Interaction Sites of the G Protein beta  Subunit with Brain G Protein-coupled Inward Rectifier K+ Channel*

Abla M. Albsoul-YounesDagger §, Pamela M. Sternweis§||, Peng Zhao**, Hiroko NakataDagger , Shigehiro NakajimaDagger , Yasuko Nakajima**, and Tohru KozasaDagger ||DaggerDagger

From the Dagger  Department of Pharmacology and the ** Department of Anatomy and Cell Biology, University of Illinois, Chicago, Illinois 60612, and the || Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, December 13, 2000, and in revised form, January 11, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-coupled inward rectifier K+ channels (GIRK channels) are activated directly by the G protein beta gamma subunit. The crystal structure of the G protein beta gamma subunits reveals that the beta  subunit consists of an N-terminal alpha  helix followed by a symmetrical seven-bladed propeller structure. Each blade is made up of four antiparallel beta  strands. The top surface of the propeller structure interacts with the Galpha subunit. The outer surface of the beta gamma torus is largely made from outer beta  strands of the propeller. We analyzed the interaction between the beta  subunit and brain GIRK channels by mutating the outer surface of the beta gamma torus. Mutants of the outer surface of the beta 1 subunit were generated by replacing the sequences at the outer beta  strands of each blade with corresponding sequences of the yeast beta  subunit, STE4. The mutant beta 1gamma 2 subunits were expressed in and purified from Sf9 cells. They were applied to inside-out patches of cultured locus coeruleus neurons. The wild type beta 1gamma 2 induced robust GIRK channel activity with an EC50 of about 4 nM. Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels. However, the ability of D1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta 1gamma 2. As to the activities to stimulate phospholipase Cbeta 2, D1 was more potent and CD2 was less potent than the wild type beta 1gamma 2. Additionally we tested four beta 1 mutants in which mutated residues are located in the top Galpha /beta interacting surface. Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels. These results suggest that the outer surface of blade 1 and blade 2 of the beta  subunit might specifically interact with GIRK and that the beta  subunit interacts with GIRK both over the outer surface and over the top Galpha interacting surface.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heterotrimeric G proteins transduce a variety of regulatory signals from a large number of heptahelical receptors to effectors such as adenylyl cyclases, phosphodiesterases, phospholipases, and ion channels (1). Each G protein oligomer contains a guanine nucleotide binding alpha  subunit and a high affinity dimer of beta  and gamma  subunits. The agonist-bound receptor activates the G proteins and generates GTP-bound alpha  subunits and free beta gamma subunits. Both GTP alpha  and beta gamma can regulate downstream effectors. The hydrolysis of GTP to GDP on alpha  subunits leads to the reassociation of alpha  and beta gamma subunits to form inactive heterotrimers.

The crystal structure of the beta gamma subunit reveals that the beta  subunit consists of an N-terminal alpha  helix followed by a symmetrical seven-bladed propeller structure based on WD repeat sequences, repeating motif of about 40 amino acids (2, 3). Each blade consists of four antiparallel beta  sheets. The top surface of the propeller structure interacts with the alpha  subunit. The bottom surface of the torus is the major site for interaction with the gamma  subunit. The outer surface of the torus is largely made up of the outer beta  strands of seven blades. beta gamma subunits directly regulate various effectors, including phospholipase Cbeta s (PLCbeta s),1 adenylyl cyclases, and ion channels. Experiments using mutated beta  subunits have demonstrated that some of the amino acid residues, which are located at the interaction sites between Galpha and -beta subunits, are crucial for the interaction between beta gamma and the effectors, and each effector demonstrates its specific domain of interaction on the beta  subunit (4, 5). Further mutational analysis showed that the activation of PLCbeta 2 also involves residues in the outer strands of blades 2, 6, and 7. However, mutations of beta  subunits that affected PLCbeta 2 activity did not influence the interaction between beta gamma and type I or type II adenylyl cyclase (6). These results suggest that beta gamma interacts with effectors through both the Galpha binding surface and the outer surface of the propeller structure and that each effector is interacting with beta gamma using different regions of its outer surface.

