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Journal of Physiology (2001), 535.1, pp. 133-143
© Copyright 2001 The Physiological Society
subunits mediates the voltage-dependent relaxation of the G protein-gated potassium channel |
ABSTRACT |
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2-adrenergic receptor.
subunit of the G protein (G) impaired the effect of RGS4.
subunits mediates the effect of RGS4 on the agonist concentration-dependent relaxation of KG channels.
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INTRODUCTION |
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Acetylcholine (ACh)-induced deceleration of heart beat and the formation of slow inhibitory postsynaptic potentials by various inhibitory neurotransmitters are mediated by G protein-gated inwardly rectifying K+ (KG) channels (Yamada et al. 1998). The cardiac KG channel is composed of Kir3.1 and Kir3.4, while neuronal channels are mainly composed of Kir3.1 and Kir3.2. Both types of channel can be reconstituted by co-expression of their respective Kir3.0 subunits and pertussis toxin (PTX)-sensitive G protein-coupled receptors in Xenopus oocytes (Kubo et al. 1993; Duprat et al. 1994; Krapivinsky et al. 1995; Lesage et al. 1995). Activation, desensitization and deactivation of the reconstituted ACh-induced KG current are accelerated by regulators of G protein signalling (RGS) proteins, thus mimicking to some extent the properties of native KG currents (Doupnik et al. 1997; Saitoh et al. 1997, 1999; Chuang et al. 1998; Herlitze et al. 1999). Therefore, RGS proteins seem to be involved in the physiological control of the KG channel system.
One of the characteristic features of ACh-induced KG current in cardiac myocytes is the property of voltage-dependent 'relaxation' (Noma & Trautwein 1978; Yamada et al. 1998). The cardiac KG current activated by ACh is composed of instantaneous and time-dependent components. The instantaneous component reflects the open probability of the KG channel at the holding potential. The time-dependent component reflects the gradual increase in channel open probability upon hyperpolarizing voltage steps (Yamada et al. 1998). The ratio between these components varies in an agonist concentration-dependent manner. Increasing the concentration of ACh ([ACh]) increases the proportion of the instantaneous component and decreases the proportion of the time-dependent component which is also accelerated (Fujita et al. 2000). In this way relaxation is a mechanism for reducing the open probability of KG channels at depolarized potentials at low [ACh]. It can therefore be important in the sino-atrial node for slowing the pacemaker depolarization without affecting the action potential configuration. We recently showed that co-expression of RGS4 restored the agonist concentration-dependent relaxation to the reconstituted KG current in Xenopus oocytes, in addition to the acceleration of activation and deactivation, although we could not reproduce the effect of RGS4 on short-term desensitization (Fujita et al. 2000). The induction of relaxation in the reconstituted KG channel is a newly identified effect of RGS proteins and replicates the ACh-induced voltage-dependent relaxation of the native cardiac KG channel. The molecular mechanism of this phenomenon, however, has not yet been determined.
In this study, we examined the signalling pathway mediating the effect of RGS4 on KG current relaxation by heterologously expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and RGS4 mutants in Xenopus oocytes. We found that the RGS domain of RGS4 and PTX-sensitive G proteins were required for RGS4-modulation of the kinetics of the KG current.
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METHODS |
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The constructs used in this study are as follows: mouse Kir3.1, rat Kir3.4, rat RGS4, porcine m2-muscarinic receptor (m2R), mouse 2-adrenergic receptor (2R), rat Gs subunit, human Kir3.1/F137S (Chan et al. 1996) and mouse Kir3.2d (Inanobe et al. 1999). Rat Kir3.4, rat RGS4, porcine m2R, mouse 2R and human Kir3.1/F137S were kindly provided by Drs D. Clapham, C. Doupnik, T. Kubo, E. Reuveny and D. Logothetis, respectively. Truncated mutants of RGS4 were produced by a polymerase chain reaction to incorporate start and stop codons with pfx polymerase (Life Technologies Inc., Gaithersburg, MD, USA). Site-directed mutagenesis in RGS4 was performed using the Quick-Change Mutagenesis kit (Stratagene, La Jolla, CA, USA). The sequence of mutants was verified using an ABI Dye terminator cycle sequencing kit with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA).
