Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates.

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.

tors (GPCRs) selectively coupled to pertussis toxin (PTX)-sensitive G␣ i/o ␤␥ proteins (1,2). The time course for GIRK channel activation elicited by application of receptor agonist can be influenced by the multiple intervening steps of the G protein cycle that begin with agonist binding to the GPCR, and end with G␤␥ binding to the GIRK channel subunits that promote gating transitions to the open state. In addition to the G protein activation steps, signal termination with GTP hydrolysis by the G␣ subunit and G␤␥ reassociation also impacts the kinetics and amplitude of agonist-activated GIRK currents. The ternary complex consisting of agonist, GPCR, and G protein influences the time course of agonist-elicited GIRK channel currents (3), supporting the notion that isoform composition of different GPCR-G␣ i/o ␤␥ protein-RGS protein-GIRK channel signaling complexes have different kinetic properties that affect their functional output (4).
We recently reported notable differences in the gating properties of GIRK channels activated by muscarinic m2 receptors coupled specifically to PTX-insensitive G␣ i isoforms (G␣ i1 , G␣ i2 , or G␣ i3 ) versus G␣ o isoforms (G␣ oA or G␣ oB ) in the Xenopus oocyte system (4). The ACh-elicited activation time course for G␣ o -coupled GIRK currents was ϳ3-fold faster than for G␣ i -coupled GIRK currents, and G␣ o expression was significantly more effective than the G␣ i isoforms at reducing receptor-independent basal GIRK channel activity. To identify the molecular determinants responsible for these differences, we constructed several PTX-insensitive G␣ oA /G␣ i2 chimeras and examined their functional properties in the Xenopus oocyte system via coupling to muscarinic m2 receptors and GIRK channels. The G␣ oA /G␣ i2 chimera experiments indicate the kinetic differences between G␣ oA and G␣ i2 involve an interdomain interaction that led us to examine a previously identified domain-domain contact in G␣ i1 that slows the basal (receptorindependent) GDP release rate compared with G␣ o in vitro (5). G␣ i2 mutations designed to disrupt the domain-domain interaction accelerated GIRK activation rates consistent with the biochemical studies of mutant G␣ i1 subunits that increased basal GDP release rates (5). Thus our findings establish experimental conditions whereby the GPCR-catalyzed G␣ GDP release rate is the rate-limiting step in receptor-dependent GIRK activation as originally proposed by Breitwieser and Szabo (6) for I K,ACh in atrial myocytes, and importantly identifies molecular determinants that mediate functional differences in GIRK channels activated by GPCRs coupled to G␣ i and G␣ o proteins that may impact the kinetics and magnitude of inhibitory GIRK channel-mediated postsynaptic currents (7). FIG. 1. Construction of PTX-insensitive G␣ i2 /G␣ oA chimera subunits. A, amino acid sequence alignment of G␣ i1 , G␣ i2 , G␣ i3 , G␣ oA , and G␣ oB subunits. The asterisk (*) at the conserved cysteine residue in the C-terminal tail (Ϫ4 position) denotes the site of PTX-mediated ADP-ribosylation. All G␣ (i/o) subunits and chimeras used in this study were rendered PTX insensitive by mutating the Ϫ4 cysteine to a glycine residue (Cys 3 Gly mutation). Black arrows (2) indicate G␣ i2 /G␣ oA junction sites in the chimeras. The red residues in the G␣ o sequences denote non-conserved amino acid differences with the three G␣ i subunits. Secondary structures (␣-helices, bars; ␤-sheets, arrows) are aligned with the amino acid sequences and are based on the G␣ i1 crystal structure (8). The color coding highlights the three major structural domains of the G␣ subunit; the ␣N domain

EXPERIMENTAL PROCEDURES
Design and Construction of PTX-insensitive G␣ oA /G␣ i2 Chimeras- Fig. 1A shows the amino acid sequence alignment of all five G␣ i/o isoforms with corresponding secondary structures determined from crystallographic studies of G␣ i1 (8). PTX-insensitive G␣ oA(C351G) and G␣ i2(C352G) subunits, which have 68% sequence identity, were used to construct PTX-insensitive G␣ oA /G␣ i2 chimeras. G␣ i/o subunits are comprised of three major domains: 1) an N-terminal ␣-helix (␣N) that contains lipid-modified residues for membrane attachment and mediates in part GPCR and G␤␥ interactions, 2) a closely packed ␣-helical domain (␣HD) that encloses the guanine nucleotide binding cleft and is thought to regulate GDP/GTP exchange rates, and 3) a Ras-like domain that binds guanine nucleotides at highly conserved "switch regions" to regulate conformational changes effecting G␤␥ association and dissociation (9). Six different PTX-insensitive G␣ oA /G␣ i2 chimeras were constructed exchanging three major regions: 1) the combined ␣N-␣HD region (with the exception of the ␣E-␣F linker and ␣F), 2) the three switch regions (SWI-III) located within the Ras-like domain, and 3) the carboxyl region containing remaining elements of the Ras-like domain (Fig. 1, B and C). The G␣ i2(C352G) or G␣ oA(C351G) sequence corresponding to these three swapped regions are indicated in the naming of each constructed chimera; e.g. G iio , G oii , G ioi , G oio , G ioo , G ooi (Fig. 1C). The two junction sites within the G␣ i2(C352G) coding region correspond to Glu 161 / Arg 162 and Asp 262 /Thr 263 (Fig. 1A). Non-conserved amino acid differences between G␣ o and G␣ i isoforms are heavily biased in the ␣HD region although differences exist in all major structural domains (see Fig. 1A).
