Towards the elucidation of the structural-functional relationship of inward rectifying K+ channel family

NEUROSCIENCE RESEARCH ELSEVIER

Neuroscience Research 21 (1994) 109-117

Update article

Towards the elucidation of the structural-functional relationship of inward rectifying K ÷ channel family Yoshihiro Kubo Department of Neurophysiology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6, Fuchu, Tokyo 183, Japan Received 3 June 1994; revision received 6 September 1994; accepted 7 September 1994

Abstract

With recent cDNA cloning of members of the inward rectifying K ÷ channel family, it was revealed that they have only 2 putative transmembrane regions with no voltage-sensor element. Based on the deduced primary structure, possible schematic models to explain their characteristic features are proposed in this article. The features are (1) blocking by intracellular Mg 2+, (2) intrinsic gating, (3) the triple barrel structure of the inward rectifier K ÷ channel and (4) the activation by the direct interaction with G-protein subunits of the muscarinic K ÷ channel. The recent findings of the mutagenesis study of voltage-gated K ÷ channels, which provide a clue for the structural-functional study of the inward rectifying K ÷ channels, are also looked at.

Keywords: Inward rectifier K ÷ channel; Muscarinic K ÷ channel; cDNA cloning; Mg 2÷ block; Intrinsic gating; Blocking particle model; K+-activated K + channel model; Triple-barrel structure; GTP binding protein; GTPase activating protein

1. Introduction

The existence of unique K ÷ channels which allows chiefly inward flow of K ÷ below the equilibrium potential of K + (Er0 has been known since 1949 (Katz, 1949). Recently, cDNAs of K ÷ channels of this inward rectifying family have been isolated. These include the ATP-regulated K + channel (ROMK1) (Ho et al., 1993), the inward rectifier K ÷ channel (IRK1) (Kubo et al., 1993a), the G-protein-coupled muscarinic K ÷ channel (GIRKI) (Kubo et al., 1993b; Dascal et al., 1993) and the ATP-sensitive K + channel (hKATP) (Ashford et al., 1994). The primary structure of these inward rectifying K ÷ channels are novel in that they harbor only 2 putative transmembrane regions, in contrast to voltagegated K ÷ channels with 6 transmembrane regions. Some interesting biophysical features of inward rectifying K + channels have been studied intensively using various cells which express them. For the inward rec-

* Corresponding author, Fax: +81 423 21 8678.

tifier K ÷ channel, it was reported that the inward rectification is caused by (1) the high sensitivity of the outward current to blocking by intracellular Mg 2+ (Matsuda et al., 1987; Vandenberg, 1987; Matsuda, 1988) and by (2) the presence of the intrinsic gating, which remains in the absence of intracellular Mg 2+ (Matsuda, 1988; Ishihara et al., 1989; Cohen et al., 1989; Silver and DeCoursey, 1990; Pennefather et al., 1992). From the analysis of substates in the presence of pore blockers, it was postulated that the inward rectifier K + channel consists of triple parallel permeation pathways (Matsuda; 1988; Matsuda et al., 1989). For the muscarinic K ÷ channels, it was reported that this channel is activated by patch-delimited interaction with activated trimer G-protein subunits (Logothetis et al., 1987; Yatani et al., 1987, Brown and Birnbaumer, 1990; Ito et al., 1991; Kurachi et al., 1992; Wickman et al., 1994). Judging from the quick turning offwhen ACh is removed, muscarinic K + channels are also postulated to be GTPase activating proteins themselves, like another G protein effector, phospholipase Cfl I (Berstein et al,, 1992).

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Y. Kubo / N eurosc i. Res. 21 (1994) 109-117

With molecular cloning, the biophysical study of the inward rectifying K + channels has entered a new stage in the structural-functional study. It is expected that the structural elements which determine the biophysical properties will be elucidated at the molecular level through mutagenesis and biochemical study in the near future. In this article, firstly, I will briefly recap on the cloning story of IRK1 and GIRK1, and then, I will summarize previous biophysical findings and propose possible schematic models based on the primary structure which explain the biophysical features of inward rectifying K + channels. In the speculation, the result of the mutagenesis study of the voltage-gated K + channel will also be discussed, because it can be expected to give a good clue for the mutagenesis study of inward rectifying K + channels. 2. Molecular cloning of inward rectifying K + channels

2.1. Isolation of the inward rectifier K + channel cDNA, IRK1 Since the electrophysiological properties of inward rectifier K + channels are distinctly different from those of voltage-gated K + channels, we expected that the similarity of their primary structures is not high enough for cloning it, based on the sequence homology with voltage-gated K + channels. Instead, we decided to isolate a eDNA clone by screening the expressed current using the Xenopus oocyte expression system. We used poly-A + RNA from a mouse maerophage cell line which is known to have the inward rectifier K + channel (MeKinney and Gallin, 1988). The obtained single clone, IRKI (428 amino acids) (Kubo et al. 1993a), exhibited properties of an authentic inward rectifier K + channel: shift of activation in accordance with EK (Hagiwara et al., 1976), time and voltage-dependent blocking by extracellular cations such as Ba 2+, Cs + and Na + (Hagiwara et al., 1976; Standen and Stanfield, 1978; Ohmori, 1980), single-channel conductance, slow kinetics (Matsuda, 1988; McKinney and Gallin, 1988) and characteristic RNA distribution. After the cloning of IRKI, some new related clones were isolated based on their sequence homology with IRK1 (Morishige et al., 1993; Stanfidd et al., 1994; Koyama et al., 1994; Morishige et al., 1994; Perier et al., 1994; Makhina et al., 1994). 2.2. Isolation of the muscarinic K + channel cDNA, GIRK1 Parasympathetic nerve stimulation causes slowing of the heart rate via activation of muscarinic receptors and the subsequent opening of muscarinic K + channels in the sinoatrial node and atrium (Trautwein and Dudel, 1958; Sakmann et al., 1983; Brcitwieser and Szabo, 1987). This K + channel is known to be coupled directly