The frogs (Xenopus laevis) were treated in accordance with the guidelines for the use of laboratory animals of Osaka University Medical School. They were deeply anaesthetized by immersion in 0.35 % tricaine solution (Sigma Chemical Co., St Louis, MO, USA). The oocytes were surgically removed from the frogs and defolliculated in 1 mg ml-1 collagenase solution (Wako Pure Chemical, Osaka, Japan). The frogs were humanely killed following the final oocyte collection. All clones were transcribed in vitro with T7, T3 or Sp6 RNA polymerases and the cRNAs were stabilized with m7G(5')ppp(5')G RNA capping analogue (Life Technologies Inc). The amounts of cRNA injected (in ng oocyte-1) were 160 for RGS4, 160 for various RGS mutants, 80 for m2R, 40-80 for 2R, 16 for Gs, 8 for Kir3.1, 8 for Kir3.4, 4 for Kir3.1/F137S, and 0.4 for Kir3.2d. The combination of injected cRNAs is indicated for each experiment in the text. Channel activity evoked by 2R stimulation was measured in the oocytes co-injected with Gs cRNA, because in the absence of co-injection isoprenaline had no effect on the KG current. After injection, the oocytes were maintained in a modified Barth's solution at 18 °C with daily solution changes. Electrophysiological studies were undertaken 72-96 h later at room temperature (20-25 °C).
Two-electrode voltage-clamp recordings of oocyte currents were performed with a commercially available amplifier (CEZ-1250; Nihon Kohden, Tokyo, Japan). Pipettes had a resistance of 0.5-1.5 M
when filled with 3 M KCl. The bath solution contained (mM): 90 KCl, 3 MgCl2, 0.15 niflumic acid and 5 Hepes-KOH (pH 7.4). Acetylcholine and isoprenaline were dissolved in the bath solution, and inward currents were obtained with voltage steps as indicated in the figure legends. Rapid perfusion was performed using a peristaltic pump. The exchange rate of solutions surrounding the oocytes was determined by changing the K+ concentration from 5 to 90 mM using the same method. The time constant of change was 460 ± 15 ms (mean ± S.E.M., n = 5). Thus, although the time constant for the deactivation phase of the ACh response could be measured accurately, that for the activation phase was approximate. Taking into account the size and anatomical complexity of Xenopus oocytes, this was the fastest rate that we could achieve under our experimental conditions.
In this series of experiments we could detect an ACh-induced K+ current in some oocytes expressing only m2R and Kir3.1. The amplitude of this current was at most ~0.2 µA at -120 mV in 90 mM K+, which was less than 5 % of that induced in oocytes injected with the cRNAs of m2R, Kir3.1 and Kir3.4. Therefore, although the KG channel subunit Kir3.5/XIR has been reported to be endogenous to Xenopus oocytes (Hedin et al. 1996), its expression level was negligible when compared with the exogenously expressed KG channels in this study.
All electrophysiological data were stored on video tapes using a PCM data recording system and subsequently replayed for computer analysis (Patch Analyst Pro; MT Corporation, Hyogo, Japan). The data were expressed as means ± S.E.M. Student's t test was used for statistical analyses. P < 0.05 was accepted as indicating a statistically significant difference.
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RESULTS |
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Comparison of the effects of RGS4 on KG channels coupled to either m2-muscarinic or 2-adrenergic receptor
When heterologously expressed in Xenopus oocytes, KG channels can be activated not only by pertussis toxin (PTX)-sensitive G protein-coupled receptors like the m2-muscarinic receptor (m2R) but also by Gs-coupled receptors such as the 2-adrenergic receptor (2R) (Doupnik et al. 1997; Müllner et al. 2000). We therefore examined the specificity of receptor-G protein signalling pathways in the influence of RGS4 on the agonist concentration-dependent relaxation of KG current (Fig. 1).