The present study was undertaken to investigate the interaction sites of beta 1 with brain GIRK channels. We focused our investigation on the outer surface of the beta 1 subunit. We made sets of beta 1 mutants in which the amino acid residues located on or near the outermost beta  strand of each blade were replaced with the corresponding residues of the yeast beta  subunit (STE4), a most distantly related member of beta  from mammalian beta  subunits. In this study we show that mutating residues located on or near the outer strands of blade 1 and blade 2 disrupt GIRK activation by beta 1gamma 2. In contrast, the same mutations did not substantially affect the ability of beta 1gamma 2 to regulate adenylyl cyclases, indicating that no global disruption of beta 1 occurred because of the mutations. It is possible that the sites of the beta 1 subunit in blade 1 and blade 2 could specifically be involved in brain GIRK activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Sf9 Cell Culture and Construction of Recombinant Baculoviruses-- Sf9 cells were cultured in suspension in IPL-41 medium containing 1% Pluronic F68 and 10% heat-inactivated fetal bovine serum at 27 °C with constant shaking (150 rpm). The site-directed mutagenesis of beta 1 cDNA was performed using the Muta-Gene in vitro mutagenesis kit (Bio-Rad). The amino acid sequence of each mutation is shown in Fig. 3. The mutations were confirmed by sequencing the mutating region. Mutated beta 1 cDNAs were subcloned into the pVL1392 transfer vector, and the resulting plasmids were cotransfected into Sf9 cells with BacPac6 viral DNA linearized with Bsu36I (CLONTECH) by LipofectAMINE (Life Technologies, Inc.). Recombinant baculoviruses were plaque-purified and amplified as described (7). Baculoviruses encoding L117A, D224S, D228R, and W332A were generated and generously provided by the late Dr. Eva J. Neer (Harvard University) (4). Recombinant baculoviruses encoding wild type beta 1, gamma 2, and His6-Galpha i1 have been described previously (8).

Purification of G Protein Subunits from Sf9 Cells-- Sf9 cells (1.5 × 106/ml) were coinfected with amplified recombinant baculoviruses encoding wild type or mutant beta 1, gamma 2, and His6-Galpha i1. Cells were harvested after 48 h, and recombinant beta gamma protein was purified from the membrane extract using an nickel-nitrilotriacetic acid column as described (8). The elute fractions from the nickel-nitrilotriacetic acid column were concentrated, and the buffer was exchanged into 20 mM NaHEPES (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and 0.5% octyl glucoside with Centricon-30 (Amicon). Recombinant Galpha i2 was purified from Sf9 cells as described previously (8).

Cultures of Locus Coeruleus Neurons-- Cultured neurons from the locus coeruleus were made from 2-4-day-old postnatal Long-Evans rats (Charles River Breeding Laboratories). The culture methods were described previously (9, 10). Rats were anesthetized with ether, their brainstems were removed, and the rats were killed by decapitation. Brain slices were made from isolated brainstems using a Vibratome (Lancer 1000). The locus coeruleus was visually identified under a dissecting microscope and excised out. The excised pieces were incubated in a papain solution, dissociated by trituration, and cultured. The culture medium contained a minimum essential medium with Earle's salts (Life Technologies, Inc., catalog no. 11430-030), modified by adding D-glucose (5 mg/ml), NaHCO3 (3.7 mg/ml), and L-glutamine (0.292 mg/ml). The medium was supplemented by heat-inactivated rat serum (2 or 5%, prepared in our laboratory), L-ascorbic acid (10 µg/ml), penicillin (50 units/ml), and streptomycin (50 µg/ml). Dissociated neurons were cultured on a squared plastic piece placed in a small well made at the center of a 35-mm culture dish. Before plating the neurons, the well, containing the plastic piece, was coated with rat collagen and a feeder layer of glia cells obtained from rat brains. The cultures were incubated at 37 °C in 10% CO2 and 90% air with saturated humidity for 13.4 ± 2.5 days (mean ± S. D.). Experiments were performed on large neurons (soma diameter, 24.8 ± 2.9 µm; mean ± S.D.).