with G protein, and shows inwardly rectifying current. Based on sequence homology with cloned inwardly rectifying K + channels, IRK1 (Kubo et al., 1993a) and ROMK1 (Ho et al., 1993), we have isolated a cDNA for a G-protein-coupled inwardly rectifying K + channel (GIRKI), from a rat heart cDNA library (Kubo et al., 1993b). The GIRK1 channel (501 amino acids) most likely corresponds to the muscarinic K ÷ channel because: (1) its functional properties (coupling with G protein, single-channel conductance, kinetics, internal Mg2+-dependent inward rectification) resemble those of the atrial muscarinic K ÷ channels (Horie and Irisawa, 1989; Kurachi et al., 1992) and (2) its mRNA is much more abundant in the atrium than in the ventricle. In addition, GIRKI mRNA is expressed not only in the heart but also in the brain. As an identical clone was obtained from a cDNA library of the brain (Kubo et al., 1993b; Dascal et al., 1993), it is likely that GIRKI functions in the brain as an effector of G-protein-coupled receptors, such as substance P, GABA, somatostatin, opioid and ACh (reviewed by Brown, 1990). 2.3. Primary structure of inward rectifying K + channels and comparison with voltage-gated K + channels The IRKI channel (428 amino acids), the ROMK1 channel (391 amino acids) and the GIRK1 channel (501 amino acids) show extensive sequence similarity (40% identity of amino acids) and constitute a new superfamily. Voltage-gated and calcium-gated outward K + channels are known to have 6 transmembrane domains (S 1, $2, $3, $4, $5 and $6). The $4 region has scattered positive charges and is known to function as a voltage sensor from the result of a mutagenesis study (Papazian et al., 1991). The region between $5 and $6, which is called H5 (reviewed by Miller, 1991), is too short to form 2 transmembrane segments, but it is thought to form the wall of the pore or the permeation pathway. It was shown that some point mutations of the H5 region affect the sensitivity to either extracellular or intracellular blockers and also alter the single-channel conductance and selectivity. From these results, the H5 region is thought to reach the intracellular side from the extracellular side and conform part of the pore. Unlike the voltage-gated and calcium-gated outward K + channels, the inward rectifying K + channels contain only 2 putative transmembrane segments (M 1 and M2). The region between 2 transmembrane segments of inward rectifying K + channels is similar to the H5 poreforming region of voltage-gated K + channels, which is reasonable because both families allow selective permeation of K + ions. In addition, M1 and M2 showed weak but significant homology with $5 and $6 of voltagegated K + channels. From these homologies, inward rectifying K + channels (M1, H5 and M2) are thought to correspond to the latter half of voltage-gated K ÷ channels ($5, H5 and $6). It is speculated that the inward rec-

Y. Kubo/Neurosci. Res. 21 (1994) 109-117

tifying K + channels have only the fundamental part and form the inner core structure ($5, H5 and $6), and in the case of voltage-gated and calcium-gated outward K + channels, they are thought to have an outer shell (S 1, $2, $3 and $4) in addition to the inner core structure (Kubo et al., 1993a; Fig. 1). It is notable that inward rectifying K ÷ channels lacked the region which corresponds to the $4 region of the voltage-gated K + channel which functions as the voltage-sensing mechanism (Papazian et al., 1991). This is a very important point when we speculate on the mechanism of intrinsic gating of inward rectifying K + channels, because it strongly suggests that the intrinsic gating is different from the conformational change which is postulated in voltage-gated K ÷ channels. There is only one negative charge in the transmembrane region of IRK1, but it is clearly insufficient to explain the steep voltage dependence of the inward rectifier K + channel. Recently, evidence that the voltage-gated K ÷ channels are composed of 4 subunits with one pore was shown by mutagenesis studies (MacKinnon, 1991; Liman et al., 1992) and the electron microscopic study of purified channel protein expressed in baculovirustransfected cells (Li et al., 1994). Li et al. estimated the Shaker tetramer protein to be a square-shaped block of 80 ~, x 80 ,A x 67 ~, with a heavily stained space in the middle, which they interpreted to be a vestibule of a channel. By analogy, since part of the pore-forming region (H5) of inward rectifying K + channels shows similarity with the voltage-gated K + channels, it is speculated that inward rectifying K ÷ channels are also composed of 4 subunits (Kubo et al., 1993a; Fig. 1).

Voltage-gated K + channel

Inward rectifier K + channel

extracellular ~ f,~ SII~S3~S4/S/ ~H5~$6membraneMll H~5~M2 N

C

N

C

Fig. 1. Proposed membrane topology of the IRKI inward rectifier K ÷ channel, (modified from Kubo et al., 1993a).