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Figure 1. Effects of RGS4 on agonist-dependent KG channel activity reconstituted with Kir3.1 and Kir3.4 A, whole-cell currents through KG channels heterologously composed of Kir3.1 and Kir3.4 were recorded from Xenopus oocytes expressing m2R (a and b) and | ||
Xenopus oocytes were co-injected with the cRNAs of Kir3.1 and Kir3.4, and either m2R or 2R. The cRNA of RGS4 was co-injected as indicated. In oocytes expressing m2R, RGS4 accelerated the activation and deactivation time courses of ACh-induced KG current as reported previously (Fig. 1Aa and b) (Doupnik et al. 1997; Saitoh et al. 1997; Herlitze et al. 1999; Fujita et al. 2000). The m2R couples to Gi and/or Go proteins in oocytes, because pre-injection of PTX A protomer (1 ng oocyte-1) into the oocytes completely abolished ACh activation of the KG channel (n = 7, data not shown). The time constants of current activation (
on) and deactivation (off) in the presence of RGS4 were almost half of those in the absence of RGS4; on values were 1.6 ± 0.3 and 3.3 ± 0.3 s, and off values were 5.5 ± 0.7 and 14.0 ± 1.2 s, respectively (n = 10 for each). It should be noted, however, that both the on- and off-rates of the ACh response, even in the presence of RGS4, are still slower than those recorded from native cells (Yamada et al. 1998). In addition, in contrast to from the reports of Doupnik et al. (1997) and Chuang et al. (1998) we could not reproduce the short-term desensitization of receptor-activated KG current under our experimental conditions, even when we increased the ACh concentration to 10 µM (not shown). This is probably not due to a slow exchange of solution, since the time constant for exchanging the solution surrounding the oocytes was ~460 ms, but is probably related to other factors such as the differential expression of intrinsic proteins among oocytes.
When 2R was expressed, isoprenaline (ISP) could induce the KG current (Fig. 1Ac; Doupnik et al. 1997) provided that the Gs subunit was co-expressed in the oocytes. Pre-injection of PTX A protomer (1 ng) into oocytes did not attenuate the 2R-evoked KG current (n = 3), while it abolished the m2R-mediated current activation. This suggests that PTX-sensitive G proteins do not mediate the 2R-KG channel signalling pathway. Activation and deactivation of the ISP-induced KG current were much slower than those of the m2R-mediated current. Both activation and deactivation of the ISP-induced KG current were unaffected by the co-expression of RGS4 (Fig. 1Ad). In the absence and presence of RGS4, on values were 22.6 ± 4.9 and 24.6 ± 2.6 s, and off values were 122.2 ± 18.7 and 110.9 ± 10.2 s, respectively (n = 5 for each). These results are consistent with those of Doupnik et al. (1997). It is currently considered that 2R-evoked stimulation activates KG current via protein kinase A-catalysed phosphorylation (Müllner et al. 2000) and/or G
released from Gs proteins (Doupnik et al. 1997). Either pathway seems to be independent of the modulation of KG current activation and deactivation by RGS4.
The voltage-dependent kinetics of agonist-induced KG current was also examined in the oocytes expressing either m2R or 2R (Fig. 1B). The oocytes were voltage clamped at 0 mV and hyperpolarizing voltage steps to -60 mV (2 s in duration) were applied every 7 s. ACh and ISP induced the KG current in a concentration-dependent fashion in oocytes expressing m2R or 2R, respectively. A slowly increasing K+ current during hyperpolarizing voltage pulses is one of the features of KG channels containing Kir3.1. The agonist-induced K+ current during a voltage pulse could be divided into instantaneous and time-dependent or relaxing components. In the absence of RGS4, at all concentrations of ACh, the ratio of instantaneous to pulse-end current (Iins/Imax) was ~0.7, this not withstanding that the current amplitude increased as the concentration of ACh ([ACh]) was increased (Fig. 1Ba, and Ca and c). This slow increase of KG current during a voltage step was recently shown to be the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel (Lancaster et al. 2000). In the presence of RGS4, the relaxation of the ACh-induced currents became dependent on [ACh] (Fig. 1Bb). At 3 
10-9 M [ACh], Iins/Imax was 0.5 and the relaxation was ~450 ms. As [ACh] was raised, the Iins/Imax increased and became smaller (Fig. 1Ca and c). At 10-6 M [ACh], both values were similar to those seen without expression of RGS4 or with over-expression of G subunits (not shown, but see Fujita et al. 2000). The superimposed normalized currents at the bottom of Fig. 1Bb clearly illustrate the alteration in the time-dependent properties of ACh-induced K+ current at various [ACh] in the presence of RGS4. Similar results were obtained when D2-dopaminergic receptors, another Gi/o-coupled receptor type, were co-expressed instead of m2R (not shown).