Electrophysiology-- Electrophysiological experiments were performed with the inside-out patch clamp techniques (11, 12). The patch pipettes were made from thoroughly washed glass capillaries. The patch pipette solution (external solution) contained 156 mM KCl (or potassium gluconate), 2.4 mM CaCl2, 1.3 mM MgCl2, 0.5 µM tetrodotoxin, and 5 mM HEPES-NaOH (pH 7.4). The GDP-containing cytoplasmic side solution (the bathing solution) contained 141 mM potassium gluconate, 8.7 mM NaCl, 5 mM EGTA-KOH, 1 mM MgCl2, 2 mM Na2ATP, 0.1 mM GDP, 5 mM HEPES-KOH, and ~5.5 mM KOH (pH 7.2). Membrane potential was corrected for the liquid junction potential between the bathing solution and the patch electrode solution.

The procedures of data analysis were similar to those previously described (12). The data were analyzed with pCLAMP programs (version 6) (Axon Instruments). The overall frequency response was set at 1 kHz (-3 db by an 8-pole Bessel filter) and digitized at 10 kHz. Transitions between the closed and the open states were registered if a level crossed a threshold and lasted for more than 100 µs. The threshold was set at 2.0 pA from the baseline when the membrane was held at -101 mV (1.6 pA when the membrane was held at -80.4 mV); this procedure excluded most of the small background channels (12). We expressed the channel activity by a variable NPo (i.e. the open probability of an elementary channel multiplied by the number of channels in the patch).

The experimental protocol was as follows. After an inside-out patch was formed, the basal channel activity (in the GDP-containing bathing solution) was recorded for a few minutes. The solution in the bath (~0.1-0.17 ml) was then exchanged with 0.5 ml of various kinds of beta gamma subunits dissolved in the same GDP-containing bathing solution. The exchange was done manually by using a pipetter wrapped up with a grounded aluminum foil. The degree of solution exchange was tested by using osmolarity changes as an indicator; the test showed that this manual method resulted in the replacement of about 86% of the original solution (using the bath volume of 0.15 ml).

When the inside-out patch configuration was established, usually the patch produced a very infrequent activity of GIRK channels (basal GIRK activity) and an activity of channels of small amplitude (of unknown origin) in the GDP-containing bathing solution. In some patches, the occurrence of large flickering channels (about 100 picosiemens with [K+]o of 156 mM), whose activity was not dependent on GTP, was observed (12). Frequent occurrence of these large background channels hindered the analysis of GIRK channel, and thus such patches were not included in our sample. Occasionally, instead of an inside-out patch, vesicle formation occurred. (The vesicle formation was inferred by the appearance of a current drooping because of the existence of membrane resistance and capacity on the opposite side of the patch.) (11). These patches were not included. Sometimes we observed rather vigorous basal activity of GIRK-like channels in the GDP-containing bathing solution before the application of beta gamma . This basal activity might have originated from the local presence of overexpressed beta gamma s or from the basal activity of the receptors. Because the present objective was to analyze the GIRK channel activation induced by the application of exogenous beta gamma proteins, we did not pursue the investigation of the patches with a high frequency basal activity (more than about once per second).

Stock solutions of beta gamma proteins were diluted with the GDP-containing bathing solution to a final concentration of 1-100 nM. At 10 nM Gbeta gamma , the solution contained the following buffer/detergent: 0.0033% octyl glucoside, 0.066 mM HEPES, 0.0033 mM EDTA, and 0.24-2.1 µM dithiothreitol. Experiments were done with a bath temperature of ~21 °C.

