclosed. The cDNAs encoding an ATP-dependent Kir channel, ROMK1 (Ho et al., 1993), and a classical Kir channel, IRK1 (Kubo et al., 1993a), were isolated by expression cloning from the outer medulla of rat kidney and a mouse macrophage cell line, respectively (fig. 7). The primary structure of these channels were similar with two putative membrane-spanning regions (M1 and M2) and one potential pore-forming region (H5). This structure resembles that of the S5, H5, and S6 segments of the voltage-gated K⁺ (K_v) channels. Because the voltage-sensor of the K_v channel subunit exists in the S4 segment that possesses repeated positively-charged amino acid residues, Kir channel subunits lack an obvious voltage-sensor region. This is consistent with electrophysiological studies that show the kinetics of Kir channels apparently depends on the difference of Vm from E_K and not on Vm itself.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Evolutionary tree of Kir subunits. The tree was made using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) Tree Window in Geneworks (IntelliGenetics, Inc., Mountain View, CA).

After the cloning of ROMK1 and IRK1, the cDNAs encoding the main subunits of K_G and K_ATP channels (GIRK1 and BIR) were also cloned (Kubo et al., 1993b; Dascal et al., 1993; Inagaki et al., 1995a). All of these Kir channel subunits exhibit basically the same primary structure. So far, at least 11 cDNAs encoding Kir channel subunits have been isolated. The evolutionary tree of this family is depicted in fig. 7.

These cloned Kir subunit cDNAs encode proteins composed of 327 to 501 amino acids. The identity of the predicted amino acid sequences is ~30 to 40% among the members of the different Kir subfamilies and more than 60% among those in the same subfamilies. The highest level of sequence identity (50 to 60%) is found in the H5 region and the proximal part of the C-terminal cytosolic domain. The cloned Kir channel subunits have been classified at least into four groups (Doupnik et al., 1995a): (a) IRK (Kir2.x) subfamily made of the classical constitutively active "inward rectifier" Kir channels: IRK1 (Kubo et al., 1993a; Morishige et al., 1993), IRK2 (Koyama et al., 1994; Takahashi et al., 1994) and IRK3 (Morishige et al., 1994; Makhina et al., 1994; Pärier et al., 1994); (b) GIRK (Kir3.x) subfamily, corresponding to G protein-regulated K⁺ channels: GIRK1 (Kubo et al., 1993b; Dascal et al., 1993), GIRK2 (Lesage et al., 1994, 1995; Isomoto et al., 1996; Tsaur et al., 1995; Stoffel et al., 1995; Bond et al, 1995; Ferrer et al., 1995), GIRK3 (Lesage et al., 1994), GIRK4 (Ashford et al., 1994; Krapivinsky et al., 1995a; Chan et al., 1996), and GIRK5 (Hedin et al., 1996); (c) K_AB subfamily of ATP-dependent K⁺ channels (Kir1.1 and Kir4.1): ROMKs (Ho et al., 1993; Zhou et al., 1994; Yano et al., 1994; Shuck et al., 1994; Boim et al., 1995; Kondo et al., 1996) and K_AB-2 (Bond et al., 1994; Takumi et al., 1995); and (d) K_ATP subfamily (Kir6.x), the ATP-sensitive K⁺ channels: uK_ATP-1 and BIR (Inagaki et al., 1995a,b; Sakura et al., 1995).

Recent progress in the molecular biology of Kir channels has enabled us to study the structure-function relationship of biophysics, physiological regulation, and pharmacology of these channels at the molecular level.

GIRK1 was first isolated from the rat atrium (Kubo et al., 1993b; Dascal et al., 1993). From a mouse brain cDNA library, two additional homologues of GIRK1 were isolated and designated GIRK2 and GIRK3 (table 2) (Lesage et al., 1994). Furthermore, it has been shown that at least three different isoforms of mouse GIRK2 are generated by alternative splicing of transcripts from a single gene, and we designated them GIRK2A, GIRK2B, and GIRK2C in the order of identification (Isomoto et al., 1997). These alternatively spliced transcripts share an N-terminal end and a central core, and differ at their C-terminal ends. GIRK2B was isolated from mouse brain cDNA library and shown to be ubiquitously expressed in various tissues (Isomoto et al., 1996). Its amino acid sequence is shorter than that of GIRK2A by 87 amino acids. The eight amino acid residues in the C-terminal end of GIRK2B are different from those of GIRK2A. GIRK2C has a C-terminus which is longer than that of GIRK2A by 11 amino acids. GIRK2C was isolated from cDNA libraries of insulinoma cells and brain (Lesage et al., 1994, 1995; Tsaur et al., 1995; Stoffel et al., 1995; Bond et al., 1995; Ferrer et al., 1995).

GIRK2C was originally termed K_ATP-2 because it was thought to be a subunit of the K_ATP channel (Stoffel et al., 1995; Tsaur et al., 1995) due to its sequence similarity to cK_ATP-1, which was isolated by Ashford et al. (1994). However, GIRK4, which is virtually identical with rat cK_ATP-1, reconstitutes cardiac K_ACh channel with GIRK1 and does not contribute to the K_ATP channel as described in the Section III.D. (Krapivinsky et al., 1995a,b). Thus, it is now clear that both cK_ATP-1 and K_ATP-2 belong to the GIRK subfamily. GIRK5 was cloned from Xenopus oocytes (Hedin et al., 1996). Although its mammalian homologue has not been reported, the amino acid sequence of GIRK5 is most homologous to that of GIRK4 among mammalian GIRKs.

The GIRK clones contain various known functional motifs in their amino acid sequences that may be important for the physiological functions of the subunits in K_G channels (fig. 8). GIRK1 possesses an amino acid sequence homologous to the G-binding domain of ARK1 in its C-terminus, which is therefore the candidate for the site of G-binding to the K_G channel (Reuveny et al., 1994). As with all the other Kir channel subunits, GIRKs possess conserved cationic residues adjacent to the C-terminal end of the M2 domain. One of these positively charged residues, arginine (R) at position 188 of ROMK1, was shown to be critically involved in PIP₂-induced activation of rundown ROMK1 channels (Huang et al., 1998). Thus, it is conceivable that the corresponding residues in GIRK subunits (R190 for GIRK1; R201 for GIRK2s; R167 for GIRK3; and R196 for GIRK4) also participate in the PIP₂-induced activation of K_G channels. All of the GIRK clones have an arginine-glycine-aspartate (RGD) motif in their linker region between M1 and H5. This motif could be an integrin receptor-site (Hynes et al., 1992), whose role in K_G channels has not been examined yet. The characteristic feature of GIRK2C is the serine/threonine-X-valine/isoleucine (S/T-X-V/I) motif at its C-terminus end (Gomparts, 1996). This motif has been shown to be important for interactions with the PSD-95/SAP90 family of anchoring proteins, not only for K_v and NMDA receptor channels (Kim et al., 1995; Kornau et al., 1995), but also for Kir channels such as IRK3 and K_AB-2 (Cohen et al., 1996; Horio et al., 1997).