The PTX-insensitive G␣ oA /G␣ i2 chimeras were constructed by PCR amplification (Vent DNA polymerase, New England Biolabs) of selected regions of the rat G␣ i2(C352G) and mouse G␣ oA(C351G) cDNAs cloned in the pCI vector (kindly provided by Stephen Ikeda, NIAAA, National Institutes of Health). Existing or new restriction sites flanking targeted regions were introduced with oligonucleotide primers that preserved the desired coding sequence (Fig. 1B). The various PCR products were gel-purified, digested with appropriate restriction enzymes, and ligated (T4 ligase, Promega) into the pcDNA3.1(ϩ) cloning vector (Invitrogen).
All primers used in the construction of the G␣ oA /G␣ i2 chimeras are provided in Table I. The full-length G␣ oA /G␣ i2 chimera sequences were confirmed by automated DNA sequencing (Molecular Biology Core Facility, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL).
Isolation and cRNA Injection of Xenopus Oocytes-All procedures for the use and handling of Xenopus laevis (Xenopus Express, Plant City, FL) were approved by the University of South Florida Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines. Methods to enzymatically isolate and culture oocytes were as described elsewhere (4). Stage V-VI oocytes were maintained at 19°C in oocyte culture medium (OCM) consisting of 82.5 mM NaCl, 2.5 mM KCl, 1.0 mM CaCl 2 , 1.0 mM MgCl 2 , 1.0 mM NaHPO 4 , 5.0 mM Hepes, 2.5 mM Na pyruvate, and 2% heat-inactivated horse serum, at pH 7.5 (NaOH).
Mixtures of cRNAs were injected the day after oocyte isolation at a final injection volume of 50 nl (Nanoliter2000, World Precision Instruments). Each 50-nl mixture consisted of cRNA's encoding the rat Kir3.1 subunit (0.5 ng), mouse Kir3.2a subunit (0.5 ng), the human m2 receptor (0.5 ng), the PTX-S1 subunit (1 ng), and a PTX-resistant G␣ subunit (5 ng). All cRNAs were synthesized in vitro from linearized cDNAs according to the manufacturer's protocol (mMessage mMachine, Ambion, Austin, TX) and concentrations determined by spectrophotometric absorbance at 260 nm. These oocyte expression conditions have been previously shown to yield roughly equivalent protein levels among the five different PTX-insensitive G␣ i and G␣ o isoforms (4).
Electrophysiological Recordings-GIRK currents were measured from cRNA injected-oocytes using the two-electrode voltage clamp recording technique (10). The oocytes were voltage clamped at a holding potential of Ϫ80 mV using 3 M KCl-filled electrodes having tip resistances of 0.8 -1.2 M⍀ (GeneClamp 500, Axon Instruments). Oocytes were initially superfused with a minimal salt solution composed of 98 mM NaCl, 1 mM MgCl 2 , and 5 mM Hepes at pH 7.5 (NaOH). The superfusion solution was then changed to a high K ϩ solution composed of 20 mM KCl, 78 mM NaCl, 1 mM MgCl 2 , and 5 mM Hepes at pH 7.5 (NaOH). The switch to high K ϩ solution produced an inward current (orange), the ␣-helical domain (purple), and the Ras-like domain (green). B, diagram illustrating the PCR strategy, restriction sites, and PCR primers used to construct PTX-insensitive G␣ oA /G␣ i2 chimeras from the rat G␣ i2(C352G) and mouse G␣ oA(C351G) cDNA templates. C, PTX-insensitive G␣ oA /G␣ i2 chimeras constructed. The three regions derived from the G␣ i2(C352G) subunit are in gray, and the three regions derived form the G␣ oA(C351G) subunit are in black. The nomenclature of each G␣ oA /G␣ i2 chimera (G oii , G ioi , G iio , G ioo , G oio , and G ooi ) denotes the amino acid composition (G␣ oA(C351G) or G␣ i2(C352G) ) of each of the three swapped regions. The secondary structure diagram is included as a reference for the chimera junction sites. consisting mostly of basal receptor-independent GIRK current (I K,basal ).