Statistical Treatment of Electrophysiological Data-- The distribution pattern of NPo was almost always non-Gaussian; this can be inferred from substantial discrepancies between the mean and the median values (see Figs. 4A and 6). This non-Gaussian distribution suggests the possible existence of more than one type of channel (e.g. solitary channels and aggregates of channels). Because the mean value in our samples is influenced greatly by a small number of patches with a large NPo, the median was a more appropriate parameter to represent the channel activity of a group. Also, statistical comparisons of NPo were done, unless otherwise noted, by using the nonparametric statistics (the Kruskal-Wallis ANOVA and a posttest using the Mann-Whitney with the Bonferroni adjustment).

We used two different types of wild type beta 1gamma 2: with and without hexahistidine tagging at the N terminus of beta 1. The median value of NPo for the channel activated by the hexahistidine-tagged wild type beta 1gamma 2 was 0.109 (n = 47), and the median value of NPo by nontagged beta 1gamma 2 was 0.099 (n = 35) (difference not significant; p > 0.5). We will refer to these two types simply as beta 1gamma 2.

In Vitro Assays for Phospholipase C and Adenylyl Cyclase Activity-- Phospholipase C activity was measured using sonicated micelles containing 50 µM phosphatidylinositol 4,5-bisphosphate, 500 µM phosphatidylethanolamine, and [inositol-2-3H]phosphatidylinositol 4,5-bisphosphate (PerkinElmer Life Sciences) (2500 cpm/assay) in a solution containing 50 mM NaHEPES (pH 7.5), 0.42 mM EDTA, 3 mM EGTA, 2 mM MgCl2, 1.7 mM CaCl2, 42 mM NaCl, 47 mM KCl, 4 µM GDP, 0.125 mg/ml bovine serum albumin, 1 mM dithiothreitol, and 0.375% octyl glucoside with 0.1 nM PLCbeta 2 and the indicated amount of beta gamma subunit. The mixture was incubated at 30 °C for 8 min, and the amount of IP3 generated was quantitated as described (4).

To measure adenylyl cyclase activity, purified beta gamma subunits were reconstituted with 10 µg of membranes from Sf9 cells expressing type I or type II adenylyl cyclase for 3 min at 30 °C in a final volume of 20 µl. Assays were then performed for 7 min at 30 °C in a total volume of 50 µl containing 4 mM MgCl2 and 0.2% octyl glucoside as described (4).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of GIRK Channels by beta 1gamma 2 in Locus Coeruleus Neurons-- When a gigaseal was formed in the on-cell mode in locus coeruleus neurons, some channel activity was usually observed. Upon making an inside-out patch, this activity started to subside, reaching a low level within a minute (presumably because the intracellular GTP was washed away in exchange of the GDP-containing solution). We then applied wild type beta 1gamma 2 (10 nM), which induced, after a latency, vigorous channel activity (Fig. 1A) (13, 14). The channels showed typical GIRK-like characteristics (12, 14) with a chord conductance of ~30-35 picosiemens, exhibiting a mixture of short openings and long openings, the latter sometimes showing bursts (Fig. 1A, record b). The current-voltage relationship of the channels showed an inward rectification (Fig. 2A). Thus, these channels activated by beta 1gamma 2 in locus coeruleus neurons probably belong to the GIRK (Kir3) subfamily (15, 16).


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Fig. 1.   Effects of wild type beta 1gamma 2 and mutant D1 on GIRK channels from locus coeruleus neurons; single channel recordings using the inside-out mode. A, the time course of GIRK channel activation (NPo) induced by wild type beta 1gamma 2. Each circle represents the NPo every 30 s. The thick, solid horizontal line indicates the period when the GDP-containing control solution (Ctr) was exchanged with wild type (Wt) beta 1gamma 2 (10 nM)-containing solution. About 2 min later, the NPo started to increase. The upper left panel (a) shows a segment of the record during the control period (Ctr). Openings of GIRK channels were observed very infrequently; the record was taken 120 s after the beginning of the record (see the main graph). The upper right panel (b) shows that the application of wild type beta 1gamma 2 (Wt) produced robust GIRK channel activity. The record was taken 501 s after the start of the record. The downward direction corresponds to inward currents. B, lack of activation of GIRK channels by the application of D1 (10 nM). The thick solid horizontal line indicates the period when the control cytoplasmic side solution (Ctr) was exchanged with a solution containing 10 nM D1. The upper left panel (a) shows a record segment during the control period (Ctr); the record was taken 159 s after the start of the record. The upper right panel shows a record (b) after the application of D1 (10 nM) (465 s after the start of the record). In both A and B, the holding potential was -101 mV, and the patch pipette contained the 156 mM KCl solution.