View larger version (67K): [in this window] [in a new window]   Fig. 8.   Alignment of amino acid sequences of GIRK1, GIRK2A, B, C, GIRK3, and GIRK4. Positions at which all six amino acid sequences are identical are boxed. The putative transmembrane segments (M1 and M2) and pore-forming region (H5) are indicated above the sequences. Overlined sequences of GIRK1, (a), the domain in the N terminus that was shown to bind G protein subunits, the GDP-form of G protein  subunits and heterotrimeric G proteins (Huang et al., 1995); (b), RGD sequence included in the integrin-binding site of fibronectin, vitronectin, and a variety of other adhesive proteins (Hynes, 1992), which is also found in other GIRK subunits; (c), the region whose amino acid sequence is similar to the G protein subunit-binding domain of the -adrenergic receptor kinase 1 (Reuveny et al., 1994); and (d) and (e), the domains in the C terminus that was shown to interact with G protein subunits (Huang et al., 1995). An underlined sequence of GIRK1: the sequence similar to that of the region of adenylyl cyclase 2 which is critical for activation of the enzyme by G protein subunits (N-X-X-E-R) (Chen et al., 1995; Huang et al., 1995). Numbered amino acids, (1) the phenylalanine (F137) of GIRK1 was shown to be responsible for the slow relaxation of GIRK1-containing KG channels (Kofuji et al., 1996a), although the interaction between F137 and the corresponding serine of other GIRK subunits may be important for the larger macroscopic current amplitude yielded with coexpression of GIRK1 and other GIRK subunits than the sum of those obtained with expression of each subunit alone (Chan et al., 1996; Wischmeyer et al., 1997); (2) the amino acid residue corresponding to aspartate at position 172 of the IRK subunit which was shown to be critically involved in the Mg2+/polyamine block of IRK1 channels (Stanfield et al., 1994; Taglialatela et al., 1994; Lu and MacKinnon, 1994; Wible et al., 1994; Lopatin et al., 1994; Ficker et al., 1994; Yang et al., 1995a); (3) the residue corresponding to the arginine of ROMK1 which was shown to be involved in interaction of ROMK1 channels with phosphatidilinositol 4,5-bisphosphate (PIP2) (Huang et al., 1998); and (4) the residues corresponding to glutamate at position 225 of the IRK1 subunit that was shown to be critically involved in the Mg2+/polyamine block of IRK1 channels (Yang et al., 1995a). The double-underlined sequence of GIRK2C: the sequence including the consensus sequence (S/T-X-V/I) for interaction with PSD-95/SAP90 anchoring protein (Gomparts, 1996; and Cohen et al., 1996), although the glutamate (E) preceding the sequence was also proposed to be important for the interaction of Shaker-type K+ channels with these anchoring proteins (Kim et al., 1995).

C. Tissue Distribution of GIRK Subunits

1. Peripheral tissues. Tissue distribution of mRNAs for GIRK subunits is summarized in table 3 (Kubo et al., 1993b; Dascal et al., 1993; Lesage et al., 1994; Stoffel et al., 1995; Bond et al., 1995; Dixon et al., 1995; Iizuka et al., 1995). In tissues other than brain, the atrium of the heart most abundantly expresses GIRK1 and GIRK4, both of which constitute the K_ACh channel. Both GIRK1 and GIRK4 proteins are diffusely immunostained in the atrium by antibodies specific for individual subunits (cf., fig. 15) (Iizuka et al., 1995). GIRK1 may be moderately expressed in the ventricle (Kubo et al., 1993b; Dascal et al., 1993; Karschin et al., 1994), although there seems to be a significant species-to-species difference in the level of expression of GIRK4 protein in the ventricle (Iizuka et al., 1995; Krapivinsky et al., 1995b). Iizuka et al. (1995) showed that GIRK4 immunoreactivities exist in subendocardial myocytes and also in dorsal atrial ganglia of rat. GIRK1 is also moderately expressed in other peripheral tissues except for spleen (table 3). GIRK2 and GIRK3 are rather brain-specific and barely found in peripheral tissues. However, GIRK2 (probably GIRK2C) exists in pancreatic islets (Stoffel et al., 1995), although GIRK2B mRNA is expressed ubiquitously in peripheral tissues (Isomoto et al., 1996). GIRK4 is also found in some other peripheral tissues.

2. Central nervous system. Detailed distribution of GIRK mRNAs in rat brain was analyzed with in situ hybridization and tabulated by Karschin et al. (1994, 1996) and DePaoli et al. (1994). Expression pattern of GIRK transcripts in the mouse brain is similar to that in the rat brain (Kobayashi et al., 1995). In general, GIRK1-3 mRNAs are abundantly expressed throughout the brain with overall similar distribution, although GIRK4 mRNA is expressed in the brain to a much lesser extent than other GIRK transcripts (Karschin et al., 1996; Iizuka et al., 1997).

When cRNAs for GIRK1 and M₂-muscarinic receptor are coinjected into Xenopus oocytes, a Kir current induced by ACh is expressed (Kubo et al., 1993b; Dascal et al., 1993). This current mimics at least some characteristics of the K_ACh channel current. GIRK1 expressed in Xenopus oocytes has been, therefore, successfully used to investigate the structure-function relationship of K_G channels (Reuveny et al., 1994; Slesinger et al., 1995; Kofuji et al., 1996a). However, Krapivinsky et al. (1995a) proposed that the K_ACh channel in cardiac atria is a heteromultimer of GIRK1 and GIRK4 rather than a homomultimer of GIRK1 because GIRK1 and GIRK4 proteins are immunocoprecipitated by both specific anti-GIRK4 and anti-GIRK1 antibodies from atrial membrane preparation (fig. 9A). Furthermore, coexpression of GIRK1 and GIRK4 in Xenopus oocytes yields greatly enhanced K_G channel currents compared with expression of either of the subunits alone (fig. 9B). Now, it is generally believed that GIRK1 is inactive by itself because expression of GIRK1 alone fails to give rise to K_G channel currents in different mammalian cell lines including CHO, COS, and HEK cells (Krapivinsky et al., 1995a; Philipson et al., 1995; Spauschus et al., 1996; Wischmeyer et al., 1997). It is likely that functional expression of GIRK1 alone is possible in Xenopus oocytes because oocytes endogenously express GIRK5 whose amino acid sequence is 78% identical with that of GIRK4 (Hedin et al., 1996). However, GIRK2 and GIRK4, but not GIRK3, may be able to form functional homomeric K_G channels, although not very efficiently (Lesage et al., 1994 and 1995; Bond et al., 1995, Krapivinsky et al., 1995a; Duprat et al., 1995; Kofuji et al., 1995; Velimirovic et al., 1996; Wischmeyer et al., 1997).

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Heteromultimeric structure of the muscarinic K+ channel. A: Immunological analysis for mouse atrial muscarinic K+ channel. Atrial membrane proteins were immunoblotted (IB) with anti-GIRK1C1 (lane 1) and anti-GIRK4N10 (lane 2) antibodies specific to GIRK1 and GIRK4 proteins, respectively. Some part of the GIRK1 protein, but not GIRK4 protein, seems to be glycosylated in the atrium. Lanes 3 and 4, the imunoprecipiants (IP) obtained from biotinylated mouse atrial membranes with the anti-GIRK1C1 and anti-GIRK4N10 antibodies, respectively. Both immunocomplexes of anti-GIRK1C1 and anti-GIRK4N10 seemed to be composed of three proteins that had molecular weights identical with GIRK1 and GIRK4 proteins on the gel. B: Acetylcholine- (Ach) induced K+ currents observed in Xenopus oocytes expressing M2-muscarinic receptors (m2R) plus mouse GIRK1 (m-GIRK1) and/or human GIRK4 (h-GIRK4). Acetylcholine- (1 µM) induced K+ currents at different membrane potentials were measured in the presence of 96 mM external K+ and are shown under the table. The voltage-clamp protocol is depicted at the left lower corner.

Heteromultimerization of GIRK subunits occurs not only between GIRK1 and GIRK4 but within any pairs of GIRK1-4 subunits (Kofuji et al., 1995; Iizuka et al., 1995; Duprat et al., 1995; Lesage et al., 1995; Spauschus et al., 1996; Velimirovic et al., 1996; Isomoto et al., 1996; Wischmeyer et al., 1997). Coexpression of GIRK1 with either of GIRKs2-4 generally yields several- to many-fold larger macroscopic currents than the sum of those obtained with each subunit alone. However, it has not been unequivocally answered whether similar synergistic interaction also occurs between GIRKs2-4. The synergistic enhancement of current expression was reported to occur with GIRK2/GIRK4 (Duprat et al., 1995; Ferrer et al., 1995) and GIRK3/GIRK4 combinations (Spauschus et al., 1996). However, these results were not necessarily confirmed by the others (Lesage et al., 1995; Velimirovic et al., 1996; Wischmeyer et al., 1997). Coexpression of GIRK3 with GIRK2 was shown to suppress GIRK2 channel currents (Kofuji et al., 1995). It is not clear whether more than two types of GIRK subunits can be assembled into a single K_G channel (Wischmeyer et al., 1997).