11 i

3. Biophysical features of the inward rectifier K ÷ channel

Firstly, a rough explanation is given below to help understand the inward rectification property in comparison with outward rectifying voltage-gated K ÷ channels. If ions permeate through the membrane passively, the direction of the net flow is determined by the electrochemical potential and the direction reverses at an equilibrium potential (inward: below, and outward: above equilibrium potential). In the case of K ÷, it (equilibrium potential of K÷; EK) is at around -80 mV under physiological conditions. Voltage-gated K ÷ channels have a voltage-sensing mechanism, and they are open only at depolarized membrane potentials. As a result, they allow chiefly outward flow of K ÷. In contrast, inward rectifier K ÷ channels lack a voltage sensor and they do not sense the membrane potential as voltagegated K ÷ channels do. They are open even at hyperpolarized potentials below EK, which allow inward flow of K ÷ current. At a depolarized potential above EK, it seems that they also allow outward flow, but this channel is highly sensitive to blocking by cytoplasmic Mg 2+, which diminishes outward K ÷ current significantly. As a result, the inward K ÷ current below EK is apparent, and there is only very diminished outward current above E K. There is also 'intrinsic gating', which is described below. 3.1. Single-file multi-ion pore The inward rectifier K ÷ channel is thought to be a multi-ion single-file pore. Hagiwara et al. (1977) showed an anomalous mole fraction using TI Z-K÷ mixture solution, which suggested the interaction of permeating ions, and Ohmori (1980) showed the interaction of permeating K ÷ ions assuming multiple binding sites. Hille and Schwarz (1978) conducted a theoretical study using the rate theory (Eyring et al., 1949) and reproduced the electrophysiological data (Hagiwara et al., 1976) based on the three-site and four-barrier model and by assuming a blocking particle which was proposed by Armstrong (1969). Their work has been a good model of the following biophysical work. 3.2. Mg 2÷ block The characteristic features of inward rectifier K ÷ current (Matsuda, 1988; Ishihara et al., 1989) are schematically shown in Fig. 2. In the presence of intracellular Mg 2+, instantaneously and slowly activating components are observed upon hyperpolarization. When depolarized above ER, the outward current decreases quickly, but there remains a small sustained outward current. In contrast, in the absence of intracellular Mg 2÷, the instantaneously activating component disappears, while the slowly activating component is

1t 2

Y. Kubo / Neurosci. Res. 21 (1994) 109-117

intracellular Mg2+ (+)

intracellular Mg2+ (-)

voltage

I .....................

zero - -

--~----

Fig. 2. Schematicdrawing showingcharacteristic features of inward rectifier K+ current. more prominent upon hyperpolarization. When depolarized, the quick decay of the outward current does not occur, and instead, the outward current decreases at a slower rate, reaching the zero-current level with no sustained outward current. This gating behavior, which is observed even in the absence of Mg 2+, is called 'intrinsic gating'. These phenomena are interpreted as follows (Matsuda, 1988; Ishihara et al., 1989) with reference to closed-open-block transition. The B-O transition is quick, and the C-O transition is slower. The channels must enter the O state to attain the C state from the B state. The instantaneously activating component and the slowly activating one correspond to the BO and the C-O transitions, respectively. The quick decay upon depolarization corresponds to the O-B transition, and the slow decay in the absence of Mg 2÷ corresponds to the O-C transition. The important findings of these investigations are as follows: intracellular Mg 2+ plays an important role of the instantaneously blocking of the outward current, and in the inward rectification property; paradoxically, intraeellular Mg 2+ also causes small but sustained outward current at depolarized potential by preventing the channels from entering the C state. Intracellular Mg 2+ apparently affects only the outward current above EK. One possible explanation is that the channel and Mg 2+ sense the direction of the net flow, and Mg 2+ is plugged into the channel by the outward flux of K + and pushed out by the inward flux. More precisely, however, the inward rectifier K + channel seems to sense extraceUular K + concentration and membrane potential, as postulated previously (Hagiwara and Takahashi, 1974; Ciani et al., 1978), rather than E r or the direction of the net flow, based on the following reasons. Hagiwara and Yoshii (1979) showed that the conductance of inward rectifiers shifts in accordance with EK when the extracellular K + concentration is changed, however, the conductance curve does not shift when the intracellular K + concentration is changed. Matsuda (1991) showed that Mg 2+ blocking

depends on extracellular K ÷ and voltage but not on intracellular K ÷ or EK. How does Mg 2÷ sense extracellular K + concentration and voltage? This was explained by assuming that the energy level of the welt for Mg 2+ in the pore changes when extracellular K ÷ binds to the channel, using the three-barrier two-site model of a multiple-ion single-file channel (Matsuda, 1991). To understand the Mg 2+ block mechanism, especially its dependence on voltage and extracellular K ÷ concentration, it is necessary to know the Mg 2÷ binding site in the channel as well as the binding site for the extracellular K ÷. If the Mg 2+ binding site is shared with permeating K ÷ ions, there is a risk that the channel becomes non-functional and will not be detected in the mutagenesis study to identify the Mg 2+ binding site. As candidates of the Mg 2+ binding site, parts of the channels which are speculated to form the pore are important, because Mg 2+ is known to be a voltagedependent pore blocker. From the mutagenesis studies of voltage-gated K + channels, it was elucidated that the H5 region forms the wall of the pore (reviewed by Miller, 1991). In addition, the contribution of the $4-$5 linker (Choi et al., 1993; Slesinger et al., 1993) and part of $6 (Kirsch et al., 1993; Lopez, 1994) to the formation of the inner part of the pore was reported. By analogy with these results, the targets may be characteristically conserved residues of H5 as well as parts of M 1 and M2. In Fig. 3, the alignments of the amino acids of the H5 region of inward rectifying K ÷ channels and various voltage-gated K ÷ channels are shown. Recently Taglialatela et al. (1994) reported surprising data of chimera experiments of IRK1 and ROMK1, which showed the involvement of the C-terminus intracellular domain, but not H5, M1, M2 domains of IRK1, in the high sensitivity to the voltage-dependent blocking by intracellular Mg 2+. The data suggest the possibility that the C-terminus domain, which is thought to be cytoplasmic, is actually folded into the membrane. Since this possibility cannot be refuted, it may be necessary to reassess the folding and topology of the inward rectifying K ÷ channel family. Needless to say, however, the data can also be interpreted as the C-terminus cytoplasmic domain interacting with the inner mouth of the pore, changing the pore property allosterically. 3.3. Intrinsic gating mechanism The intrinsic gating which is observed even in the absence of intracellular Mg 2+ cannot be due to a voltagesensing mechanism similar to that of a voltage-gated K ÷ channel (Papazian et al., 1991), because the $4 voltage-sensor region is lacking. There are 2 possibilities, namely an intrinsic blocking particle model, and a K*-activated K + channel model (Pennefather et al., 1992). An intrinsic blocking particle model is shown schematically in Fig. 4., which is similar to the ball and