To evoke receptor-dependent GIRK currents (I K,ACh ), a computer controlled superfusion system (SF-77B, Warner Instruments) was used to rapidly apply and washout various concentrations of acetylcholine (ACh, Sigma-Aldrich) in high K ϩ solution (11). All recordings were performed at room temperature (21-23°C).
Data Analysis-GIRK current kinetics were analyzed using nonlinear curve fitting software that fit single exponential functions to derive the activation time constant ( act ) and the deactivation time constant ( deact ) (pCLAMP software, Axon Instruments). ACh doseresponse relations were analyzed by fitting peak I K,ACh amplitudes with the Hill function shown in Equation 1, where the ACh concentration producing a 50% response (EC 50 ) and the Hill coefficient (n H ) were derived from a non-linear least-squares best fit (Origin 6.0 software, OriginLab Corp., Northampton, MA). Statistical comparisons between the various experimental groups were performed by one-way analysis of variance where p Ͻ 0.05 was considered significant. Each experiment was replicated in oocytes derived from at least two separate oocyte batches (dissections).

RESULTS
Functional Expression of G␣ oA /G␣ i2 Chimeras-Each of the six PTX-insensitive G␣ oA /G␣ i2 chimeras effectively rescued m2 receptor-activated GIRK currents in PTX-S1-expressing oocytes (Fig. 2). PTX-mediated uncoupling of endogenous G␣ i/o subunits (Ͼ95%) was confirmed in parallel groups of oocytes that did not receive cRNA encoding a PTX-insensitive G␣ subunit (data not shown), indicating the ACh-activated GIRK currents were evoked via coupling to the PTX-insensitive G␣ subunits. As reported previously (4), expression of the parental G␣ oA(C351G) subunit produced smaller I K,basal amplitudes and larger receptor-activated currents (I K,ACh ) compared with G␣ i2(C352G) , and expression of each of the PTX-insensitive G␣ oA / G␣ i2 chimeras yielded I K,basal and I K,ACh amplitudes that ranged between the properties of the G␣ oA(C351G) and G␣ i2(C352G) subunits (Fig. 2). Since each chimera was expressed under identical conditions (see "Experimental Procedures"), and expression of the parental G␣ oA(C351G) and G␣ i2(C352G) sub-units produce equivalent protein levels (4), the differences in receptor-independent and receptor-dependent GIRK channel activity with the different G␣ oA /G␣ i2 chimeras is not readily attributable to differences in G␣ subunit protein levels but instead to differences in intrinsic G␣ function. Moreover, the results demonstrate that each G␣ oA /G␣ i2 chimera expresses a functional PTX-insensitive G␣ subunit capable of m2 receptor coupling, receptor-catalyzed GDP/GTP exhange, endogenous G␤␥ association/dissociation, and intrinsic GTPase activity.
Regions of G␣ o That Accelerate ACh-evoked GIRK Activation Kinetics-The time course for ACh-elicited GIRK current activation and the ACh dose-dependence varied among the six G␣ oA /G␣ i2 chimeras tested, yet ranged between the properties of the parental G␣ oA(C351G) and G␣ i2(C352G) subunits as anticipated. Replacing either the ␣N/␣HD region of G␣ i2(C352G) with the corresponding G␣ oA sequence (G oii chimera), or replacing the G␣ i2 switch regions with G␣ o switch regions (G ioi chimera), both accelerated the GIRK activation time course compared with G␣ i2(C352G) -coupled responses (Fig. 3A). The effects of each G␣ oA region substitution, however, was only partial and did not fully reconstitute the activation kinetics of the G␣ oA(C351G)coupled response. The effects on GIRK activation kinetics were also reflected in the steady-state ACh dose-response curves, where G oii -coupled GIRK responses were leftward shifted and more characteristic of G␣ oA(C351G) -coupled GIRK currents than G␣ i2(C352G) -coupled GIRK currents (Fig. 3A). Differences in EC 50 values with G␣ i2(C352G) coupling (0.43 Ϯ 0.07 M, n ϭ 9) versus G␣ oA(C351G) coupling (0.21 Ϯ 0.05 M, n ϭ 10) are small yet significantly different (Fig. 4B) and have comparable Hill coefficients (G␣ i2(C352G) 1.11 Ϯ 0.04; G␣ oA(C351G) 1.44 Ϯ 0.07). Replacing the G␣ i2(C352G) carboxyl region with the G␣ oA(C351G) carboxyl region (G iio chimera) had no significant effect on GIRK activation kinetics or the ACh dose-reponse curve, and was indistinguishable from the G␣ i2(C352G) -coupled kinetics (Fig.  3A). Thus isoform differences in the last 92 residues do not mediate the kinetic differences between G␣ i2(C352G) -coupled and G␣ oA(C351G) -coupled GIRK currents (12).