GIRK1/GIRK4 heteromultimeric K_G channels are likely to correspond to cardiac K_ACh channels as mentioned in this Section. GIRK1/GIRK2 channels, however, may represent some neuronal type of K_G channels for the following reasons: (a) GIRK2 mRNA is preferentially expressed in the brain (table 3); (b) GIRK1 and GIRK2 exhibit overlapping distribution in many areas of the brain at both the mRNA and protein levels (Karschin et al., 1996; Liao et al., 1996); (c) both specific anti-GIRK1 and anti-GIRK2 antibodies coimmunoprecipitate GIRK1 and GIRK2 proteins from membrane preparations of the brain (Liao et al., 1996); (d) in the mice whose GIRK2 genes are genetically deleted (GIRK2 -/-), the substantial amount of GIRK1 proteins is concomitantly lost in the brain (Signorini et al., 1997); and (e) the hippocampal CA1 and CA3 pyramidal neurons of these mice fail to exhibit postsynaptic inhibitory K_G channel currents in response to different inhibitory neurotransmitters (Lüscher et al., 1997). Some neuronal K_G channels may also be composed of GIRK1 and GIRK3 because their transcripts are also expressed together in various regions of the brain (Karschin et al., 1996). It is also possible that GIRK4 is included in neuronal K_G channels (Spauschus et al., 1996; Iizuka et al., 1997) and that some K_G channels are homomultimers of GIRKs2 or 4 or heteromultimers of GIRKs2-4.

Chan et al. (1996) found that the synergistic interaction between GIRK1 and GIRK4 for K_G channel current expression can be at least in part ascribed to interaction between phenylalanine at position 137 (F137) in the H5 region of GIRK1 and serine at the corresponding site (S143) in GIRK4 (fig. 8). GIRKs2 and 3 also have conserved serine at this site. They found that coexpression of the wild-type GIRK4 with the mutant GIRK4 whose S143 was replaced with phenylalanine [GIRK4(S143F)] yielded the significantly larger macroscopic current amplitude than the sum of those obtained with either of the subunits alone, as is the case for GIRK1/GIRK4 coexpression. At the single-channel level, GIRK4(S143F)/GIRK4 channels, like GIRK1/GIRK4 channels, opened in clearer bursts and exhibited a significantly longer open time than GIRK4 homomeric channels (Krapivinsky et al., 1995a; Chan et al., 1996). Thus, F137 of GIRK1 may be responsible for the larger macroscopic current amplitude of GIRK1/GIRK4 than GIRK4 channels by stabilizing the channel's open-state conformation. Wischmeyer et al. (1997) also obtained similar results by using a mutant GIRK3 subunit bearing a mutation corresponding to GIRK4(S143F) [i.e., GIRK3(S114F)].

However, neither GIRK4(S143F) nor GIRK3(S114F) synergistically interacted with GIRK1 (Chan et al., 1996; Wischmeyer et al., 1997). GIRK1 whose F137 was substituted with serine [GIRK1(F137S)] could form a functional homomeric channel (Chan et al., 1996; Wischmeyer et al., 1997), although coexpression of GIRK1(F137S) with the wild-type GIRK1 yielded current amplitudes intermediate between those obtained with either of the subunits alone (Wischmeyer et al., 1997). These results indicate that F137 of GIRK1 on its own is inhibitory for the K⁺ ion flux through the channel pore possibly because of its bulky aromatic side chain (Wischmeyer et al., 1997). Thus, serine derived from other types of GIRK subunits might somehow attenuate this inhibitory effect of F137 and yield the larger current amplitudes of the heteromeic GIRK1-containing channels than those obtained with expression of GIRK1 alone.

It is, however, difficult to explain whole the aspect of the synergistic interaction between GIRK1 and other GIRK subunits only in terms of the interaction between complementary phenylalanine/serine residues in the heteromeric channel pore. For example, GIRK1(F137S)/GIRK4 channels exhibit much larger currents than the GIRK4 homomeric channels (Chan et al., 1996). GIRK1/GIRK4 channels have ~2 times larger macroscopic currents than GIRK4(S143F)/GIRK4 channels. Thus, some region(s) other than F137 of GIRK1 must also be significantly involved in the synergistic interaction between GIRK1 and GIRK4. Similarly, GIRK1(F137S)/GIRK4 channels have substantially larger current amplitudes than homomeric GIRK1(F137S) channels (Chan et al., 1996), indicating that GIRK4 also has some effect(s) on GIRK1 which cannot be ascribed to the serine/phenylalanine interaction.

Kennedy et al. (1996) found that GIRK1 cannot translocate to the cell membrane in the absence of GIRK4. They expressed epitope-tagged GIRK1 and GIRK4 in COS cells alone or in combination and examined the localization of the subunits with immunofluorescence labeling. When expressed alone, GIRK1 was localized to the cytosol associated with intracellular intermediate filament proteins, whereas GIRK4 was primarily on the plasma membrane. GIRK1 was detected on the plasma membrane when coexpressed with GIRK4. Therefore, GIRK4 may facilitate the membrane-translocation of GIRK1 subunits. It has, however, not been shown whether GIRK2 and GIRK3 also have similar effect of translocation of GIRK1 to the membrane.

Yang et al. (1995a) found that the inward rectification of the IRK1 channel was substantially attenuated by replacement of aspartate (D) at position 172 in the M2 region with asparagine (N) and substitution of glutamate (E) at position 225 in the proximal carboxyl terminal region with lysine (K). They analyzed the subunit stoichiometry of the IRK1 channel by using the double mutation. They formed tandem tetramers or trimers consisting of different numbers of wild-type subunits and/or subunits bearing the mutation. When either the tetramer or trimer of the wild-type subunits was expressed in Xenopus oocytes, the resultant channel currents exhibited the same extent of inward rectification as those obtained from expression of the wild-type monomers. This was also the case when the tetramer was coexpressed with an excess amount of the mutant monomers, indicating that the number of subunits required for a functional IRK1 channel does not exceed four. In contrast, the inward rectification of the channel currents resulting from expression of wild-type trimers was significantly attenuated by coexpression of the mutant monomers. In this case, the channel currents exhibited approximately the same extent of inward rectification as those obtained from expression of a tetramer consisting of three wild-type subunits and one mutant subunit. These data suggest that a functional IRK1 channel is formed by a tetramer of IRK1 subunits.

Biochemical measurement of the molecular weight of brain K_G channel proteins suggested that these channels also have tetrameric structure (Inanobe et al., 1995a). As stated earlier, GIRK2 and GIRK4 can form functional homomeric channels (fig. 10A) (Lesage et al., 1995; Krapivinsky et al., 1995a; Duprat et al., 1995; Bond et al., 1995; Kofuji et al., 1995; Velimirovic et al., 1996). However, GIRK1 requires other GIRK subunits to form functional K_G channels (Krapivinsky et al., 1995a; Duprat et al., 1995; Velimirovic et al., 1996; Hedin et al., 1996; Chan et al., 1996; Wischmeyer et al., 1997). Tucker et al. (1996) assessed the stoichiometry and relative subunit positions in the GIRK1/GIRK4 heteromeric K_G channel by using tandemly linked tetramers consisting of GIRK1 and GIRK4. They found that the most efficient channel comprises two subunits of each type in an alternative array within the tetramer (fig. 10Ba). Through a similar approach, Silverman et al. (1996) found that more than one kind of subunit arrangement including (GIRK1) (GIRK1) (GIRK4) (GIRK4) may also be viable (fig. 10Bb). They also obtained similar data with GIRK1/GIRK2 heteromers.

View larger version (52K): [in this window] [in a new window]   Fig. 10.   Tetrameric structure of IRK(Kir2.x) and GIRK(Kir3.x) channels. A putative tetrameric structure of the channels composed of IRK or GIRK subunits in the cell membrane is schematically represented according to the functional and structural studies reported by Lesage et al. (1994 and 1995), Krapivinsky et al. (1995a), Velimirovic et al. (1996), Yang et al. (1995b), Silverman et al. (1996), and Tucker et al. (1996).

Tinker et al. (1996) studied the molecular mechanism of homomeric assembly of IRK1. They concluded that among IRK1, IRK2, and IRK3, the proximal C-terminus and the M2 region equally contribute to polymerization. The proximal C-terminus plays a more significant role in prevention of heteromultimerization between more distantly related channel subunits, such as IRK1 and ROMK1. Tucker et al. (1996), however, found that the core region of the GIRK subunit (M1-H5-M2) but neither the C- nor N-terminal domain was important for subunit assembly between GIRK1 and GIRK4. Thus, the mechanism of heteromultimerization of GIRK subunits may not be the same as that of IRK subunits.