Y. Kubo/NeuroscL Res. 21 (1994) 109-117 1

2

3

4

5

6

7

8

113

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

inward rectifying

IRK1 GIRK1 ROMK1

VNSFTAAFLFS VYNFPSAFLFF I NGMTSAFLFS

~ i ~ i . GY GF~-]Ci~DE~-~P AT I GYGYIRIY DK P I GYGFL~F EQ A

voltage-gated

Kvl.1 (Shaker) Kv1.2 (RCK2) Kv1.4 (RCK4) Kv2.1 (drkl) Kv3.1 (NGK2) Kv4.1 (mShal)

F-K SII F P SII F Q SII F K SII FKNI FTSl

P DA F WIwA V V T MT T V GY GD MT PDAFWIWAV TMTTVGYGDMY P DA F W~WA V V T MT T V G Y G D MK P A S F W1WAT -/ T MTTMGYGDI Y PI G F W I W A V V T M T T LGYGDMV P A A F W1YT I V T M T T L G Y G D M V I

P-IV G V ~ G ii MTVGG PI T V G G PKTLLG PQTWSG STI AG

I

H5 Fig. 3. Alignment of deduced amino acid sequences of various inward rectifying K ÷ channels and voltage-gated K ÷ channels. The alignment of H5 and the surrounding region is shown. Conserved sequences in each family is boxed, and conserved sequences in both families are shaded. Residues of the voltage-gated K ÷ channel family which were reported to determine pore properties are printed in bold letters. The mutations of TI 6 and T17 of Kvl. 1 are reported to affect the ionic selectivity (Yool and Schwarz, 1991), and the sensitivity to blocking by intracellular TEA (Yellen et al., 1991). The mutations of D6 and T24 of Kvl.l are reported to affect the sensitivity to blocking by extracellular TEA (Yellen et al., 1991). Mutations of K24 of Kvl.4 are reported to affect the dependence on the extracellular K ÷ (Pardo et aL, 1992) and the sensitivity to blocking by the extracellular TEA and the intracellular Mg 2÷ (Ludewig et al., 1993). Mutations of V I3 and L18 of Kv3.1 are reported to affect the singlechannel conductance, and the sensitivity to blocking by intracellular Ba 2÷ or intracellular TEA (Kirsch et al., 1992; Taglialatela et al., 1993a,b). Y20 and G21 are reported to be critically important for K + selectivity by comparison with cGMP-gated channels (Heginbotham et al., 1992). Mutation of a residue of cGMP-gated channel, which corresponds to D22, is reported to affect the ionic selectivity, the rectification property and the sensitivity to blocking by extracellular Ca 2÷ (Eismann et al., 1994) or extracellular Mg 2÷ (Root and MacKinnon, 1993).

chain model of the inactivation of the voltage-gated K ÷ channel (Hoshi et al., 1990; Zagotta et al., 1990). It postulates a kind of gating particle in the intracellular chain, which plugs the channel pore (closed state). If we assume that the blocking particle cannot approach the hyperpolarized potential below E K open state

extracellular

r,

intracellular

depolarized potential above E~ blooked state

closed state

Fig. 4. Schematic representation of an intrinsic blocking particle model to explain the intrinsic gating of the inward rectifier K ÷ channel. The interaction of Mg 2÷ and the intrinsic blocking particle with the channel is thought to be affected by extracellular K ÷, but it is not included in this scheme.

channel when a Mg 2+ ion is in the pore, this model explains the delayed closing of the channel in the presence of intracellular Mg 2+. For the Na + channel, it was reported that not only the blocking particle of the cytoplasmic region of the subunit, but also the/3 subunit affects the inactivation (Isom et al., 1992). Recently a fl subunit of the voltagegated K + channel was cloned (Scott et al., 1994) and shown to affect the inactivation property (Rettig et al., 1994). If there is also a/3 subunit for inward rectifier K + channels, the possibility can be raised that the gating particle is not a cytoplasmic domain of the IRK1, but a fl subunit itself. If this is the case, the fl subunit might be distributed in various types of cells, since a similar intrinsic gating was also observed when only the cloned IRK1 was expressed in cells which lack the inward rectifier K + channel (Stanfield et al., 1994; Ishihara and Hiraoka, 1994). Another possibility could be that the intrinsic gating particle is a ubiquitously distributed cytoplasmic molecule which is actually 'extrinsic' to the channel. It has been known that the conductance of inward rectifier K + channels depends on membrane potential and extracellular K + concentration (Hagiwara and Takahashi, 1974; Hagiwara and Yoshii, 1979). Matsuda and Noma (1984) reported that the conductance of the inward rectifier K + channel is abolished when the extracellular K + is removed. The possibility that the inward rectifier K + channel is a K+-activated K + channel

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Y. Kubo/Neurosci. Res. 21 (1994) 109-117

has been postulated (Ciani, 1976; Cohen 1989; Pennefather, 1992). In this case, the intrinsic gating could be explained by the conformational change determined by the presence or absence of extracellular K + bound to the external part of the channel (Fig. 5). By assuming that the bound K ÷ changes the energy profile of the pore, it may be possible to explain the regulation of permeation or gating. From this aspect, the identification of the binding site for the extracellular K ÷ would be very important for the understanding of the intrinsic gating mechanism of the inward rectifier K ÷ channel. A voltage-gated K + channel, RCK4, also shows dependence on extracellular K +. Pardo et al. (1992) identified a site which is involved in the regulation of this channel by extracellular K ÷. By mutating K to Y at 24 (Fig. 3), the RCK4 channel lost its dependence on extracellular K +. The same site was also found to affect the sensitivity to blocking by external TEA as well as intracellular Mg 2+ (Ludwig et al., 1993). They postulated that the extracellular K + bound to the channel modulates the energy profile of the outer part of the pore. Using the results of this work, it might be possible to identify this site of the IRK1 channel. This work also suggests a possible link between the Mg 2+ blocking mechanism and intrinsic gating, both of which sense extracellular K + and voltage. Cohen et al. (1989) showed that the activation kinetics of an intrinsic gate slows down when intracellular K + is decreased. They explain-