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Fig. 2.   A, current-voltage relation of GIRK channels from locus coeruleus neurons; single channel recordings using the inside-out mode. The channel activity was induced by 10 nM wild type beta 1gamma 2. The solid line is the fit of the data to a second order polynomial. Five patches (represented by different symbols) were used. The inset records show single channel currents at different membrane potentials. The experiment was done in the 156 mM potassium gluconate pipette solution. B, dose-response relationship of wild type beta 1gamma 2. For each patch, two or three different concentrations (including the standard concentration, 10 nM) of wild type beta 1gamma 2 were applied in ascending order. The channel activity was determined by averaging NPo during the 5-9 min of introducing a new concentration of beta 1gamma 2 (except that values at zero concentration were determined by NPo during 1 min before introducing beta 1gamma 2). For each patch, the activity was normalized to the NPo value at the standard concentration (10 nM). The mean NPo at the standard concentration (solid square; 100%) was 0.265. The vertical lines are S.E. The continuous line is drawn by fitting to a logistic equation using EC50 = 3.78 nM and Hill's coefficient = 2.03. The number of patches was 7 at 30 nM, 22 at 10 nM, 7 at 3 nM, 5 at 1 nM, and 5 at 0.3 nM.

10 nM Is an Almost Saturated Concentration for beta 1gamma 2-- Fig. 2B shows a concentration-response relation in wild type beta 1gamma 2. Values of NPo over 5 to 9 min after the introduction of beta 1gamma 2 were plotted. The wild type beta 1gamma 2 activated the brain GIRK with an EC50 of about 4 nM, and the activation almost saturated with 10 nM. These results are in approximate agreement with previous studies on cardiac and cloned GIRK channels (17-20).

beta 1 Mutations at Outer Blades: K+ Channel-- Within the beta  subunit family, the STE4 gene product of Saccharomyces cerevisiae is most distantly related to mammalian beta  subunits. So far, no evidence has been presented to suggest the regulation of adenylyl cyclase, PLC, or K+ channel activity by yeast beta gamma subunits. Therefore, we suspected that exchanges of the effector-interacting domains of the beta  subunit with the corresponding sequences from STE4 would result in mutant proteins, which are incapable of interacting with mammalian effectors. Indeed, Peng et al. (21) have recently reported that STE4 does not activate GIRK channels.

To test the hypothesis that regions on the side surface of the beta gamma torus are important for the regulation of effectors, we mutated residues of an outer strand of each blade into the corresponding sequence of STE4 (Fig. 3). Each beta  mutant was coexpressed with His6-Galpha i1 and gamma 2 in Sf9 cells, and mutant beta gamma subunits were purified as described under "Experimental Procedures."


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Fig. 3.   Mutation of outer blades of beta 1 subunit. The amino acid sequence of bovine beta 1 (beta 1) is aligned with the corresponding STE4. For each type of mutation (D1 through D7, except D2R), the amino acid sequence of the outer blade of the bovine beta 1 subunit (underlined) was replaced with the corresponding sequence of STE4 (aligned underneath, underlined). WD represents WD repeat motif. Because blade 5 (WD 6) is interacting extensively with the gamma 2 subunit, it was excluded from the mutational analysis. In mutant D2R, valine was changed into glycine; this mutation has been shown to inhibit the ability of STE4 to transmit mating signaling (25). The names of the mutants are indicated in bold letters above the sequence (such as D1 and CD2). The empty parentheses in WD6 of STE4 represents the following sequence: (LFRGYEERTPTPTYMAANMEYNTAQSPQTLKSTSSSYLDNQ).