F. Molecular Mechanism Underlying G Protein Activation of G Protein-Gated K⁺ Channels

1. Interaction between G protein subunits and subunits of G protein-gated K⁺ channels. a. THE G PROTEIN SUBUNIT-BINDING DOMAINS IN GIRK1 SUBUNITS. GIRK1 has a significantly longer C-terminus than the constitutively active Kir channel subunits such as IRK1 (Kubo et al., 1993a,b). Reuveny et al. (1994) first suggested that the C-terminus of GIRK1 includes an amino acid sequence (between positions 318 and 455) exhibiting a certain level of similarity (~26%) with that of the G-binding site of ARK1 (fig. 8). They also found that truncation of the C-terminus of GIRK1 at leucine (L) at position 403 but not at proline (P) at position 462 resulted in loss of functional expression of a K_G channel in Xenopus oocytes coexpressing G₁₂. Inanobe et al. (1995b) directly demonstrated that G bound to a glutathione S-transferase (GST) fusion protein including the whole C-terminal domain of GIRK1 (between positions 180 and 501). The fusion protein also bound G when incubated with purified trimeric G_i in the presence of GTPS but not in the presence of GDP (see also Inanobe et al., 1995a). Furthermore, the binding of G to the fusion protein was prevented by G_-GDP but not G_-GTPS, indicating that the C-terminal domain of GIRK1 cannot bind with G included in the trimeric form of the G protein.

View larger version (35K): [in this window] [in a new window]   Fig. 11.   Domains of GIRK1 involved in G protein-mediated regulation of GIRK1-containing KG channels. Schematic representation of approximate positions of identified domains of GIRK1 which are responsible for interaction of GIRK1 and G protein subunits or phosphatidilinositol 4,5-bisphosphate (PIP2). The approximate positions of three identified G protein -binding sites (one in the N- and two in the C-terminal domain) and one trimeric G protein and/or GDP-from of G protein  subunit-binding site in the N-terminal domain are depicted (Huang et al., 1995, 1997; Slesinger et al., 1995). R is the residue corresponding to the arginine of ROMK1 that was reported to play a critical role in interaction of ROMK1 channels with PIP2 (Huang et al., 1998).

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b. THE G PROTEIN SUBUNIT-BINDING DOMAINS IN OTHER SUBUNITS OF G PROTEIN-GATED K⁺ channels. Homomeric GIRK2 or GIRK4 channels are also activated by G (Krapivinsky et al., 1995a

2. Mechanism underlying G protein subunitinduced activation of G protein-gated K⁺ channels. As described in Sections II.B.3. and 4., functional analyses of the K_ACh channel indicate that the G protein-mediated activation of the channel results from an increase in the functional number of the channels due to the cooperative interaction of G_K and GIRK subunits (Hosoya et al., 1996). However, the molecular mechanism responsible for this phenomenon has not been clearly identified.

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3. Interaction between subunits of G protein-gated K⁺ channels, G proteins, and membrane agonist receptors. Huang et al. (1995) found that G_-GDP and the entire heterotrimeric G protein can bind to fusion proteins containing the N-terminal domain of GIRK1 (fig. 11). This observation raises the possibility that membrane agonist receptors, G_K, and K_G channels, might form a functional complex in native K_G channel systems. Namely, once G_K associated with a GIRK1 subunit is activated, the dissociated G_K would promptly access the G_K-binding site on the GIRK1 subunit. The G_K-GTP dissociated from the G_K, however, may be quickly converted to G_K-GDP by the aid of RGS proteins, leading to reassociation of G_K-GDP with the GIRK1 subunit. This G_K-GDP would effectively sequestrate the G_K from the G_K-binding site on the GIRK1 subunit, and the cycle would be complete. Indeed, it has been shown that K_G channels coexpressed with RGS proteins in Xenopus oocytes are deactivated much faster than expected from the intrinsic rate of G dissociation from GIRK subunits (Doupnik et al., 1997; Krapivinsky et al., 1995c; Saitoh et al., 1997). In the classical view of receptor/G protein interaction (Levitzki, 1981), association of an agonist-bound receptor with G_-GDP is the ratelimiting step for the receptor-mediated activation of effectors. However, the fast conversion of G_K-GTP to G_K-GDP due to the enhanced GTP_i hydrolysis rate evoked by RGS proteins might not provide a sufficient time for G_K-GTP to be dissociated from receptor. In the continuous presence of an agonist bound to the receptor, therefore, the receptor would promptly restart the next round of the G protein cycle. The resultant increase in GDP/GTP exchange might balance the accelerated GTPase activity and thereby maintain the steady-state concentrations of G_K-GTP and G_K at a certain level, which would explain why RGS proteins do not significantly decrease the steady state response of the K_G channel system (Doupnik et al., 1997; Saitoh et al., 1997). A distinct but similar hypothesis of a functional receptor/G protein/effector complex was recently proposed by Ross' group based on an extensive analysis of the reconstituted M₁-muscarinic/G_q/phospholipase C-1 system (Biddlecome et al., 1996).

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4. Possible mechanisms underlying specific signal transduction in the receptor/G protein/G protein-gated K⁺ channel system. In atrial myocytes, the K_ACh channel is activated by stimulation of M₂-muscarinic and A₁ adenosine receptors. However, ₁-adrenergic stimulation, which would also induce dissociation of G from G_s, never activates the K_ACh channel in cardiac atrial myocytes.

Such specific signal transduction cannot be explained in terms of the different affinities of distinct types of G for K_G channels. Wickman et al. (1994)

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	IV. Voltage-Dependent Properties of G Protein-Gated K+ Channels

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In previous sections, we considered the G protein-mediated activation of K_G channels. In this section, we will deal with voltage-dependent properties of K_G channels. As described in the section II.B.1., all K_G channels exhibit the inward rectification property where ionic currents flow through K_G channels more readily in the inward than the outward direction. The mechanism of inward rectification has been mainly studied with the constitutively-active, classical Kir channels such as IRK1 channels. Most of our understanding of the mechanism of inward rectification of K_G channels is, therefore, based on these results. However, the inward rectification of K_G channels is not identical with that of the classical Kir channels especially in terms of its voltage-dependency and kinetics. In this section, we first review the present understanding of the mechanism of inward rectification of the classical Kir channels, and then deal with specific issues regarding K_G channels.

A. Inwardly-Rectifying K⁺ Channels

1. Voltage-dependent change in inwardly rectifying K⁺ channel activity. K⁺ channels mediate a flow of K⁺ ions depending on an electrochemical gradient of K⁺ ions across the plasma membrane (i.e., V_m - E_K) (Hille, 1992b). Thus, the macroscopic K⁺ current flowing through K⁺-selective channels can be expressed as:

(3)

where I is a macroscopic K⁺ current, and gK is a macroscopic chord conductance of the K⁺ channels. Comparison of equations 2 and 3 indicates that gK is N*P_o*. If gK is constant, the I-V relationship should be linear. In the presence of the physiological transmembrane gradient of K⁺ concentration; however, it slightly bends outward as predicted by the constant field theory (Hille, 1992b).