Regions of G␣ i2 That Slow GIRK Activation Kinetics-For the reciprocal set of experiments where G␣ i2(C352G) regions replaced the equivalent regions of G␣ oA(C351G) , only substituting the G␣ i2 switch regions (G oio chimera) caused a significant yet partial slowing in the GIRK activation kinetics (Fig. 3B). Neither the G␣ i2 ␣N/␣HD region (G ioo chimera) nor the carboxyl region (G ooi chimera) significantly slowed the GIRK activation characteristics of G␣ oA(C351G) -coupled responses. Yet the combined presence of both the G␣ i2 ␣N/␣HD region plus the G␣ i2 switch regions (G iio chimera), was sufficient to fully reproduce the slower G␣ i2(C352G) -coupled GIRK activation kinetics (Fig. 3A). Similarly, the combined presence of the G␣ oA ␣N/ ␣HD region plus the switch regions (G ooi chimera) was sufficient to fully reproduce the faster G␣ oA(C351G) -coupled GIRK activation kinetics (Fig. 3B). Thus in both cases the ␣N/␣HD region plus the switch regions are necessary and sufficient to fully reproduce the parental G␣ subunit effects on GIRK activation kinetics.
Effects of G␣ oA /G␣ i2 Chimera Expression on Basal GIRK Channel Activity-In addition to receptor-dependent GIRK activation kinetics, the receptor-independent basal GIRK channel activity (I K,basal ) associated with expression of each G␣ oA / G␣ i2 chimera was also examined. A reduction in I K,basal caused by G␣ subunit expression in parallel with an increased I K,ACh amplitude, is largely a consequence of G␣ sequestration of free endogenous G␤␥ dimers and increased heterotrimeric G␣ (GDP) ␤␥ formation (4, 13). For comparisons among the different G␣ oA /G␣ i2 chimeras, I K,basal was expressed as the fraction of the total GIRK current elicited from each oocyte (I K,total ϭ I K,basal ϩ maximal I K,ACh ). As reported previously (4), G␣ oA(C351G) reduced I K,basal amplitudes significantly greater than G␣ i2(C352G) under equivalent expression conditions, with I K,basal representing ϳ10% of the total GIRK current with G␣ oA(C351G) expression compared with ϳ45% with G␣ i2(C352G) expression (Fig. 4A). The fractional I K,basal amplitudes for each of the G␣ oA /G␣ i2 chimeras indicated a significant effect of the switch region substitutions (Fig. 4A). Expression of the G ioi chimera produced a significantly lower fractional I K,basal amplitude (22 Ϯ 4%, n ϭ 6) compared with the parental G␣ i2(C352G) expression (45 Ϯ 6%, n ϭ 9). And conversely, expression of G oio significantly increased the fractional I K,basal amplitude to 28 Ϯ 6% (n ϭ 9) compared with 11 Ϯ 1% (n ϭ 10) for parental G␣ oA(C351G) expression (Fig.  4A). Again, the effects of the individual domain substitutions were partial, where substitutions of both the ␣N/␣HD region plus the switch regions (G iio and G ooi ) reconstituted the parental I K,basal amplitude (Fig. 4A). Interestingly, the effects of the switch regions on I K,basal correlate well with the effects on receptor-evoked GIRK activation kinetics (Fig. 4C), with the exception of the G␣oA ␣N/␣HD region chimera (G oii ), which displayed a slightly accelerated receptor-evoked GIRK activation time course without significantly effecting the fractional I K,basal amplitude.