ed this by assuming that intracellular K ÷ allosterically modifies the interaction of extracellular K ÷ with a binding site that regulates the gating of the channel. 3.4. Triple barrel structure Based on the analysis of substates revealed by poreblocking ions, Matsuda et al. (1989) advocated a model in which the inward rectifier K + channel is composed of three-parallel barrels. By analogy with voltage-gated K + channels (Li et al., 1994), the inward rectifier K + channel is also likely to be a tetramer with a single pore in the center. If the channel in fact has the triple barrel structure, then it might be necessary to postulate a superassembly of 3 tetramers. In this case, the next question is the mechanism of the concerted gating of superassembled tetramers which explains the finding that the closures of these 3 permeation pathways occur in a concerted manner. To prove the superassembly model, it is necessary to perform not only electrophysiological studies, but also electron microscopic studies of the purified channel protein. The study by Li et al. (1994), which found the Shaker K + channel to be a square-shaped protein of 80 A, serves as a good reference for this purpose. An alternative possibility is to postulate that the substates are determined by the number of blockers bound to a single pore of the channel.

C--O--B--B--B--(B) 0

(4) (number of Mg 2+ bound to the channel pore) 3/3 2/3 1/3 0/3 (0/3)(conductance)

hyperpolarized potential below E K open state

®

intracel~ular

depolarized potential above E K blocked state

J

closed state

/

Fig. 5. Schematic representation o f K+-activated K + channel model tO explain the intrinsic gating of the inward rectifier K + channel. The interaction of Mg 2+ with the channel is reported to be affected by extracellular K + (Matsuda, 1991), but it is not included in this scheme. Mg 2+ blocking is reported to be more intense with low extracellular K + concentration. Based on this finding, it may be more appropriate to assume that the affinity of Mg 2+ to the channel is higher when there is no K + bound to the channel. This model does not necessarily mean that the binding site for extracellular K + ions located outside the permeation pathway.

1

2

3

By assuming three equal and independent binding sites in a single pore, this model explains the report of Matsuda (1988) and Matsuda et al. (1989), which fitted the probability of the presence in substates by binomial distribution in 3 barrels. It may be natural, however, to postulate 4 rather than 3 equal Mg 2+ binding sites when we consider the high possibility that there are 4 subunits, and that there is one binding site in each subunit. In this case, we need to assume 2 blocked states in the nonconducting level. Once the binding site for intracellular Mg 2+ is identified by mutagenesis study, it is possible to construct a channel with a reduced number of Mg 2+ binding sites. Using these constructs, for example a construct with only one binding site in the tetramer, this hypothesis can be proven if the disappearance of the 1/3 and 0/3 blocked substates is observed. The binomial distribution to 3 equal substates was also observed in the presence of extracellular cation blockers such as Cs + and Rb + (Matsuda et al., 1989). According to the model shown above, it is necessary to postulate again 3 (or 4) independent and equal binding sites in a single pore for these blockers.

Y. Kubo/Neurosci. Res. 21 (1994) 109-117

Shioya et al. (1993) reported that there are various binding sites for extracellular cation blockers. Extracellular Cs ÷, which shows flickering blocking, binds to the deep part of the pore, while the binding sites for extracellular Mg 2÷ and Ca 2÷, which decrease the singlechannel conductance probably by very fast blocking, are in the shallow part of the pore. To elucidate blocking and permeation, and to obtain an image of the pore, a mutagenesis study to map these binding sites will also be necessary.

4. Electrophysiological properties of the G-protein coupled muscarinic K + channel 4.1. Coupling with G protein subunits There has been a debate as to whether f13' (Logothetis et al., 1987; Ito et al., 1991; Kurachi et al., 1992) or o~ subunits (Yatani et al., 1987; Brown and Birnbaumer, 1990) of the trimer G protein activate the muscarinic K + channel. Recently Wickman et al. (1994) reported the activation of this channel by recombinant /33' subunits proteins. Reuveny et al. (1994) obtained similar results using the expressed GIRK1 channel together with recombinant f13' subunits. /3-Adrenergic receptor kinase (/3 ARK), another G protein effector protein, is known to be activated by /37 subunits (Inglese et al., 1992). There is a limited but significant homology of the C-terminus intracellular chain of the GIRKI channel with/3 ARK. Reuveny et al. (1994) further showed that the/3T-associated part of/3 A R K inhibits activation of the fiT-activated GIRKI channel, probably by acting as an absorbing molecule for free/33, subunits. They also showed that a-GDP also inhibit the activated GIRK1 channel, whereas a-GTP does not. They could not observe any clear effect of c~-GTP as an activator of the channel. With such evidence, it now seems almost certain that/33, subunits function as the activator of the Gprotein-coupled muscarinic K ÷ channel, and that the binding site for/33, is in the intracellular chain, although mutagenesis studies are required to confirm this. The next aim may be to confirm that the interaction is not merely patch-delimited but truly direct. For this purpose, use of a reconstitution system such as a lipid bilayer and recombinant proteins will be required. If the activation of GIRKI is observed in the presence of only flT-subunit recombinant proteins, the interaction can be concluded to be truly direct. 4.2. Mechanism of muscarinic K ÷ channel opening by f13" subunits How is the information that/37 subunits have bound to the channel transmitted to the pore to activate the channel? Judging from the putative binding site of/37 subunits, it is unlikely that the information is transmitted as a deformation force to the membranespanning region. Instead, it is more likely that f17 bind-

115

hyperpolarized potential below E K not activated

activated

':~jK ) extracelM, .....

intracellular

, ---~-- ~,

j[ I,

I" '

I,~jIl~

,¢

/

~

I-

L ,'

--~,j

Fig. 6. Schematic representation of a model to explain the mechanism of the activation of the muscarinic K + channel by interaction with 87 subunits.

ing prevents the blocking particle of the intracellular chain from plugging the channel, as illustrated in Fig. 6. When no/33' subunits are activated, the blocking particle plugs the channel, but through the interaction with fit subunits, the particle is trapped and removed from the pore. As supporting evidence of this model, Kirsch and Brown (1989) reported that the application of trypsin to the intracellular side of the muscarinic K ÷ channel activated this channel. A similar finding was reported also for ATP-sensitive K ÷ channels (Furukawa et al., 1993). This might be due to the cleavage of the intraceUular chain. Deletion experiments of the intracellular chain will give further evidence for this idea.