(4)

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2. Mg²⁺ and polyamine block. The voltage-dependent change in gK of strong inward rectifiers recently turned out to be caused by blockade of the channel pore by intracellular cations such as Mg²⁺_i and polyamines (Matsuda et al., 1987; Vandenberg, 1987; Matsuda, 1988 and 1991; Lopatin et al., 1994; Ficker et al., 1994; Fakler et al., 1994). In other words, the apparent gating of Kir channels results from exogenous elements. Consistent with this, they lack in their primary structure the domain corresponding to the voltage-sensing S4 region of voltage-dependent K⁺ channels (Ho et al., 1993; Kubo et al., 1993a). Polyamines are aliphatic amines such as putrescine, spermidine and spermine, which are endogenous metabolic intermediates derived from arginine. They normally exist in submillimolar concentrations in the cytosol of almost all cell-types (Watanabe et al., 1991). Putrescine, spermidine, and spermine bear 2, 3, and 4 positive charges per molecule, respectively. Thus it is likely that polyamines and Mg²⁺_i interact with Kir channels via the electrostatic force created between their own positive charges and negatively charged amino acid residues within the channel pore (Stanfield et al., 1994; Taglialatela et al., 1994; Lu and MacKinnon, 1994; Wible et al., 1994; Lopatin et al., 1994; Ficker et al., 1994; Fakler et al., 1995; Yang et al., 1995a)

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3. Mg²⁺/polyamine block sites in the inwardly rectifying K⁺ channel pore. The molecular mechanism underlying the Mg²⁺/polyamine block has been elucidated through comparison of the molecular structures of the strong inward rectifier IRK1 and the weak inward rectifier ROMK1 channels. At E_K 20 mV, the K_d value of Mg²⁺ and spermine for IRK1 channels are respectively ~1 mM and ~10 µM (Lopatin et al., 1994), which are ~1000 and ~1000,000 times smaller than the corresponding values for ROMK1 channels (Nichols et al., 1994; Yang et al., 1995a). Mutagenesis studies revealed that at least two negatively-charged amino acid residues found in IRK1 (aspartate (D) at position 172 in the M2 region and glutamate (E) at position 224 in the proximal C-terminus) but not in ROMK1 are responsible for this discrepancy (fig. 12) (Stanfield et al., 1994; Taglialatela et al., 1994; Lu and MacKinnon, 1994; Wible et al., 1994; Lopatin et al., 1994; Ficker et al., 1994; Yang et al., 1995a). For convenience, we shall designate these two positions R1 and R2, respectively. The two acidic residues are likely to interact with the blocking particles independently of each other (Yang et al., 1995a). Other residues may also participate in the blocking to a lesser extent because ROMK1 exhibits weak but significant inward rectification (Ho et al., 1993). IRK2 and IRK3 also have the aspartate (D) and glutamate (E) residues at the analogous positions, although both BIR and uK_ATP-1 lack acidic residues at either of the sites (Takahashi et al., 1994; Morishige et al., 1994; Ho et al, 1993; Inagaki et al., 1995a and b).

View larger version (41K): [in this window] [in a new window]   Fig. 12.   Proposed Mg2+/polyamine interaction sites in Kir subunits. A schematic representation of proposed Mg2+/polyamine interaction sites in Kir subunits. R1 and R2 indicate the approximate position of the negatively charged amino acid residues in IRK1 that are responsible for the high sensitivity of the channel to intracellular Mg2+ and polyamine: aspartate at position 172 and glutamate at position 224, respectively (Stanfield et al., 1994; Taglialatela et al., 1994; Lu and MacKinnon, 1994; Wible et al., 1994; Lopatin et al., 1994; Ficker et al., 1994; Yang et al., 1995a). The table shows the amino acid residues at the sites analogous to R1 or R2 in different types of Kir subunits. N indicates asparagine; G, glycine; D, aspartate; E, glutamate; and S, serine.

B. Inward Rectification of G Protein-Gated K⁺ Channels

1. Inward rectification of the muscarinic K⁺ channel. The K_ACh channel exhibits clear inward rectification irrespective of ACh concentrations and thus G_K activity (figs. 13Aa and b). The G-V relationship of the K_ACh channel can be fitted with Eq. 4 with V_h and v of ~0 and ~20 mV, respectively (fig. 13Ac). Both the values are significantly larger than those of strong inward rectifiers but much smaller than those of weak inward rectifiers. The Boltzmann's fit deviates from the measured values at V positive to ~+40 mV. Thus, the actual G-V relationship is less steep at depolarized potentials than predicted from the data at more negative potentials as is the case for strong inward rectifiers (Ishihara et al., 1989)

View larger version (25K): [in this window] [in a new window]   Fig. 13.   Inward rectification of the muscarinic K+ channel. These data were obtained from the whole-cell recording of the muscarinic K+ channel current in a single sinoatrial node cell of rabbit. A: a, The channel current induced by 5 µM acetylcholine applied to the bath. The extracellular K+ concentration was 5.4 mM. Five hundred msec command pulses (c.p.) were applied from the holding potential of 53 mV to the potentials indicated above each trace. Note that in each trace, the command pulse induced an instantaneous jump followed by a time-dependent change (relaxation) of the channel current. This was also the case when the membrane potential was returned to the holding potential. b, The relationship between the membrane potential and the channel currents induced by different concentrations of acetylcholine. The magnitude of the currents was measured immediately (closed symbols) or 500 msec (open symbols) after application of the command pulses. The reversal potential was ~80 mV under each condition. c, The relationship between the membrane potential relative to the K+ equilibration potential (~80 mV under these conditions) (V) and the macroscopic chord conductance (gK) of the muscarinic K+ channel current induced by 5 µM acetylcholine (symbols). In this graph, gK is normalized to its maximum value (gKmax) predicted by the fit of the data with Boltzmann's equation (Equation 4 in the text) (line in the graph). The best fit was obtained with Vh and v of 0.32 and 21.0 mV, respectively. B: The voltage-dependence of the time constants of the relaxation of the muscarinic K+ channel currents induced by 0.2 µM (squares), 1 µM (triangles), and 5 µM (circles) of acetylcholine. The voltage-clamp protocol was the same as in A. The relaxation of each acetylcholine-induced current could be fitted with a biexponential function with a smaller (open symbols) and a larger (closed symbols) time constant. The inverted triangle on the voltage-axis indicates the reversal potential of the channel currents. C: The channel currents observed in the sinoatrial node cell in the presence and the absence of different concentrations of acetylcholine. Command steps were applied from 53 mV to various membrane potentials indicated to the right of each current trace, and the obtained currents are superimposed. Note that the higher concentration of acetylcholine induced not only larger currents but accelerated relaxation at every membrane potential. [Reproduced with permission from Kurachi (1990)].

2. Mg²⁺/polyamine block of G protein-gated K⁺ channels. Fig. 14A shows the effect of Mg²⁺_i and intracellular polyamines on the single K_ACh channel current (Yamada and Kurachi, 1995). In these experiments an inside-out patch was obtained from a rabbit atrial myocyte with pipette solution containing 145 mM K⁺ and 0.5 µM ACh. The patch was excised in a solution containing 145 mM K⁺ and 1.4 mM free Mg²⁺. Application of a nonhydrolyzable GTP analogue, GTPS induced inward K_ACh channel currents at 60 mV. This channel activity continued even after washout of GTPS because the nucleotide irreversibly activated G_K in the membrane. Depolarization of the membrane to +40 mV resulted in appearance of small outward currents. The unitary amplitude of these outward currents was increased after removal of Mg²⁺_i. Under these conditions, the single-channel I-V relationship was virtually linear, indicating that  had been reduced by Mg²⁺ block at the depolarized potential (Horie and Irisawa, 1987, 1989). The IC₅₀ of Mg²⁺_i has been reported to be ~300 µM at V of +40 mV (Horie and Irisawa, 1987 and 1989), whereas cytosolic Mg²⁺ concentrations are estimated to be in millimolar range (Hess et al., 1982; Gupta et al., 1984; Alvarez-Leefmans et al., 1986, Blatter and McGuigan, 1986). Thus, Mg²⁺ block is likely to underlie the physiological inward rectification of the single K_ACh channel conductance ()