The deactivation time course of GIRK currents after rapid ACh washout, a process dependent on G␣ GTPase activity, was previously found to be similar among all the PTX-insensitive G␣ i/o isoforms (4). However in the current set of experiments, the GIRK deactivation time constant with G␣ oA(C351G) expression ( deact ϭ 14.7 Ϯ 0.4 s, n ϭ 10) was different (p Ͻ 0.05) from the deactivation time constant derived with G␣ i2(C352G) expression ( deact ϭ 23.2 Ϯ 1.6 s, n ϭ 9). Introducing either the G␣ i2 ␣N/␣HD region (G ioo ) or the switch regions (G oio ) into G␣ oA(C351G) was sufficient to convert the deactivation time constant to a G␣ i2(C352G) -like value (Fig. 4D).
G␣ i2 Interdomain Contacts Affecting GIRK Channel Gating Properties-The G␣ oA /G␣ i2 chimera results indicate the molecular determinants that mediate differences in G␣ oA(C351G) and G␣ i2(C352G) -coupled GIRK currents reside in both the ␣N/␣HD region and the switch regions. When both are present together from the same subunit (i.e. G iio and G ooi ), the chimeras behave like their parental subunit. These findings lead us to examine a previously reported interdomain interaction in G␣ i1 , between the ␣D-␣E loop located in ␣HD and the switch III loop located in the Ras-like domain (Fig. 5A), that mediates the 4-fold slower GDP release rate of recombinant G␣ i1 versus G␣o subunits in vitro (5,14). To disrupt the interdomain contact in G␣ i2(C352G) , the switch III loop was mutated to the corresponding G␣ o switch III loop (G␣ i2 (G oSW3 ) chimera) that consists of a four amino acid substitution (G␣ i2 L233Q/A236H/E240T/ M241T). In addition, a conserved arginine residue in the G␣ i2 ␣D-␣E loop that forms a hydrophobic contact with the switch III loop was also mutated to an alanine (G␣ i2(R145A) ) to disrupt the interdomain interaction. Both of these mutations when introduced into G␣ i1 accelerate the basal GDP release rate to levels similar to G␣ o (5).
Shown in Fig. 5B, mutating the four switch III residues of G␣ i2(C352G) to the corresponding G␣ o switch III amino acids (L233Q/A236H/E240T/M241T) partially accelerated the AChevoked GIRK activation rate similar to the G ioi chimera (Fig.  6C), indicating isoform differences in the switch III loop contribute a significant part of the kinetic differences in G␣ oA and G␣ i2 -coupled GIRK currents. More dramatically, expression of the G␣ i2(R145A) mutant fully replicated G␣ oA(C351G) -coupled GIRK activation kinetics (see Figs. 5B and 6C). Both the G␣ i2 switch III loop mutation and the ␣D-␣E loop R145A mutation reduced I K,basal amplitudes (Fig. 6A), consistent with the observations of the G ioi chimera, yet neither effected the GIRK current deactivation time constant (Fig. 6D). Thus in conjunction with the previous structure-function analysis of GDP release differences in G␣ i and G␣ o subunits (5,14), the results of the G␣ i2(R145A) and switch III mutations on GIRK channel activation kinetics indicate G␣ i /G␣ o isoform differences are attributable to differences in receptor-catalyzed GDP release rates that determine the time course of G␤␥ production and activation of GIRK channels. DISCUSSION The goal of this study was to identify molecular determinants in G␣ i and/or G␣ o subunits that mediate differences in GIRK channel gating kinetics associated with selective G␣-GPCR coupling (4). Our G␣ i2 /G␣ oA chimera results implicated an interdomain interface previously linked to G␣ i /G␣ o isoform differences in basal GDP release rates (5,14,15). They also ruled out the possible role of the carboxyl region known to be involved in GPCR coupling and previously implicated in G␣ i2 / G␣ o differences in nucleotide exchange (12). Isoform differences in basal GDP release rate among G␣ i and G␣ o subunits (14) correlate well with the kinetic profile we observed with receptor-activated GIRK currents coupled to individual PTX-insensitive G␣ i and G␣ o subunits (4). That is, the three G␣ i isoforms that have a significantly slower basal GDP release rate compared with G␣ o subunits in vitro (14), also produce slower receptor-activated GIRK currents compared with G␣ o subunits in vivo (4). Thus, these findings are consistent with the ratelimiting step in GPCR activation of GIRK channels being receptor-catalyzed GDP release from associated G␣ subunits (6).