4.3. ls the muscarinic channel itself a G TPase-activating protein (GAP) ? When the agonist stimulation is turned off, the muscarinic K ÷ channel closes very quickly. Since the GTPase activity of the ct subunit itself is slow, it is natural to expect that the channel activity will last for some time even after the removal of agonist stimulation, but this is not the case. One idea to explain this is to postulate that the muscarinic K + channel itself is a GAP, similarly to phospholipase C-ill (Bernstein et al., 1992). Once activated, the muscarinic K + channel functions not only as an inward-rectifying K ÷ channel but also as a GAP to turn off the G protein system. If this is the case, the putative interaction site may be elucidated by comparing the turn-off speed of wild-type and some mutant GIRK1 channels. 4. Concluding remarks Some schematic models to explain characteristic features of inward rectifying K ÷ channels have been proposed based on the deduced primary structure. These models are expected to serve as a starting point for mutagenesis studies toward the elucidation of the structural-functional relationship of the inward rectifying K ÷ channels. Through the accumulation of knowledge from such mutagenesis experiments, precise and

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Y. Kubo / Neurosci. Res. 21 (1994) 109-117

more detailed pictures are expected to be drawn in the near future.

Acknowledgments The author is currently supported by research grants from the Naito Foundation, the Human Frontier Science Program Organization and the Ministry of Education, Science and Culture of Japan.

References Armstrong, C.M. (1969) Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons. J. Gen. Physiol., 54: 553-575. Ashford, M.L.J., Bond, C.T., Blair, T.A. and Adelman J.P. (1994) Cloning and functional expression of a rat heart KATP channel. Nature, 370: 456-459. Bernstein, G., Blank, J.L., Jhon, D.Y., Exton, J.H., Rhee, S.G. and Ross, E.M. (1992) Phospholipase C-/51 is a GTPase-activating protein for Gq/11, its physiological regulator. Cell, 70:411-418. Breitwieser, G.E. and Szabo, (3. (1987) Uncoupling of cardiac muscarinic and /~-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature, 317: 538-540. Brown, D.A. (1990) G-proteins and potassium currents in neurons. Annu. Rev. Physiol., 52: 215-242. Brown, A.M. and Birnbaumer, L. (1990) Ionic channels and their regulation by G protein subunits. Annu. Rev. Physiol., 52: 197-213. Chi, K.L., Mossman, C., Aube, J. and Yellen, G. (1993) The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron, 10:533-541 Ciani, S., Krasne, S., Miyazaki, S. and Hagiwara, S. (1978) A model of anomalous rectification: electrochemical-potential-dependent gating of membrane channels. J. Membr. Biol., 44: 103-134. Cohen, I.S., DiFrancesco, D., Pennefather, P. and Mulrine, N.K. (1989) Internal and external K help gate the inward rectifier. Biophys. J., 55: 197-202. Dascal, N., Schreibmayer, W., Lira, N.F., Wang, W., Chavkin, C., DiMagno, L., Labarca, C., Kieffer, B.L., Gaveriaux-Ruff, C., Trollinger, D., Lester, H. and Davidson, N. (1993) Atrial Gprotein-activated K + channel: expression cloning and molecular properties. Proc. Natl. Acad. Sci. USA, 90: 10235-10239. Eismann, E., Muller, F., Heinemann, S. and Kaupp, U.B. (1994) A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca 2+ blockage, and ionic selectivity. Proc. Natl. Acad. SCi. USA, 91: 1109-1113. Eyring, H., Lumry, R. and Woodbury, J.W. (1949) Some applications of modern rate theory to physiological systems. Rec. Chem. Prog., 10: 100-114. Furnkawa, T., Fan, Z., Sawanobori, T. and Hiraoka, M. (1993) Modification of the adenosine 5 '-triphosphate-sensitive K + channel by trypsin in guinea-pig ventricular myocytes. J. Physiol., 466: 707-426. Hagiwara, S., Miyazaki, S., Krasne, S. and Ciani, S. (1977) Anomalous permeabilities of the egg cell membrane of a starfish in K +TI + mixtures. J. Gen. Physiol., 70: 269-281. Hagiwara, S., Miyazaki, S. and Rothenthal, N.P. (1976) Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J. Gen. Physiol., 67: 621-638. Haglwara, S. and Takahashi, K. (1974) The anomalous rectification and cation selectivity of the membrane of a starfish egg cell. J. Membr. Biol., 18: 61-80.