View larger version (40K): [in this window] [in a new window]   Fig. 14.   Effect of spermine on the muscarinic K+ channel. All data shown were obtained from muscarinic K+ channels in inside-out patch membranes of rabbit atrial myocytes exposed to symmetrical 145 mM K+ solutions. The pipette also contained 0.5 µM acetylcholine. A: Effect of 10 µM spermine applied to the internal side of the patch membrane. The protocol of perfusion of the intracellular side of the patch membrane and the change in holding membrane potential are indicated above and below the current trace, respectively. Arrowheads indicate the zero current levels. Large and small spikes observed at + 40 mV in the presence of spermine were occasional opening of ATP-sensitive K+ and muscarinic K+ channels, respectively. B: Concentration-dependent inhibitory effect of internal spermine on outward muscarinic K+ currents at + 40 mV. a, After the muscarinic K+ channels were irreversibly activated by GTPS the indicated concentrations of spermine was applied to the intracellular side of the patch membrane in the absence of intracellular Mg2+. The N·Po of the muscarinic K+ channels was 0.096, 0.084, 0.046, and 0.014 in the presence of 0, 10, 30, and 100 nM spermine, respectively. The bars and numbers below the current trace correspond to the numbers in the graph c. b, High-speed current traces obtained from the same patch as a under the conditions indicated to the right of each current trace. c, The current amplitude histogram obtained from the same current record as a. The numbers in the graph correspond to those in a and indicate the portions of the record from which each histogram was constructed. Bin width is 0.02 pA, and the number of points in each bin is expressed as a percentage of the total number of points recorded. d, Concentration-dependent effect of spermine on the unitary current amplitude (open circles) and N·Po (closed circles). Data were normalized to the values obtained in the absence of spermine. Symbols and bars indicate the mean ± SE. Lines were drawn by eye. Unitary channel current amplitude in 1 and 10 µM spermine was measured by eye because very few openings of muscarinic K+ channels were available in this concentration range. C: Voltage-dependent change in N·Po of muscarinic K+ channels in the presence (closed circles) and absence (open circles) of 100 nM spermine. ATP (1 mM) was added to the intracellular side of patch membranes. N·Po is expressed as a percentage of the value recorded at 60 mV. Symbols and bars indicate the mean ± SE. Lines were drawn by eye. D: The time-dependent change in muscarinic K+ currents in the presence or absence of 300 nM, 1 µM, and 3 µM spermine. Muscarinic K+ channels in an inside-out patch membrane were preactivated with 10 µM GTPS, and then Mg2+ was removed from the intracellular side of the patch membrane. As shown at the top, 1 sec duration command steps to + 40 mV were applied every 5 sec from a 60 mV holding potential. Reading from left to right, progressively higher concentrations of spermine (300 nM3 µM) were applied to the intracellular side of the patch membrane. The membrane was depolarized ~30 to 40 times in each condition. The first and second rows show representative current traces. In each of the traces, the baseline current was subtracted with a computer. Arrowheads indicate the zero current level. Time and current scales are shown to the right beneath the second row. The third row shows the ensemble average current obtained from 20 successive pulses under each condition. The zero current level is indicated by a straight line. Each of the ensemble average current traces could be fitted by a single exponential both at + 40 and 60 mV. At + 40 mV, the time constant obtained with the best fit was 9.9 sec in the control; 1.3 sec in the presence of 300 nM spermine; 0.38 sec, 1 µM; 0.18 sec, 3 µM. [Reproduced with permission from Yamada and Kurachi (1995)].

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3. The Mg²⁺/polyamine-binding sites in G protein-gated K⁺ channels. The K_G channels have an unique arrangement of negative charges at the R1 and R2 sites (figs. 8 and 12). GIRK1 has negatively-charged aspartate at R1 (D173) but uncharged serine (S) at R2 (Kubo et al., 1993b; Dascal et al., 1993). However, all GIRKs 2-5 have uncharged asparagine (N) at R1 and negativelycharged glutamate (E) at R2 (Lesage et al., 1994 and 1995; Isomoto et al., 1996; Krapivinsky et al., 1995a). Recently, Kofuji et al. (1996a) examined the functional significance of D173 of GIRK1 by replacing this residue with non-charged asparagine [GIRK1(D173N)]. The G-V relationship of K_G channels in Xenopus oocytes expressing GIRK1(D173N) was much less steeply voltagedependent and shifted leftward along the voltage-axis compared with that for the wild-type GIRK1: v and V_h were respectively ~40 and ~50 mV for GIRK1(D173N), and ~20 and ~30 mV for the wild-type GIRK1. These data indicate that the Mg²⁺_i/polyamine block at D173 of GIRK1 is in fact crucial for the inward rectification of K_G channels that contain GIRK1.

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4. Slow relaxation of G protein-gated K⁺ channels containing GIRK1. No mammalian strong inward rectifiers show such slow relaxation as the K_ACh channel does (fig. 13) (Leech and Standfield, 1981; Kurachi, 1985; Tourneur et al., 1987; Harvey and Ten Eick, 1988; Cohen et al., 1989; Silver and DeCoursey, 1990). GIRK2 and GIRK4 homomultimeric channels show fast relaxation kinetics similar to those of IRKs (Lesage et al., 1995; Krapivinsky et al., 1995a; Iizuka et al., 1995; Duprat et al., 1995; Bond et al., 1995; Kofuji et al., 1995; Velimirovic et al., 1996). The heteromeric K_G channels containing GIRK1, however, exhibit the slow relaxation after a voltage step similar to that of the K_ACh channel (i.e., a native GIRK1/GIRK4 heteromer) (Kubo et al., 1993b; Krapivinsky et al., 1995a; Iizuka et al., 1995; Duprat et al. 1995; Kofuji et al., 1995; Doupnik et al., 1995b; Velimirovic et al., 1996; Isomoto et al., 1996), although the relaxation kinetics of GIRK1/GIRK2 channels may be slightly faster than those of GIRK1/GIRK3 or GIRK1/GIRK4 channels (Wischmeyer et al., 1997). Therefore, it is likely that GIRK1 is responsible for the slow relaxation.

	V. Pharmacological Properties of G Protein-Gated K+ Channels

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The pharmacological properties of K_G channels including the K_ACh channel have not been extensively investigated. However, it is well known that like other Kir channels (Hagiwara and Takahashi, 1974; Hagiwara et al., 1976, 1978; Gay and Stanfield, 1977; Standen and Stanfield, 1978; Constanti and Galvan, 1983; Harvey and Ten Eick, 1989), cesium and barium ions effectively block various native K_G channels (Carmeliet and Mubagwa, 1986; Gähwiler and Brown, 1985; Inoue et al., 1988; Mihara et al., 1987; Surprenant and North, 1988; Pennefather et al., 1988; Penington et al., 1993; Lacey et al., 1987 and 1988; Gerber et al., 1991; Dousmanis and Pennefather, 1992; Sodickson and Bean, 1996). These cations, when applied extracellularly, block K_G channels in a steeply voltage-dependent manner with higher potency at more negative membrane potentials (but see Sodickson and Bean, 1996). Thus, the cations seem to go through the transmembrane electric field before binding to their receptor sites in the channel pore. Depending on experimental protocols and cell type, the reported K_d values of these cations for K_G channels vary but largely fall into a concentration range of 10^-3-10^-2 M for cesium ions and 10⁵ to 10⁴ M for barium ions at negative membrane potentials. These values are ~10 times more than those required to block the classical Kir channels (Hagiwara and Takahashi, 1974; Hagiwara et al., 1976, 1978; Gay and Stanfield, 1977; Standen and Stanfield, 1978; Constanti and Galvan, 1983; Harvey and Ten Eick, 1989). Similar values were reported for the cesium or barium blockade of the recombinant K_G channels (Velimirovic et al., 1996; Lesage et al., 1995; Bond et al., 1995). Although barium ions are known to block the classical Kir channel in a clear time-dependent manner (Hagiwara et al., 1978; Constanti and Galvan, 1983), some K_G channels were reported to be instantaneously inhibited by the ions (Carmeliet and Mubagwa, 1986; Dousmanis and Pennefather, 1992, but see Velimirovic et al., 1996). K_G channels are also known to be blocked by millimolar concentrations of rubidium ions (Katayama et al., 1997; Mihara et al., 1987; Surprenant and North, 1988).

Recent studies indicated that cesium, rubidium, and strontium ions block a recombinant strong inward rectifier IRK1 channel at least in part through binding to a negatively charged aspartate at the R1 position in the putative channel pore (D172) (Reuveny et al., 1996; Abrams et al., 1996). As we have already seen in Section IV.A.3., this acidic residue is conserved among various constitutively active Kir channels and may serve as a receptor site for the intracellular Mg²⁺ and polyamines as well (fig. 12) (Stanfield et al., 1994; Taglialatela et al., 1994; Lu and MacKinnon, 1994; Wible et al., 1994; Lopatin et al., 1994; Ficker et al., 1994; Yang et al., 1995a). GIRK1 but not GIRKs2-4 bear this residue. This may at least in part explain the lower sensitivity of K_G channels to these cation blockers than classical Kir channels.