The ISP-induced KG current in oocytes expressing 2R could also be divided into the instantaneous and relaxing components (Fig. 1Bc). In both absence and presence of RGS4, at all concentrations of ISP, Iins/Imax was ~0.65 and the relaxing time-dependent components of ISP-induced KG currents were unaffected by the presence of RGS4 (Fig. 1Bc and d and Cb and d). These results indicate that RGS4 acts specifically on the signalling pathway from the PTX-sensitive G protein-coupled receptor to the KG channel to modulate not only turn-on and turn-off rates of the ACh response but also the agonist concentration-dependent relaxation.
Effects of RGS4 on KG channels composed of homomeric Kir3.0 subunits
We next examined whether the effect of RGS4 would depend upon the subunit composition of KG channels. For this purpose, cRNA for either human Kir3.1 with phenylalanine exchanged for serine at position 137 (Kir3.1/F137S) or mouse Kir3.2d was injected with that for m2R into oocytes (Fig. 2). Although Kir3.1 alone is known not to form a functional KG channel, the mutated Kir3.1/F137S subunit will form a functional homomeric KG channel (Chan et al. 1996). Kir3.2d is an isoform of Kir3.2, cloned from mouse testis which expresses a fairly good receptor-dependent KG channel activity in the Xenopus oocyte expression system with entirely different voltage-dependent kinetics (Inanobe et al. 1999). Therefore, we used Kir3.1/F137S and Kir3.2d in this study.
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Figure 2. Effects of RGS4 on KG channel activity reconstituted with homomeric assemblies A, m2R-evoked whole-cell currents through homomeric KG channels composed of Kir3.1/F137S (a and b) or Kir3.2d (c and d) were recorded with the same protocol described in the legend of Fig. 1. In the presence of RGS4 (b and d), both turn-on and turn-off time courses of ACh-induced KG channels were accelerated, compared with those in the absence of RGS4 (a and c). B, the time constants of ACh-induced activation and deactivation phases ( | ||
Expression of these homomeric Kir3.0 subunits gave rise to ACh-induced K+ currents (Fig. 2A). The homomeric Kir3.1/F137S current was less sensitive to RGS4-induced modulation (Fig. 2Aa and b and B). The deactivation time course of the ACh response was significantly faster in the presence of RGS4, while acceleration of activation was not significant. On the other hand, the turn-on and turn-off phases of the Kir3.2d homotetramer were clearly accelerated in the presence of RGS4 (Fig. 2Ac and d and B).
The amplitude of the homomeric KG currents of either Kir3.1/F137S or Kir3.2d increased as [ACh] was raised. In the absence of RGS4, the Kir3.1/F137S currents showed only a small time-dependent component (Fig. 2Ca). The Kir3.2d current decreased rapidly to a steady state after the instantaneous current jump during a hyperpolarizing step (Fig. 2Cc). Thus, in contrast to that from the Kir3.1/Kir3.4 heteromeric channel, the currents of both homomeric channels did not exhibit slow activation during hyperpolarizing voltage steps. Nonetheless, the co-expression of RGS4 conferred an agonist concentration-dependent relaxation on both types of homomeric KG channels (Fig. 2Cb and d). This effect decreased as [ACh] was increased. Both Iins/Imax values for Kir3.1/F137S and Kir3.2d currents in the absence and presence of RGS4 (Fig. 2D) indicated that RGS4 influences the gating behaviour of homomeric KG channels in a similar manner to that of the heteromeric assembly of Kir3.1 and Kir3.4 (Fig. 1C).