The ␣HD-Switch III Interaction in G␣ Subunits and Receptor-catalyzed GDP Release-Domain-domain interactions involving the ␣D-␣E loop and the switch III loop have previously been implicated in regulating basal and receptor-activated nucleotide exchange for different G␣ subunits (5,(15)(16)(17)(18). Indeed, inherited mutations in the human G␣ s switch III domain enhance GDP release and cause Albright hereditary osteodystrophy characterized by skeletal and developmental abnormalities (19). Mutational analysis of G␣ subunits indicate that substitution of G␣ i switch III residues (conserved in all three G␣ i isoforms) into G␣ s impairs ␤-adrenergic receptor-mediated G protein activation (15), and substitution of corresponding G␣ o switch III residues into G␣ i1 (L232Q/A235H/E239T/M240T) speed up the basal GDP release rate similar to wild-type G␣ o (5). Together these findings indicate the conserved G␣ i switch III loop slows the rate of GDP release in G␣ i subunits through interactions with the ␣-helical domain, principally through hydrophobic interactions between Leu 232 in switch III and Arg 144 in the ␣D-␣E loop (5). In our experiments, m2 receptoractivated GIRK currents coupled to G␣ i2(C352G) containing the G␣ o switch III loop (i.e. the G␣ i2 (G oSW3 ) chimera) displayed accelerated activation kinetics consistent with a faster G␣ GDP release rate. Introducing the R145A mutation in the ␣D-␣E loop of G␣ i2(C352G) to disrupt the domain-domain interaction, more dramatically accelerated the GIRK activation kinetics which were indistinguishable from G␣ oA(C351G) -coupled GIRK kinetics. Since the equivalent mutation in G␣ i1 (R144A) increases basal GDP release rates equivalent to the kinetics of wild-type G␣ o (5), the PTX-insensitive G␣ i2(R145A/C352G) subunit appears to similarly have faster receptor-stimulated GDP release rates compared with the parental G␣ i2(C352G) subunit as reflected in the GIRK activation time course. Since GIRK channels are activated by G␤␥ subunits, the different ACh-evoked GIRK activation time courses reflect differences in receptorcatalyzed production of G␤␥ subunits that are rate-limited by GDP release from the heterotrimeric G␣(GDP)␤␥ complex. The  FIG. 3. Receptor-dependent GIRK activation kinetics via coupling to PTX-insensitive G␣ i2 /G␣ oA chimeras. A, effects of introducing G␣ oA regions into G␣ i2(C352G) . Left panels, representative ACh-evoked GIRK currents from oocytes expressing PTX-insensitive G oii , G ioi , and G iio subunits (red traces). GIRK current activated by m2 receptors coupled to G␣ i2(C352G) is superimposed (gray traces) on each G␣ chimera-coupled response for comparison. The peak currents were normalized for kinetic comparisons. The horizontal bar above the traces indicate the 15 s period of ACh (1 M) application. Middle panels, GIRK activation time constants ( act ) derived from exponential fits of the ACh-evoked GIRK current activation time course at four different ACh concentrations. Values are the mean Ϯ S.E. (n Ն 8). Red symbols are values for the corresponding PTX-insensitive G␣ oA /G␣ i2 chimera indicated in the adjacent left panel. Activation time constants from G␣ i2(C352G) -coupled GIRK currents (gray symbols) and G␣ oA(C351G) -coupled GIRK currents (black symbols) are included for comparisons with the chimera values. Right panels, ACh dose-response relations with m2 receptor coupling to G oii , G ioi , and G iio chimeras. GIRK current (I K,ACh ) amplitudes from the application of five different ACh concentrations were normalized to the maximal current (I max ) elicited within each oocyte (10 M ACh). The mean values were fit with much faster GTP binding step, G␣(GTP)-G␤␥ dissociation step, and GIRK channel gating steps apparently do not affect the receptor-dependent GIRK activation time course under our expression conditions. Interestingly, native GIRK currents in hippocampal neurons activated by either GABA B or adenosine A1 receptors in G␣ o -knockout mice have significantly slower activation kinetics (7), analogous to the slower G␣ i -coupled GIRK currents reconstituted in Xenopus oocytes. Thus G␣ o versus G␣ i -coupled GIRK currents clearly have distinct kinetics that could impact the kinetics of GIRK-mediated postsynaptic currents in neurons and atrial myocytes. Our findings reported here indicate this is attributable to the G␣ isoform differences in GDP release kinetics. Further swapping of isoform-specific residues in the switch III loop and ␣HD will better refine and ultimately resolve all the key residues that contribute to the slower G␣ i -coupled GIRK channel kinetics, including the switch III leucine residue (5).