K_G channels are also fully blocked by quinidine and quinine with the IC₅₀ of 10 µM (Kurachi et al., 1987a; Nakajima et al., 1989; Katayama et al., 1997). Verapamil also inhibits the ACh-activated cardiac K_ACh channel with the IC₅₀ of 1 µM although its effect is partially mediated by suppression of the M₂-muscarinic receptor/G_K system (Ito et al., 1989). To our knowledge, K_G channels are rather insensitive to other well-known channel blockers such as tetraethylammonium, 4-aminopyridine, apamine, charybdotoxin, disopyramide, and procainamide (Inoue et al., 1988; Lacey et al., 1987 and 1988; Nakajima et al., 1989; Katayama et al., 1997).

	VI. Localization of the G Protein-Gated K+ Channel in Different Organs

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G_K seems to be the physiologically functional arm of G_K activating K_G channels not only in the heart but also in the brain and endocrine organs. However, the molecular mechanisms of G protein-regulation of ion channels have been found to be more complicated than we had thought. In AtT20 cells which had been transfected with the _2A-adrenergic receptor, adrenergic agonists can inhibit the Ca²⁺ current and adenylyl cyclase and activate a K⁺ current (Surprenant et al., 1992). A point mutation of the receptor removes activation of the K⁺ current, but not inhibition of Ca²⁺ current and adenylyl cyclase. This indicates that the G protein coupling to the K⁺ channel is different from that to the Ca²⁺ channel and adenylyl cyclase although the receptor is the same. G proteins may thus be more specific to each receptor and to each signaling system than we are currently assuming or than we can determine in vitro. In Xenopus oocytes, however, when ₂-adrenergic receptors, G_s protein, and GIRK1 are coexpressed, -adrenergic agonists could induce activation of K_G channel current (Lim et al., 1995). Accordingly, the affinity of particular G protein subunits for the K_G channel may not be sufficient to explain specific activation of K_ACh channel by G_K. Actually, various combinations of recombinant G (except for G₁₁) have similar efficacy and potency in activating K_ACh channels (Wickman et al., 1994). However, the receptor specificity in cardiac atrial myocytes is well documented by extensive studies (Kurachi, 1995). It is often argued that receptor specificity could arise from compartmentalization of the appropriate receptors and channels, although little evidence has been shown for such compartmentalization. We do not know whether different mechanisms underlie receptor specificity in different organs. In other words, we have not yet fully answered the question how information specifically passes from a membrane receptor to the effector, the K_G channel, via G proteins.

Because the signal transduction mechanisms are not necessarily the same among heart, neurons, and endocrine cells, it is worthwhile at present to summarize the observations on the localization of K_G channels in these organs. Apparently, GIRK1 and GIRK4 immunoreactivities diffusely distribute in the cell membrane of cardiac myocytes, whereas those of GIRK1 and/or GIRK2 are localized to specialized segments of neuronal membrane, such as presynaptic axonal termini and postsynaptic dendritic regions (fig. 15). Thus, we may tentatively classify the system based on the apparent distribution into two categories: (a) homogeneously distributed system and (b) localized system. In these systems, different mechanisms may underlie signal transduction.

View larger version (140K): [in this window] [in a new window]   Fig. 15.   Different subcellular localization of GIRK1 proteins. A and B: Immunohistochemical analyses for the GIRK1 proteins in the rat atrium (A) and ventricle (B). Homogeneous immunoreactivity was found on the plasma membranes of the atrial, but not of the ventricular myocytes. C and D: Electron microscopic analyses for the GIRK1 immunoreactivity in the rat paraventricular nucleus of the hypothalamus. Obvious GIRK1 immunoreactivity was present at the axonal terminals (probably on the vesicles) neighboring upon a dendrite (D) (reproduced with permission from Morishige et al., 1996). All GIRK1 immunoreactivities were developed with diaminobenzidine-horseradish peroxidase method.

The cardiac K_ACh channel is the prototype of K_G channels and is a heteromultimer of GIRK1 and GIRK4 (Krapivinsky et al., 1995a). Immunohistochemistry using a specific antibody showed that GIRK1 is homogeneously localized on the cell membrane of atrial but not ventricular myocytes (fig. 15A). This is consistent with the electrophysiological studies of cardiac myocytes. The electrophysiological experiments also suggested that some topological restriction may exist in cardiac atrial myocytes, because K_ACh channels in the cell-attached membrane patch are activated by ACh or adenosine when they are applied to the pipette solution, but not when they are added to the bathing solution (Soejima and Noma, 1984).

It was found that either G_i- or G_s-coupled receptors, when expressed together with GIRK channels in Xenopus oocytes, can activate the channels (Lim et al., 1995). This may indicate that under the conditions where compartmentalization does not exist, as in Xenopus oocytes, the subunits released from G_s may be able to activate K_G channels. Because -adrenergic agonists never activate these channels in cardiac myocytes, there must be some mechanism to guarantee the specificity of the native G_K/K_ACh channel system in cardiac atrial myocytes. Further studies are needed to elucidate the mechanism.

B. Neurons

1. Differential cellular and subcellular distribution of GIRK subunits. Cellular and subcellular localization of GIRK1, 2, and 4 subunits in the rat and mouse brains has been studied with specific antibodies against individual GIRK proteins (Ponce et al., 1996; Liao et al., 1996; Iizuka et al., 1997). No corresponding studies have been done on GIRK3 proteins to our knowledge. The followings are the summary of currently available information.

2. Functional significance of differential subcellular distribution of GIRK subunits. Differential subcellular distribution of GIRK subunits in neurons would be intimately correlated with the functional task of K_G channels in each neuron. For example, in the CA3 hippocampal pyramidal neurons, a selective GABA_B agonist baclofen causes both presynaptic and postsynaptic inhibition in the wild-type mice but only presynaptic inhibition in GIRK2 -/-mice (Lüscher et al., 1997). These results indicate that GIRK1/GIRK2 channels in these neurons are selectively activated by the postsynaptic GABA_B receptor because of the postsynaptic localization of the GIRK subunits in these neurons. In hippocampal CA1 pyramidal neurons, Drake et al. (1997) found that GIRK1 immunoreactivity is found mainly associated with the dendritic membranes in the vicinity of the asymmetric (stimulatory) but not symmetric (inhibitory) type of synapses. These observations raise the possibility that the GIRK1-containing K_G channels in these neurons might serve to modulate the propagation of neuronal inputs originated at the excitatory synapses to the soma, as well as the back propagation of the action potential from the soma to the synapses on a synapse-to-synapse basis. To more concretely identify the functional roles of K_G channels in these neurons, it is important to determine the type(s) of neurotransmitter receptors, G proteins and nerve terminals associated with these channel subunits.

Electrophysiological studies indicate that K_G channels activated by somatostatin and/or dopamine exist in endocrine cells of anterior pituitary lobe (Pennefather et al., 1988; Einhorn and Oxford, 1993). This system would be essential for the inhibitory regulation of hormone secretion. However, there is no information available on subcellular distribution of GIRK subunits in these and other endocrine cells so far.

	VII. Weaver Mutant Mice and the GIRK2 Gene

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Weaver mice have been studied intensively over the past 25 years for insights into the normal processes of neuronal development and differentiation (Hess, 1996). Homozygous animals (wv/wv mice) suffer from severe ataxia due to death of cerebellar granular cells. The animals also represent a model of Parkinsonism because dopaminergic input to the striatum is lost during the first few weeks after birth due to the death of dopaminergic neurons in the substantia nigra. Male wv/wv mice are sterile: spermatogenesis fails to proceed normally past the third postnatal week leading to a complete failure of sperm production.