Hagiwara, S. and Yoshii, M. (1979) Effects of internal potassium and sodium on the anomalous rectification of the starfish egg as examined by internal perfusion. J. Physiol., 292: 251-265. Heginbotham, L., Abramson, T. and MacKinnon, R. (1992) A functional connection between the pores of distantly related ion channels as revealed by mutant K + channels. Science, 258:1152-1155. Hille, B. and Schwarz, W. (1978) Potassium channels as multi ion single file pores. J. Gen. Physiol., 72: 409-442. Ho, K., Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V. and Hebert, S.C. (1993) Cloning and expression of an inwardly rectifying ATP-regnlated potassium channel. Nature, 362: 31-38. Horie, M. and Irisawa, H. (1989) Dual effects of intracellular magnesium on muscarinic potassium channel current in guinea-pig atrial ceils. J. Physiol., 408: 313-332. Hoshi, T., Zagotta, W.N. and Aldrich, R.W. (1990) Biophysical and molecular mechanisms of Shaker K + channel inactivation. Science, 250: 533-538. lnglese, J., Koch, W.J., Caron, M.G. and Lefkowitz, R.J. (1992) Isoprenylation in regulation of signal transduction by G-proteincoupled receptor kinases. Nature, 359: 147-150. Ishihara, K., Mitsuiye, T., Noma, A. and Takano, M. (1989) The Mg 2+ block and intrinsic gating underlying inward rectification of the K + current in guinea pig cardiac myocytes. J. Physiol., 419: 297-320. Ishihara, K. and Hiraoka, M. (1994) Gating mechanism of the cloned inward rectifier K + channel from mouse heart. J. Membr. Biol., (in press). Isom, L.L., DeJongh, K.S., Patton, D.E., Reber, B.F.X., Offord, J., Charbonneau, H., Walsh, K., Goldwin, A.L. and Catterall, W.A. (1992) Primary structure and functional expression of the bl subunit of the rat brain sodium channel. Science, 256: 839-842. Ito, H., Sugimoto, T., Kobayashi, I., Takahashi, K., Katada, T., Ui, M., and Kurachi, Y. (1991) On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K + channel in atrial myocytes of guinea pig heart. J. Gen. Physiol., 98: 517-533. Katz, B. (1949) Les constantes electriques de la membrane du muscle. Arch. Sci. Physiol., 2: 285-299. Kirsch, G.E. and Brown, A.M. (1989) Trypsin activation of atrial muscarinic K + channels. Am. J. Physiol., 257: H334-H338. Kitsch, G.E., Drewe, J.A., Hartmann, H.A., Taglialatela, M., Biasi, M.D., Brown, A.M. and Joho, R.H. (1992) Differences between the deep pores of K + channels determined by an interacting pair of nonpolar amino acids. Neuron, 8: 499-505. Kirsch, G.E., Shieh, C.C., Drewe, J.A., Vener, D.F. and Brown, A.M. (1993) Segmental exchanges define 4-aminopyridine binding and the inner mouth of K + pores. Neuron, 11: 503-512. Koyama, H., Morishige, K., Takahashi, N., Zanelli, J.S., Fass, D.N. and Kurachi, Y. (1994) Molecular cloning, functional expression and localization of a novel inward rectifier potassium channel in the rat brain. FEBS Lett., 341: 303-307. Kubo, Y., Baldwin, T.J., Jan, Y.N. and Jan, L.Y. (1993a) Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature, 362: 127-133. Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N. and Jan, L.Y. (1993b) Primary structure and functional expression of a rat G protein coupled muscarinic potassium channel. Nature, 364: 802-806. Kurachi, Y., Tung, R.T., Ito, H. and Nakajima, T. (1992) G protein activation of cardiac muscarinic K ÷ channels. Prog. Neurobiol., 39: 229-246. Li, M., Unwin, N., Stauffer, K.A., Jan, Y.N. and Jan L.Y. (1994) Images of purified Shaker potassium channels. Curr. Biol., 4: 110-115. Liman, E.R., Tytgat, J. and Hess, P. (1992) Subunit stoichiometry of

Y. Kubo/Neurosci. Res. 21 (1994) 109-117

a mammalian K ÷ channel determined by construction of multimeric cDNAs. Neuron, 9: 861-871. Logothetis, D.E., Kurachi, Y., Galper, J., Neer, E.J. and Clapham, D.E. (1987) The beta, gamma subunits of GTP-binding proteins activate the muscarinic K ÷ channel in heart. Nature, 325: 321-326. Lopez, G.A., Jan, Y.N. and Jan, L.Y. (1994) Evidence that the $6 segment of the Shaker voltage-gated K ÷ channel comprises part of the pore. Nature, 367: 179-182. Ludewig, U., Lorra, C., Pongs, O. and Heinemann, S.H. (1993) A site accessible to external TEA ÷ and K ÷ influences intracellular Mg 2÷ block of cloned potassium channels. Eur. Biophys. J., 22: 237-247. MacKinnon, R. (1991) Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature, 350: 232-235. Makhina, E.N., Kelly, A.J., Lopatin, A.N., Mercer, R.W. and Nichols, C.G. (1994) Cloning and expression of a novel human brain inward rectifier potassium channel. J. Biol. Chem., (in press). Matsuda, H. (1988) Open-state substructure of inwardly rectifying potassium channels revealed by magnesium block in guinea-pig heart cells. J. Physiol., 397: 237-258. Matsuda, H. (1991) Effects of external and internal K ÷ ions on magnesium block of inwardly rectifying K ÷ channels in guinea pig heart cells. J. Physiol., 435: 83-99. Matsuda, H., Matsuura, H. and Noma, A. (1989) Triple-barrel structure of inwardly rectifying K ÷ channels revealed by Cs ÷ and Rb ÷ block in gninea-pig heart cells. J. Physiol., 413: 139-157. Matsuda, H. and Noma, A. (1984) Isolation of calcium current and its sensitivity to monovalent cations in dialyzed ventricular cells of guinea-pig. J. Physiol., 357: 553-573. Matsuda, H. Saigusa, A. and Irisawa, H. (1987) Ohmic conductance through the inwardly rectifying K ÷ channel and blocking by internal Mg 2÷. Nature, 325: 156-159. McKinney, L.C. and GaUin, E.K. (1988) Inwardly rectifying wholecell and single-channel K currents in the murine macrophage cell line J774.1. J. Memb. Biol., 103: 41-53. Miller, C. (1991) 1990: Annus mirabilis of potassium channels. Science, 252: 1092-1096. Morishige, K., Takahashi, N., Findlay, I., Koyama, H., Zanelli, J.S., Peterson, C., Jenkins, N.A., Copeland, N.G., Mori, N. and Kurachi, Y. (1993) Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain. FEBS Lett., 336: 375-380. Morishige, K., Takahashi, N., Jahangir, A., Yamada, M., Koyama, H., Zanelli, J.S. and Kurachi, Y. (1994) Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel. FEBS Lett., (in press). Ohmori, H. (1980) Dual effects of K ions upon the inactivation of the anomalous rectifier of the tunicate egg cell membrane. J. Memb. Biol., 53: 143-156. Papazian, D.M., Timpe, L.C., Jan, Y.N. and Jan, L.Y. (1991) Alteration of voltage-dependence of Shaker potassium channel by mutations in the $4 sequence. Nature, 349: 305-310. Pardo, L.A., Heinemann, S.H., Terlau, H., Ludewig, U., Lorra, C., Pongs, O. and Stumer, W. (1992) Extracellular K ÷ specifically modulates a rat brain K + channel. Proc. Natl. Acad. Sci. USA, 89: 2466-2470. Pennefatber, P., Oliva, C and Mulrine, N. (1992) Origin of the potassium and voltage dependence of the cardiac inwardly rectifying Kcurrent. Biophys. J., 61: 448-462. Perier, F., Radeke, C.M. and Vandenberg, C.A. (1994) Primary structure and characterization of a small-conductance inwardly rectifying potassium channel from human hippocampus. Proc. Natl. Acad. Sci. USA., 91: 6240-6244. Rettig, J., Heinemann, S.H., Wunder, F., Lorra, C., Parcej, D.N., Dolly, J.O. and Pongs, O. (1994) Inactivation properties of