Effects of truncated RGS4 on KG channel gating
RGS proteins contain a conserved region, the RGS domain, which is composed of 120-130 amino acid residues. Analyses of the structures of the RGS4-Gi1 complex (Tesmer et al. 1997), the Axin-adenomatous polyposis coli protein complex (Spink et al. 2000) and GAIP (de Alba et al. 1999) indicate that their RGS domains share similar protein folding composed of nine -helices. It has been also noted that the regions outside of the RGS domain in the RGS protein family are divergent and might be responsible for regulation of subcellular localization of the proteins (Zheng et al. 1999). Therefore, we next examined the effects of truncated mutants of RGS4 to identify the critical region for the modulation of KG channel gating (Fig. 3).
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Figure 3. Requirement of RGS domain for modulation of KG channel activity Truncated mutants of RGS4 were constructed as presented in the left side panel of B. The filled bars indicate the RGS domain included in the mutants. Whole-cell currents through KG channels composed of Kir3.1 and Kir3.4 without and with co-expression of RGS4, RGS4/51-177 or RGS4/51-165 are presented in Aa. Their normalized ACh-induced inward KG currents are also shown in Ab. Bar graphs (Ba and b) summarize the activation and deactivation time constants of KG channel currents activated by ACh ( | ||
Various truncated mutants of RGS4 were expressed in oocytes with m2R, Kir3.1 and Kir3.4. Representative current recordings of the time course of the ACh-induced KG current and voltage-dependent relaxation are shown in Fig. 3A. The primary structures of truncated mutants are depicted in the left panel of Fig. 3B. The time constants for activation and deactivation of the ACh-induced KG current and the relative potencies with which the mutants induced relaxation are indicated in Fig. 3Ba-c. As shown in Fig. 3A, the mutant RGS4/51-177, which is composed of only the RGS domain, could accelerate both activation and deactivation time courses and confer relaxation on the ACh-induced KG current. The mutants RGS4/1-177 and RGS4/51-205, which also contained the full RGS domain, showed almost the same effects as the wild-type RGS4 (Fig. 3B). However, even a small deletion of the RGS domain, as in mutants RGS4/85-165, RGS4/85-177 or RGS4/51-165, abolished the effects of RGS4 not only on the time courses of stimulation but also on relaxation. Mutations of regions outside of the RGS domain, such as in RGS4/1-50 and RGS4/178-205, had no effect. Because we injected excess amounts of cRNAs, we might have missed possible differences in modulation of KG channel activity between RGS4/1-177, RGS4/51-205, RGS4/51-177 and the wild-type RGS4. Overall, however, these results clearly indicate that the intact RGS domain is mandatory for the expression of the RGS4-induced modulation of channel activity.
Effects of point mutations in the RGS domain on the relaxation
Analysis of the crystal structure of the RGS4-Gi1 complex has revealed the amino acid residues in the RGS domain that are important for association with Gi1 (Tesmer et al. 1997). These residues are localized on the loops between the 3-4, 5-6 and 7-8 helices and also in the 4, 7 and 8 helices (see Fig. 4A). On the other hand, RGS4 interacts with the Ca2+-calmodulin complex in the region (residues 99-113) of 4 and 5 helices, which is on the face opposite the Gi1-binding site (Popov et al. 2000). To identify the critical residues in the RGS domain for the induction of KG channel relaxation, we examined the effects of RGS4 whose amino acid residues at various sites were mutated (Fig. 4A).