First, we surveyed all these outer blade mutants (eight mutants altogether) for their ability to activate GIRK channels. We used beta 1gamma 2 mutants at a concentration of 10 nM, which is, for the wild type beta 1gamma 2, an almost saturating dose for activating the K+ channel (Fig. 2B). The variability of NPo among different patches was quite large (see "Experimental Procedures") (Fig. 4A). Nevertheless, the mutant D1 clearly showed far less ability to activate the GIRK channel than did the wild type (p < 0.001) (Figs. 1B and 4A). The mutant CD2 was also significantly less effective in activating the channels compared with the wild type (p < 0.05). Mainly because of the large variation of NPo, we could not obtain significant differences in NPo between the wild type and each of the other mutants (D2F, D2R, D3, D4, D6, and D7) (Fig. 4A). We, therefore, focused on D1 and CD2 mutants and tested a higher concentration for their ability to activate GIRK channels. Even at 100 nM, both D1 and CD2 produced only a small amount of channel activity. Comparison of the channel responses by D1 and CD2 with the wild type dose-response curve clearly indicates the impaired ability of these two mutants (CD1 and CD2) to activate the K+ channel (Fig. 4B).


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Fig. 4.   A, effect of replacing residues on the outermost strands of beta 1 with the corresponding sequences of yeast STE4 on GIRK activity in locus coeruleus neurons. The thick horizontal lines represent median values, and the heights of the columns represent mean values of NPo. The vertical lines represent S.E. The number in parentheses indicates the number of patches; for each patch, the channel activity was determined by averaging NPo during the 5-9 min after introducing various types of beta 1gamma 2. The mean basal activity (before applying various types of beta 1gamma 2) was 0.0051 in NPo (n = 74) and mainly originated from the small background channel activity (12). The 156 mM KCl pipette solution was used. Comparison with the wild type: *, p < 0.05 (p = 0.035); ***, p < 0.001 (nonparametric ANOVA and the posttest). B, comparison between wild type dose-response relation with the responses to D1 and CD2 mutants at 10 and 100 nM. Square symbols represent the dose-response relationship of the wild type. For each patch, the data were normalized to the response at the standard beta 1gamma 2 concentration (10 nM). This figure was derived from the same experiment as in Fig. 2B; here, NPo was averaged during the 5-7 min (instead of 5-9 min) of the solution exchange (see legend for Fig. 2B for details). Circles and triangles, respectively, represent data for D1 and CD2 mutants (at 10 and 100 nM) obtained in separate experiments from those in A. The response (NPo) was averaged during the 5-7 min of the exchange of the solution. The data were not normalized. Solid symbols represent the mean values, whereas the open symbols represent the median values. For D1 at 10 nM, S.E. = 0.010 and n = 9. For D1 at 100 nM, S.E. = 0.007 and n = 7. For CD2 at 10 nM, S.E. = 0.024 and n = 7. For CD2 at 100 nM, S.E. = 0.0167 and n = 5. The mean of NPo for the wild type obtained during the D1 experiments was 0.105 (median 0.067; n = 18). The mean obtained during the CD2 experiments was 0.145 (median 0.106; n = 16). The difference between 10 nM wild type and 10 nM D1 was significant (p < 0.01; nonparametric ANOVA and posttests). The difference between 10 nM wild type and 10 nM CD2 was marginally significant (p = 0.13). Because we performed two independent tests on the differences between wild type and CD2 (10 nM) (A and B), we calculated the overall significance level by which wild type and CD2 (10 nM) are different; it gives p = 0.03. In contrast, no significant differences were obtained (ANOVA, posttest) between 10 nM D1 and 100 nM D1 or between 10 nM CD2 and 100 nM CD2.

beta 1 Mutations at Outer Blades: Adenylyl Cyclases and PLCbeta 2-- Despite the impaired ability of D1 and CD2 to activate GIRK channels, these two mutants were as active as the wild type to stimulate type II adenylyl cyclase (Fig. 5A). They could also inhibit type I adenylyl cyclase, similarly to wild type beta 1gamma 2 (data not shown). Both D1 and CD2 were capable of stimulating PLCbeta 2. The mutant D1 was, however, more active and the mutant CD2 was less active than the wild type within the concentration range of the assays (Fig. 5B).