117

voltage-gated K + channels altered by presence of b-subunit. Nature, 369: 289-294. Reuveny, E., Slesinger, P.A., Inglese, J., Morales, J.M., Iniguez-Lluhi, J.A., Lefkowitz, R.J., Bourne, H.R., Jan, Y.N. and Jan L.Y. (1994) Activation of the cloned muscarinic potassium channel by G protein/~' subunits. Nature, 370: 143-146. Root, M.J. and MacKinnon, R. (1993) Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron, I l: 459-466. Sakmann, B., Noma, A. and Trautwein, W. (1983) Acetylcholine activation of single muscarinic K + channels in isolated pacemaker cells of the mammalian heart. Nature, 303: 250-253. Scott, V.E.S., Rettig, J., Parcej, D.N., Keen, J.N., Findlay, J.B.C., Pongs, O. and Dolly, J.O. (1994) Primary structure of a b-subunit of a dendrotoxin-sensitive K + channels from bovine brain. Proc. Natl. Acad. Sci. USA, 91: 1637-1641. Shioya, T., Matsuda, H. and Noma, A. (1993) Fast and slow blockades of the inward-rectifier K + channel by external divalent cations in guinea-pig cardiac myocytes. Pflugers Arch., 422: 427-435. Silver, M.R., and DeCoursey, T.E. (1990) Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence and absence of internal Mg 2+. J. Gen. Physiol., 96: 109-133. Slesinger, P.A., Jan, Y.N. and Jan, L.Y. (1993) The $4-$5 loop contributes to the ion-selective pore of potassium channels. Neuron, I l: 739-749. Standen, N.B. and Stanfield, P.R. (1978) A potential- and timedependent blockade of inward rectification in frog skeletal muscle fibres by barium and strontium ions. J. Physiol., 280: 169-191. Stanfield, P.R., Davies, N.W., Shelton, P.A., Khan, I.A., Brammar, W.J., Standen, N.B. and Conley, E.C. (1994) The intrinsic gating of inward rectifier K ÷ channels expressed from the murine IRK1 gene depends on voltage, K ÷ and Mg 2+. J. Physiol., 475: 1-7. Tagiialatela, M., Drewe, J.A. and Brown, A.M. (1993a) Barium blockade of a clonal potassium channel and its regulation by a critical pore residue. Mol. Pharmacol., 44: 180-190. Taglialatela, M., Drewe, J.A., Kirsch, G.E., Biasi, M.D., Hartmann, H.A. and Brown, A.M. (1993b) Regulation of K+/Rb ÷ selectivity and internal TEA blockade by mutations at a single site in K ÷ pores. Pfiugers Arch., 423:104-112. Tagiialatela, M., Wible, B.A., Caporaso, R. and Brown, A.M. (1994) Specification of pore properties by the carboxyl terminus of inward rectifying K ÷ channels. Science, 264: 844-847. Trautwein, W. and Dudel, J. (1958) Zum mechanismus der Membranwirkung des Acetylcholin an der Herzmuskelfaser. Pflugers Arch. ges. Physiol., 266: 324-334. Vandenberg, C.A. (1987) Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions. Proc. Natl. Acad. Sci. USA, 84: 2560-2564. Wickman, K.D., lniguez-Llihi, J.A. Davenport, P.A., Taussig, R., Krapivinsky, G.B., Linder, M.E., Gilman, A.G. and Clapham, D.E. (1994) Recombinant G-protein /3~,-subunits activate the muscarinic-gated atrial potassium channel. Nature, 368: 255-257. Yatani, A., Codina, J., Brown, A.M. and Birnbaumer, L. (1987) Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein GK. Science, 235:207-211. Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R. (1991) Mutations affecting internal TEA blockade identify the probable pore-forming region of a K ÷ channel. Science, 251: 939-942. Yool, A.J. and Schwarz, T.L. (1991) Alteration of ionic selectivity of a K ÷ channel by mutation of the H5 region. Nature, 349: 700-704. Zagotta, W.N., Hoshi, T. and Aldrich, R.W. (1990) Restoration of inactivation in mutants of Shaker K ÷ channels by a peptide derived from ShB. Science, 250: 568-571.