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Figure 4. Effects of mutagenesis in the RGS domain on RGS4-induced modulation of KG channel activity A, ribbon diagram of the RGS domain complexed with Gi | ||
Among the residues mutated in RGS4 in this study, those supposed to be involved in the binding to Gi1 are E87, N88, N128, L159, R167 and F168, while L66, I67, S103 and P144 are thought not to be involved (Fig. 4A). Substitution of amino acids was performed as indicated in Fig. 4A and C. Representative current traces were shown in Fig. 4B. The mutant RGS4/N128H, which should interfere with the interaction with Gi1, attenuated the potency with which RGS4 accelerated the deactivation of the ACh-induced KG current. In addition, this RGS4 mutant did not confer relaxation on the channel. On the other hand, the mutation at P144, which is not involved in the contact between RGS4 and Gi1 (RGS4/P144A), had a comparable effect to wild-type RGS4. The results thus obtained are summarized in Fig. 4C.
Figure 4C showed that it was possible to separate the effects of RGS4 on the on- and off-rates of the muscarinic response. It was also clear that mutations that result in the slowing of the off-rate of the muscarinic response abolished agonist concentration-dependent relaxation. Furthermore, it is noteworthy that these effects of the mutants correlated with their GTPase-activating protein (GAP) activity (Natochin et al. 1998; Srinivasa et al. 1998). Only one of the series of mutations shown in Fig. 4C, N88S, had an effect on the on-rate of the ACh response, while it had almost no effect on the off-rate and partially reduced the effect on relaxation. Otherwise the results obtained with mutations of RGS4 were divided into two groups, those with wild-type characteristics, E87A, N88A, S103E and P144A, and those where the effects of RGS4 on the off-rate and the relaxation of ACh currents were abolished, N128A, N128H, L159F, R167A and F168A. Of the former group the effects of S103E and P144A were perhaps not surprising since these locations are not thought to be involved in the interaction between RGS4 and the G subunit. The lack of an effect of either E87A or N88A may be due to the fact that, although lower than the wild-type RGS4, these mutants retain higher GAP activity than RGS4/N128A, N128H, L159F and R167A (Natochin et al. 1998; Srinivasa et al. 1998). Because we injected excess amounts of cRNAs, we might have missed relatively small effects in this study. A double mutant (E87A/N88A), which strongly impaired the activities of both binding to G and acceleration of the hydrolysis of GTP in G (Srinivasa et al. 1998), did show disruption of the off-rate and relaxation. Double mutations at the sites which are supposed not to make contact with G subunit, RGS4l-166R/I67K, also abolished the effects on the deactivation time course and relaxation. This might be due to a conformational change in the RGS domain caused by double mutations. Most of these results, however, support the notion that an interaction between the RGS domain and G subunits is required for the time-dependent relaxation of KG current as well as for the acceleration of deactivation upon washout of the agonist.
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DISCUSSION |
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The major findings in this study are as follows: (1) the effect of RGS4 on the agonist concentration- and time-dependent relaxation of the KG current is specific to PTX-sensitive G proteins and not to the type of KG channel; (2) the RGS domain in RGS4 is essential for induction of relaxation; (3) mutations of the RGS domain at sites of contact between RGS4 and Gi1 impair relaxation. We conclude that the effect of RGS4 on the agonist concentration-dependent relaxation of the KG channel is mediated exclusively by the association between the RGS domain and a PTX-sensitive G subunit.
Specificity of the effect of RGS4 on KG channel gating
RGS4 conferred the agonist concentration-dependent relaxation on KG channels activated by Gi/o-coupled receptors but not by a Gs-coupled receptor in Xenopus oocytes. The gating kinetics of either a heteromeric assembly of Kir3.1 and Kir3.4 or homomeric channels composed of Kir3.1/F137S or Kir3.2d was affected by RGS4. Therefore, the specificity of the effect of RGS4 on gating resides at a step involving the PTX-sensitive G protein and not at the channel protein itself. We previously showed that RGS4 could bind to the KG channel protein complex composed of Kir3.1 and Kir3.4 (Fujita et al. 2000). We have re-examined these biochemical experiments and found that RGS4 could form a protein complex with KG channels in the crude cell lysate, but not with partially purified KG channel proteins (not shown). Thus, it might be the case that some unidentified cellular component(s) may be required for the formation of a complex between RGS4 and KG channels. However, it remains unclear at present what the component is and whether the component is involved in the modulation of KG channel activity by RGS4.