Receptor-independent Basal GIRK Channel Activity and G␣specific GDP Release Rates-Receptor-independent basal GIRK channel activity is largely due to the high level of free G␤␥ FIG. 4. Effects of PTX-insensitive G␣ oA /G␣ i2 chimera expression on receptor-independent basal GIRK channel activity and receptor-evoked GIRK deactivation kinetics. A, receptor-independent basal GIRK channel activity associated with the expression of each PTX-insensitive G␣ oA /G␣ i2 chimera. Left panel, effects of introducing G␣ oA regions into G␣ i2(C352G) (open bars) compared with the parental G␣ i2(C352G) subunit (gray bar). Right panel, effects of introducing G␣ i2 regions into G␣ oA(C351G) (open bars) compared with the G␣ oA(C351G) subunit (black bar). I K,basal amplitudes are expressed as a fraction of the total GIRK current (I K,basal ϩ I K,ACh ) elicited from each oocyte expressing the indicated G␣ subunit. B, derived EC 50 values from GIRK currents elicited by a range ACh concentrations as shown in Fig. 3, via muscarinic m2 receptor coupling to each PTX-insensitive G␣ oA /G␣ i2 chimera. C, receptor-dependent GIRK activation time constants ( act ) associated with expression of each PTX-insensitive G␣ oA /G␣ i2 chimera. Time constant values are derived from exponential fits of ACh-elicited GIRK current responses with maximal receptor stimulation (100 M ACh). Data are also presented in Fig. 3  a Hill function, excluding the 100 M ACh response, which was typically lower and reflects the degree of desensitization over the course of the experiment. The red symbols and lines correspond to the PTX-insensitive G␣ oA /G␣ i2 chimera indicated in the adjacent left and middle panel. ACh dose-response curves with G␣ i2(C352G) coupling (gray symbols) and G␣ oA(C351G) coupling (black symbols) are included for comparison. B, effects of introducing G␣ i2 regions into G␣ oA(C351G) . The left, middle, and right panels are as described in Fig. 2A for the G ioo , G oio , and G ooi chimeras. All values are the mean Ϯ S.E. (n Ն 8).
subunits in Xenopus oocytes, important for regulated oocyte maturation (20,21). Exogenous expression of G␣ subunits effectively reduce I K,basal by sequestering the free G␤␥ subunits, and increasing the pool of G␣(GDP)␤␥ complexes available for receptor-dependent GIRK channel activation (13). Expression of PTX-insensitive G␣ i versus G␣ o subunits differentially reduce I K,basal , with G␣ o isoforms being more effective than G␣ i1 , G␣ i2 , or G␣ i3 subunits (4). In light of our findings reported here implicating G␣ i /G␣ o isoform differences in GDP release kinetics, we propose the I K,basal differences are due to G␣ i /G␣ o isoform differences in GDP binding affinity (5). Although the basal rate of GDP dissociation from G␣ o is ϳ4 times faster than G␣ i isoforms (14), the measured equilibrium binding affinity for GDP to G␣ o is ϳ2 times greater than G␣ i1 , indicating a faster GDP association rate as well (5). Thus under equilibrium conditions in our oocyte expression experiments, the PTX-insensitive G␣ o isoforms are expected to be more effective than the G␣ i isoforms at reducing basal GIRK channel activity by more readily occupying the GDP-bound state and sequestering free G␤␥ dimers. This notion is contrary to that originally proposed by Remmers et al. (5), who suggested the faster G␣ o GDP release rate would promote a higher level of basal G protein activity versus G␣ i1 . Our findings correlating GIRK channel basal activity, receptor-dependent GIRK activation rates, with the transient and steady-state biochemical GDP binding kinetics suggest that, alternatively, the basal G protein activity is determined instead by the steady-state GDP binding affinity.