Recently, it was shown that wv/wv mice have their neurological abnormalities because of a point mutation of guanine 953 to adenine in the GIRK2 gene (Patil et al., 1995). This mutation causes a change of the corresponding amino acid from glycine (G) at position 156 to serine (S), which is in the ion selectivity filter of the potassium channel in the H5 region. In this way, the "finger print" K⁺ channel sequence of glycine-tyrosine-glycine (G-Y-G) is altered to serine-tyrosine-glycine (S-Y-G) in GIRK2 with the weaver mutation (GIRK2 wv). This results in a striking change in the selectivity of homomultimeric GIRK2 channels. Wild-type GIRK2 channels are highly selective for K⁺ ions with the permeability ratio P_Na/P_K of <0.05, and are virtually impermeable to Cs⁺ (Lesage et al., 1994; Slesinger et al., 1996; Kofuji et al., 1996b). However, homomultimeric GIRK2 wv channels allow K⁺, Na⁺, Rb⁺, and Cs⁺ to permeate with P_Na/P_K of 0.5 to 0.95, P_Rb/P_K of ~0.8 and P_Cs/P_K of 0.9 to 1.0, but are still impermeable to Ca²⁺, N-methyl-D-gulcamine, and anions (Slesinger et al., 1996; Kofuji et al., 1996b; Navarro et al., 1996). Thus, the weaver mutation renders GIRK2 channels nearly nonselective among monovalent cations.

The weaver mutation also has another unexpected effect on GIRK2 channels. Compared with wild-type GIRK2 channels, GIRK2wv-channels have a large G protein-independent basal current and a small G protein-induced increase in a current amplitude (Slesinger et al., 1996; Kofuji et al., 1996b; Navarro et al., 1996). This may be because the pore mutation impairs the gating which is crucial for the regulation of channel activity in response to G protein activity. However, Slesinger et al. (1996) found that as a larger amount of GIRK2wv cRNA was injected into Xenopus oocytes, expressed GIRK2wv-channels exhibited a larger basal current and a smaller response to G protein stimulation. Thus, it is also possible that the Na⁺ influx through GIRK2wv-channels increases the intracellular Na⁺ concentration, which in turn directly activates GIRK2wv-channels and occludes the channels' response to G protein stimulation (Lesage et al., 1995; Sui et al., 1996).

As with the wild-type GIRK2 subunits, GIRK2wv subunits form a heteromultimeric channels with GIRK1 (Slesinger et al., 1996; Liao et al., 1996). However, coexpression of GIRK1 and GIRK2wv in Xenopus oocytes yields current amplitudes not larger than the sum of those obtained with either of the subunits alone. Liao et al. (1996) found that in the wv/wv brain, the amounts of both GIRK1 and GIRK2 proteins were reduced although the amount of the unglycosylated form of GIRK1 proteins increased compared with the control. Thus, some fraction of the GIRK1/GIRK2wv complex might be retained in the endoplasmic reticulum and not be expressed to the membrane. Furthermore, GIRK1/GIRK2wv channels also lack K⁺ selectivity and have an impaired response to G protein stimulation (Slesinger et al., 1996; Kofuji et al., 1996b; Navarro et al., 1996; Liao et al., 1996). All these in vitro studies indicate that the effect of the weaver mutation can be pleiotropic depending on the expression level of GIRK2wv subunits and the presence of other types of GIRK subunits coexisting with GIRK2wv subunits.

During the early postnatal development of the mouse cerebellum, the external granule cell layer (EGL) consists of a mitotically active outer layer and a later developing inner postmitoic layer (Goldowitz and Smeyne, 1995). As development proceeds, the postmitotic granule cells migrate out of the EGL to the internal granule cell layer (IGL). In the wv/wv cerebellum, the granule cells undergo an apoptotic process before the migration. GIRK2 mRNA can be already detected in granule cell precursors in the prenatal mouse cerebellum (Kofuji et al., 1996b). Both GIRK1 and GIRK2 immunoreactivities are found in the mouse cerebellum by the postnatal day (PND) 3 and in the EGL and the newly forming IGL on PND4 (Slesinger et al., 1996). On PND19 and PND27, an anti-GIRK2 and anti-GIRK1 antibodies give a nearly uniform staining in the cerebellum of wv/wv mice compared with the discrete staining pattern in the wild-type littermates (Liao et al., 1996). This abnormal staining pattern of the wv/wv cerebellum corresponds to a loss of granule cells in the mutant mice by these ages. Therefore, the temporal expression pattern of GIRK2 and GIRK1 in the mouse cerebellum consists with the time course of the development of the neuronal deficits in the wv/wv cerebellum.

However, how the malfunction of GIRK2-containing K_G channels leads to the death of the cerebellar granule cells has not been unequivocally identified. The simplest explanation is that the basal or neurotransmitter-induced Na⁺ influx caused by the mutation imposes a heavy metabolic burden on granule cells and thereby causes the premature death or prevention of differentiation of the neurons. Kofuji et al. (1996b) found that charged channel blockers MK-801 and QX-314 and a Ca²⁺ channel blocker verapamil more potently inhibited GIRK1/GIRK2wv channels than wild-type GIRK1/GIRK2 channels. They also found that these blockers potently inhibited the aberrant, constitutively-active Na⁺ conductance in cultured wv/wv granule cells and promoted the survival and differentiation of the neurons in vitro. These results strongly suggest the causal relationship between the Na⁺ current caused by the weaver mutation and the deterioration of wv/wv granule cells. Slesinger et al. (1996) reported that Xenopus oocytes injected with GIRK2 wv cRNA also died much faster than those injected with the wild-type GIRK2 cRNA and that the survival period of oocytes was shorter when the larger amount of GIRK2 wv cRNA was injected. However, Surmeier et al. (1996) reported that they could not detect such aberrant Na⁺ currents in their cultured wv/wv granule cells. Instead, they found that somatostatin and a metabotropic glutamate receptor agonist trans-ACPD induced significantly smaller K_G channel currents in these cells than in the wild-type granule cells. Accordingly, they speculated as follows. In the postnatal cerebellum, the postmitotic, premigratory granule cells are known to be exposed to elevated levels of extracellular glutamate, which may trigger the migration of granule cells from the EGL. On this occasion, the GIRK2-containing K_G channels in the wild-type granule cells may be activated by glutamate and serve to counteract the depolarization caused by stimulation of N-methyl-D-aspartate glutamate receptors. However, wv/wv granule cells may be continuously depolarized by glutamate and die due to excessive Ca²⁺ entry because they lack the K_G channels. It is difficult to reconcile these different two observations. However, Signorini et al. (1997) found that the morphology of the cerebellum and midbrain dopaminergic neurons and the fertility of GIRK2 -/- mice are different from those of the wv/wv mice and indistinguishable from those of the wild-type (GIRK2 +/+) mice. Thus, the loss of GIRK2-containing K_G channels in the wv/wv mice may not be the primary cause of the weaver phenotype. They further argued that the cerebellum of the heterozygous wv/- mice is histologically more similar to wv/+ mice than that of +/+ or wv/wv mice. This observation favors the hypothesis that the gain-of-function and gene dosage mechanisms are responsible for the developmental defects in weaver mutants.

GIRK2 proteins are widely expressed in different regions in the brain as described in the section VI.B. However, only limited regions such as the cerebellar cortex, substantial nigra, and hippocampus are severely affected by the weaver mutation (Hess, 1996). Even within the same regions, damages are not homogeneous. For example, granule cells in the lateral cerebellar hemispheres are more resistant to the weaver mutation than midline neurons. Such inhomogeneity could be accounted for by several factors including the pleiotropic effects of the mutation depending on the expression levels of GIRK2 and the other GIRK subunits (Slesinger et al., 1996) as well as the environmental factors that determine the inherent vulnerability of each neuron (Hess, 1996). Further studies are necessary to answer why some neurons are more susceptible to the weaver mutation.

	VIII. Conclusions

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Until 1993, the G protein-activation of inwardly rectifying K⁺ channel systems was mainly studied in cardiac myocytes with electrophysiological techniques. The recent rapid progress in the molecular biology of K_G channels has disclosed an unimagined complexity of this channel system. Although many aspects of regulation of the K_G channels have been elucidated by the efforts of many laboratories listed in this review, there also have emerged many unclarified but possibly important mechanisms that may underlie the physiological regulation of K_G channels in organs that include heart, brain, and endocrine tissues. We cannot yet explain the molecular mechanisms responsible for receptor-specific control of K_G channels. Because the expression of the different GIRK genes and coupling to different receptor subtypes occurs throughout the central nervous system, the role of the K_G channel and its regulation by G proteins in neural systems requires more attention than it has received to date. Phenomena described for K_ACh channels in cardiac atrial myocytes, such as desensitization, deactivation, and cross-talk with other signaling systems have not yet been examined at all in other tissues.