The RGS domain and regulation of KG channel activity
Truncation of RGS4 clearly showed that the intact RGS domain is essential for reproducing the effects of wild-type RGS4 on the KG current. Neither the N- nor the C-terminus conveyed any effects in our experiments (Fig. 3). Zeng et al. (1998), however, reported that the RGS domain alone was less potent than the wild-type RGS4 in accelerating GTPase activity during m1-muscarinic stimulation of the Gq protein system reconstituted in phospholipid vesicles. This was attributed to the lack of the N-terminus of RGS4, because the palmitoylation of the N-terminus at its amphipathic -helix was shown to be essential for recruiting the RGS protein into the membrane (Tu et al. 1999; Bernstein et al. 2000). Although the RGS domain alone is less potent, it has been shown that it can still accelerate GTPase activity (Zeng et al. 1998). Because we injected excess amounts of the cRNAs of various mutants into the oocytes, it may be the case that we missed such relatively small differences caused by the N- or the C-terminus. In contrast, even a small deletion of ~20 amino acids from the RGS domain (residues 51-85 and 165-177) completely abolished its effects (Fig. 3). Therefore, it can be concluded that the intact RGS domain is mandatory for the effect of RGS4 on the KG channel system, although further modulation by either the N- or C-terminus cannot be excluded. This agrees with the report by Popov et al. (1997) that the RGS domain is the minimal sequence required for the GAP activity of RGS4 for Gi1 and also for RGS4-binding to Gi1-GDP-AlF4-.
Residues 51-85 include helices 1-3, while residues 165-177 include helices 8-9 (Fig. 4). Structural analysis indicates that R167 in helix 8 forms salt bridges with E83 in the loop between helix 3 and 4 and with D163 in helix 7. These interactions are supposed to stabilize the conformation of the RGS domain (Tesmer et al. 1997). Therefore, the deletion at residues 51-85 and 165-177 may disrupt these salt bridges in the RGS domain. Thus the mutants might be unstable in oocytes (Popov et al. 1997; Srinivasa et al. 1998).
The role of amino acids in the G subunit-binding pocket of RGS4 in the regulation of KG channel activity
Crystal structure analysis of RGS4 has shown that N128 is supposed to be a key residue for promoting the hydrolysis of GTP in Gi1. A hydrogen bond between the amide nitrogen in the side chain of N128 in RGS4 and a carboxylate group in the side chain of E204 in the switch II subunit of Gi1 controls the orientation of a water molecule for nucleophilic attack upon the GTP phosphate (Tesmer et al. 1997). We therefore substituted N128 in RGS4 for alanine or histidine. Both mutants reproduced the effect of wild-type RGS4 on the activation of the ACh-induced KG current but not on either the deactivation time course or time-dependent relaxation (Fig. 4). Similar studies showed that the residue N128 is crucial for the function of RGS4 in terms not only of GAP activity but also of binding to Gi1 (Srinivasa et al. 1998; Natochin et al. 1998). Therefore, it is strongly suggested that both the RGS4-G subunit association and the subsequent acceleration of GTP hydrolysis are involved in KG channel relaxation.
It has been shown that multiple residues (S85, E87, N88, L159, D163, S164 and R167) of RGS4 form a binding pocket for T182 on the switch I of Gi1 (Fig. 4A; Tesmer et al. 1997). These residues are also known to be important for expressing the effects of wild-type RGS4 on G protein signalling (Srinivasa et al. 1998). Most mutations of these residues in this study consistently reduced the effects of RGS4 on the deactivation phase and agonist concentration-dependent relaxation. The mutations of residues S103 and P144, which are located away from the binding pocket, did not modulate the effects of RGS4 except in the case of one double mutant (RGS4/L66K/I67K). Several mutations in the binding pocket (RGS4/E87A, RGS4/N88A), which partially reduce their GAP activities (Srinivasa et al. 1998), did not impair the effects of RGS4 but maintained the effects of the wild-type on the off-phase of the ACh response and relaxation. This is probably because excess amounts of cRNAs for the mutants were injected in this study. Therefore, our overall assessment indicates that the amino acids in the G-binding pocket of the RGS domain are important for RGS4 induction of relaxation and acceleration of deactivation.