Implications for the RGS-accelerated G Protein Cycle and GIRK Channel Gating Kinetics-RGS proteins accelerate both the activation and deactivation phase of receptor-activated GIRK currents by accelerating the GTPase activity of G␣ subunits (22,23). Yet since RGS proteins do not reduce steadystate GIRK current amplitudes during enhanced GTPase activity (signal termination), other processes must promote G protein activation to maintain steady-state GIRK current amplitudes. RGS proteins do not affect the basal (receptor-independent) GDP/GTP exchange rates for G␣ subunits in vitro (24), but do increase receptor-dependent G protein cycling (e.g. GTPase activity) in reconstituted cell membranes containing GPCRs, G proteins, and RGS proteins (25)(26)(27)(28). These findings have led to the hypothesis that the RGS-accelerated GTPase activity allows for rapid heterotrimeric G␣(GDP)␤␥ re-formation and receptor re-activation, thus enabling multiple G protein cycles from an agonist-bound receptor (25,28). This process requires 1) receptor-catalyzed GDP release to be sufficiently fast to sustain the multiple cycling with agonistbound receptor, and 2) proximity or association of the agonist-GPCR-G protein ternary complex with the effector molecule (GIRK channel) for efficient signal transduction (G␤␥ gating of the GIRK channel) over multiple G protein cycles. These events may also be facilitated by the ability of RGS proteins to increase the pool of G␣ subunits to the plasma membrane (29, 30) through a process not very well understood but requiring the RGS N-terminal domain (31).
ally dissociate during agonist activation, but instead undergo subunit rearrangements within the macromolecular complex (35). Native GPCR-G protein-GIRK channel complexes are expected to include RGS proteins (22,36). Whether RGS proteins within a GPCR-G protein-GIRK channel signaling complex affect receptor-catalyzed GDP/GTP exchange is still not clear, but plausible since RGS proteins interact with the switch domains including the switch III loop (37,38).
The potential influence of endogenous oocyte RGS proteins in our experiments is also worth noting. Preliminary studies using RGS-resistant and PTX-insensitive G␣ subunits (39) indicate endogenous oocyte RGS proteins do indeed impact receptor-activated GIRK currents primarily by slowing the deactivation phase. 2 Characterizing the kinetic properties of RGS-resistant G␣ o and G␣ i subunit coupling to GIRK channels in oocytes is currently underway and will determine whether endogenous oocyte RGS proteins selectively regulate G␣ isoform function. Despite this caveat, the dramatic effects of the G␣ i2(R145A) mutant and G␣ i2 (G oSW3 ) chimera on GIRK channel gating kinetics, together with the previous biochemical characterization of these mutations in recombinant G␣ i1 nucleotide exchange, strongly implicates intrinsic G␣ isoform differences in GDP release kinetics as the molecular mechanism involved, versus isoform-specific interactions with endogenous RGS proteins.
The impact of G␣ isoform-specific GDP release kinetics are also of interest in light of our previous observations of RGS4modulated GIRK currents coupled selectively to G␣ i and G␣ o subunits (4). These reconstitution experiments demonstrated that G␣ i -coupled GIRK currents are appreciably suppressed by RGS4 (Ͼ60%), whereas G␣ o -coupled GIRK currents are not (4). We interpreted these findings as a consequence of the difference in m2 receptor-G protein precoupling, which is better promoted by G␣ o subunits given their greater ability to sequester endogenous G␤␥ and form more functional G␣(GDP)␤␥ heterotrimers. The inability of all three G␣ i isoforms to enhance G protein signaling sufficient to maintain GIRK current amplitudes during RGS4-stimulated GTPase activity, may be a consequence of the intrinsically slower GDP release rate from G␣ i subunits. That is, under conditions where RGS4 accelerates the GTPase activity of G␣ i subunits (evidenced by faster GIRK channel deactivation rates), G protein cycling may be limited by the slow rate of GDP release from G␣ i subunits that actually prevents multiple cycling. It is not clear whether higher m2 receptor expression levels could overcome this, which would be expected to catalyze higher GDP release rates. At high levels of ␣ 2 -adrenergic receptor expression in a CHO cell line, RGS4 stimulated GTPase activity enables GDP-bound G␣ i subunits to undergo multiple rounds of activation to enhance steady-state G protein activation (28). Clearly the kinetics of GDP release and the GTPase activity of G␣ i/o subunits can each rate-limit the G protein cycle under different experimental conditions. Examining RGS modulation of G␣ oA /G␣ i2 chimeras and G␣ i2 mutants with biochemically defined GDP release kinetics will help to better understand the role G␣ isoforms, GDP release kinetics, and RGS accelerated GTPase activity on GIRK channel gating properties.
Summary-Identifying the molecular determinants responsible for the different kinetic properties of G␣ i -and G␣ o -coupled GIRK channels provides new insight and opportunity to further investigate the molecular mechanisms that control G protein activation of GIRK channels, as well as other G protein effector molecules. An appreciation of these fundamental differences will be important for a full understanding of both the molecular and cellular mechanisms that determine GPCR and RGS selectivity, an active area of investigation with broad therapeutic implications (40,41).