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Fig. 5.   Activation of type II adenylyl cyclase and PLCbeta 2 by purified mutant beta gamma subunits. A, the indicated amount of each beta gamma mutant was reconstituted with 10 µg of Sf9 cell membrane expressing type II adenylyl cyclase in the presence of 100 nM GTPgamma S-Gsalpha . Adenylyl cyclase activity was measured as described under "Experimental Procedures." B, the indicated amount of each beta gamma mutant was reconstituted with 0.1 nM PLCbeta 2, and the synthesis of IP3 was measured over 8 min at 30 °C as described under "Experimental Procedures." The data of each panel show the average of duplicate determinations from a single experiment that is representative of three such experiments. The vertical bars indicate S.E.

Effects of Detergent-- We also tested whether channel activities were affected by the detergent in which the protein was dissolved. We tested the buffer with 0.033% octyl glucoside, 0.66 mM HEPES, 0.033 mM EDTA, and 6-17 µM dithiothreitol, corresponding to those used for 100 nM beta 1gamma 2. This buffer alone did not induce channel activity during ~10 min of application (mean NPo: 0.000189 before application; 0.000232 after application; n = 5; p > 0.7; paired t test). We also compared the effect of 10 nM wild type beta 1gamma 2 in the standard buffer with that in the high detergent buffer. Again, no marked difference was observed. The NPo during the 4-6 min after the start of application of the wild type beta 1gamma 2 in the standard buffer was 0.233 (mean, n = 7), and that in the high buffer/detergent was 0.14 (n = 5). (The medians were 0.065 and 0.17, respectively; p > 0.6.)

beta 1 Mutations at Galpha /beta Interaction Sites-- beta 1 mutants (L117A, D228R, D246S, and W332A), which are mutated at a residue of the interface between beta gamma and alpha  subunits, were previously tested for their ability to regulate PLCbeta s and adenylyl cyclases (4). We also tested these four mutants at 10 nM for their ability to activate the GIRK channels. Fig. 6 summarizes the values of NPo during the 5-9 min after the start of beta 1gamma 2 application. Two of these mutants (D246S and W332A) produced channel activity significantly lower than the wild type (p < 0.01 and p < 0.001, respectively).


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Fig. 6.   Effect of mutating some of the Galpha /beta interaction sites on GIRK activity in locus coeruleus neurons. The thick horizontal lines represent the median values, and the heights of the columns represent the mean values of NPo. The vertical lines represent S.E. The number in parentheses indicates the number of patches. For each patch, the channel activity was determined by averaging NPo over 5-9 min after introducing various types of beta 1gamma 2 (wild-type and mutants). The mean basal activity (NPo) (before applying various types of beta 1gamma 2) of this sample was 0.0057 (n = 69). The 156 mM KCl pipette solution was used. Comparison with the wild type: **, p < 0.01; ***, p < 0.001 (nonparametric ANOVA and posttest).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GIRK channels are activated directly by the G protein beta gamma subunit. The interaction sites of beta gamma with GIRK have been investigated on the Galpha interacting surface of the beta gamma subunit (5). In the present study, we have demonstrated that regions outside of the Galpha /beta interaction surface of the beta  subunit also participate in the interaction with GIRK channels. We characterized the interaction of beta  with brain GIRK by using beta 1 mutants on the outer strands of the seven-bladed beta -propeller structure. Mutations of certain residues on the outer strands of blade 1 (D1) and blade 2 (CD2) resulted in the severe disruption of their ability to activate GIRK channels. However, these mutants could regulate adenylyl cyclases similarly to the wild type, suggesting that the mutations of the D1 and CD2 areas did not produce a global disruption of beta 1 structure. Thus, the results suggest that the mutated residues on the side of blade 1 and blade 2 of the beta gamma torus might be specifically involved in the regulation of GIRK channels.