The replacement of N88 with serine caused slowing of the activation phase while deactivation and relaxation were the same as with wild-type RGS4. Herlitze et al. (1999) showed that replacement of the corresponding asparagine residue with serine in RGS2 lessens the accelerated turn-on and turn-off time courses of KG channel activity. Human RGS2 has been reported to be less effective in the modulation of the ACh-evoked KG current (Doupnik et al. 1997). It was also shown that the protein possesses a higher affinity for Gq than Gi, while rat RGS4 associates equally with both G subunits (Hepler 1999). Therefore the discrepancy between the effects of these mutations in RGS4 and RGS2 on the response to ACh of the KG current might be due to the differences in the efficacy with which these RGS proteins couple with endogenous G subunits and/or in the expression level in oocytes. Nevertheless, the result with RGS4/N88S and those with N128A, N128H, L159F, R167A and F168A all suggest that the molecular mechanism responsible for accelerating the activation phase of the ACh response is different from that or those for deactivation and relaxation. This is consistent with the proposal that a particular and unidentified action of RGS proteins is responsible for the acceleration of the activation phase (Saito et al. 1999). Further studies are needed to clarify the molecular mechanism for RGS protein-induced acceleration of the activation phase of the ACh response.
Modulation of receptor-dependent KG channel activity by RGS proteins
It has been shown that RGS proteins accelerate activation, desensitization and deactivation of the reconstituted ACh-induced KG current (Doupnik et al. 1997; Saitoh et al. 1997, 1999; Chuang et al. 1998; Bünemann & Hosey 1998; Herlitze et al. 1999). RGS proteins are also responsible for the agonist concentration- and time-dependent relaxation of the KG current (Fujita et al. 2000). Therefore, RGS proteins are an essential component of many physiological properties of KG channels.
This study has clearly shown that the interaction between the RGS domain of RGS4 and PTX-sensitive G subunits exclusively mediates both the relaxation of the KG current and the acceleration of the deactivation time course of the ACh response and also that this interaction cannot account for the acceleration of the activation time course of the ACh response. Because truncated mutants containing the RGS domain alone could reproduce the effects of wild-type RGS4 not only on deactivation and relaxation but also on the activation phase, sites in the RGS domain that are not involved in the interaction with G might be responsible for acceleration of the activation phase. Further studies are needed to resolve this question.
It was also found that RGS4 could control the properties of various types of KG channels. Therefore, RGS proteins may have physiological roles in the regulation of KG channels not only in cardiac myocytes but also in endocrine cells and neurons. RGS proteins may differentially regulate the strength and duration of G protein signalling to various downstream effectors, based on differences in their specificity and affinity for various G subunits, expression level among various cell types and subcellular localization in each cell (for review, see Zheng et al. 1999; Hepler 1999).
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Acknowledgements
The authors thank Dr Ian Findlay (Tours University, Tours, France) for his critical reading of this manuscript, Ms Kazue Takahashi and Miho Fukui for their technical assistance, and Ms Keiko Tsuji for secretarial work. This work was supported by the grants to Y.K. from the Ministry of Education, Science, Sports and Culture of Japan for priority areas (B) (12144207), from the Research for the Future Program of the Japan Society for the Promotion of Science (96L00302), and from the Human Frontier Science Program (RG0158/1997-B).
A. Inanobe and S. Fujita contributed equally to this work.
Corresponding author
Y. Kurachi: Department of Pharmacology II, Graduate School of Medicine, A7, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Email: ykurachi{at}pharma2.med.osaka-u.ac.jp
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