It should be noted that in this study, we have concentrated on analyzing mutations that produced a large functional deterioration in the K+ channel activation. This study does not exclude the possibility that other mutants may have a moderate defect in their ability to interact with K+ channels.

It was previously shown that the activation of PLCbeta 2 involves the outer strands of blades 2, 6, and 7 (6). Interestingly, CD2 also showed a defect in its ability to activate PLCbeta 2. The results further support the importance of blade 2 for the interaction with PLCbeta 2. As shown in Fig. 5, D1 was 2-3-fold more potent than the wild type in its ability to stimulate PLCbeta 2. The exact reason for this difference is currently unclear.

Our results on beta  mutations over the Galpha /beta interaction surface indicate that W332A showed far less ability to activate GIRK channels. Although only one concentration (10 nM) of beta 1gamma 2 was tested, we observed a very large difference in their activity between W332A and the wild type. Because 10 nM is an almost saturating dose for the wild type, this result suggests that W332A is much less active than the wild type in its ability to stimulate GIRK channels. Thus, beta /GIRK interaction sites partially overlap with the Galpha /beta interaction sites, but they are not identical. This is similar to the case of beta  subunit interaction with PLCbeta 2 or adenylyl cyclase (4, 5).

It is known that beta gamma can interact with effectors only if Galpha is dissociated from beta gamma . X-ray crystallographic studies have shown that the beta  subunit does not undergo conformational changes when it is dissociated from Galpha (3). It has been demonstrated that GDP-bound Galpha could sever the beta /GIRK association quickly (22, 23). Because the interaction sites of GIRK overlap with the Galpha interaction surface on Gbeta gamma , this effect of Galpha could be explained by a simple spatial (three-dimensional) competition on the beta  subunit between Galpha and GIRK (4). It is also possible that the association of GIRK to beta gamma induces a conformational change of the beta  subunit, and this change would favor the binding of beta gamma and GIRK. Conformational changes of the beta gamma subunit are demonstrated in the complex of beta gamma with phosducin (24). The binding of phosducin to the beta gamma subunit produces a distinct conformational change in blade 6 and blade 7 of the beta  subunit. The phosphorylation of phosducin on Ser-73 reduces its affinity for beta gamma and the released beta gamma subunit and then switches back to the conformation of free beta gamma or that of the heterotrimer (24). In the case of beta gamma complexed with GIRK, when Galpha i interacts with a certain region of Galpha /beta interaction sites, the conformation of beta gamma could return to the resting state (before GIRK was attached), and this could decrease the affinity of GIRK with beta gamma . The determination of the structure of beta gamma complexed with GIRK will be necessary to answer this question.

    ACKNOWLEDGEMENTS

We thank Dr. Alfred. G. Gilman for support and encouragement throughout this study. We thank the late Dr. Eva J. Neer (Harvard University) for baculoviruses encoding mutant beta 1 subunits (L117A, D228R, D246S, and W332A), Dr. Paul C. Sternweis for PLCbeta 2 protein and valuable comments on the manuscript, and Dr. Marlos A. G. Viana (University of Illinois at Chicago) for advice on statistics.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AG06093 (to Y. N.) and GM61454 (to T. K.) and American Heart Association Texas Affiliate Grant 96G-105 (to T. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Present address: Dept. of Clinical Pharmacy and Pharmaceutical Technology, College of Pharmacy, University of Science and Technology, Irbid, Jordan.

Dagger Dagger To whom correspondence should be addressed. 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-413-0111; Fax: 312-996-1225; E-mail: tkozas@uic.edu.

Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011231200

    ABBREVIATIONS

The abbreviations used are: PLCbeta , phospholipase Cbeta ; GIRK, G protein-coupled inward rectifier K+; ANOVA, analysis of variance; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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