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Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways
Neuron,
Vol. 12, 109-116,
January,
1994, Copyright
0 1994 by Cell Press
Bradykinin Modulates Potassium and Calcium Currents in Neuroblastoma Hybrid Cells via Different Pertussis Toxin-Insensitive Pathways M. A. Wilk-Blaszczak, S. Gutowski, and F. Belardetti Department of Pharmacology University of Texas Southwestern at Dallas Dallas, Texas 75235
P. C. Sternweis,
Medical
Center
Summary In NGlOB-15 cells, bradykinin (BK)activatesapotassium current (Ix,Bx) and inhibits the voltage-dependent calcium current (I&. BK also stimulatesa phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK,through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by C, and/or GI1, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of G@,, in the electrical responses to BK. GTPTS mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-G@,,. antibody into the pipette suppressed IK,BK, but not the inhibition of Ica,” by BK. Thus, BK couples to Ix,~x via GM,,, but coupling to IcI,v is most likely via a different, pertussis toxin-insensitive G protein. Introduction CTP-binding proteins (G proteins) are membraneassociated proteins that couple receptors for neurotransmitters and hormones to effector proteins. They have been implicated in the regulation of ion channel activities by many neurotransmitters (Brown, 1990; Sternweis and Pang, 1990; Hille, 1992). Although the putative G proteins in many of these pathways have been characterized with respect to their sensitivity to the bacterial toxins, pertussis toxin (PTX) and cholera toxin, the identification of the specific G proteins utilized in vivo has been rare. This is especially true for the systems that are insensitive to PTX. The receptor-sensitive G proteins are heterotrimers of three unique subunits (Gilman, 1987; Ross, 1989; Birnbaumer, 1990). The a subunit contains the guanine nucleotide-binding site and, when present, the ADP ribosylation sites for the bacterial toxins. When receptors bind agonists, they can activate G proteins by catalyzing the exchange of GTP for GDP bound to the a subunit of the G protein. This activation also promotes dissociation of the a from its by subunits, thus generating two potential regulatory molecules. The a subunit is generally thought to provide the specificity for coupling to the effector, although recently defined actions of the 8~ subunits indicate that the
picture may be more complex (Sternweis and Smrcka, 1992; Birnbaumer, 1992; tiiiguez-Lluhi et al., 1993). Several types of novel a subunits that lack sites for ADP ribosylation by PTX have recently been identified (Simon et al., 1991; Hepler and Gilman, 1992). Two of these, the highly homologous G,, and GIla, have been shown to regulate phosphatidylinositol-specific phospholipase C (PI-PLC) activity in vitro (Smrcka et al., 1991; Gutowski et al., 1991; Taylor et al., 1991) and mobilization of calcium in vivo (LaMorte et al., 1993). The importance of G, or G,, in mediating the PTXinsensitive regulation of several ion channels has not been established. In the neuroblastoma-glioma hybrid cell line, NG108-15 (Hamprecht et al., 1985), bradykinin (BK) transiently activates a potassium current (IK,BK;Brown and Higashida, 1988a, 1988b) and inhibits the voltagedependent calcium current (Ica,“; Brown and Higashida, 1988b). In the same cells, BK stimulates PI-PLC activity (Yano et al., 1984) via the G,,,, proteins (Gutowski et al., 1991). PI-PLC generates inositol 1,4,5trisphosphate (IP3, which in turn raises intracellular calcium. This BK-triggered calcium signal is thought to activate a calcium-dependent potassium current, which would account for, or at least contribute to, theobserved IK,BKin thesecells (Brown and Higashida, 1988a, 1988b, 1988c). The PTX sensitivity of potassium channel regulation by BK in NG108-15 cells is controversial. One group has reported that the inhibitory responses elicited by BK, as well as stimulation of GTPase activity by BK, are sensitive to PTX; this led to the proposal that G, mediates the response to BK (Higashida et al., 1986). However, other groups have claimed that the stimulation of GTPase by BK (Grandt et al., 1986) and the pathway that mediates Ica,v inhibition by BK (Taussig et al., 1992) are insensitive to PTX. Furthermore, in NG108-15 cells (Gutowski et al., 1991) and in a related cell line (Jackson et al., 1987), activation of PI-PLC by BK was PTX insensitive. A third action of BK is to produce long lasting increases of excitability in NG108-15 cells. This is thought to be mediated by inhibition of the potassium M-current (Brown and Higashida, 1988b; Robbins et al., 1992). Under our experimental conditions, this action was modest and highly variable (Schafer et al., 1991; see also Discussion in Robbins et al., 1993). In addition, the modulation of the M-current may have also been obscured by the simultaneous activation of an inward current, not previously described (see Figure 2Q. This effect of BK was not studied further. In the present work, we have used whole-cell recording and perfusion with a specific antibody (Gutowski et al., 1991) to investigate the role of G,,,, in mediating the activation of IK,BKand the inhibition of I ca.v by BK.
B
B
30s
4
BK
Cl
c2
+
GTP’)‘S
,... .,. COll
Wash 1Oms
Figure cludes
1. The Perfusion with GTPyS the Responses to BK
First Mimics
and Then
Oc-
(A) Rapid activation of IK,BKby BK (IO nM) in a cell dialyzed with GTP (0.1 mM) for more than 10 min. (B,-B,) Slow activation of IK,~K in 2 cells. The pipette was perfused with CTPyS (0.25 mM), and the perfusion was started 20 s after the beginning of the record (left). In the first cell shown (B,-BZ), BK was applied after 24 min of perfusion with GTPyS. In the second cell (B,), BK was applied at the peak of the GTPyS effect. The arrows (A-B3 mark the application of BK. The continuous lines mark the zero current. (C,-C2) Effect of the pipette perfusion with GTPyS (0.5 mM) on the peak ICa,” and on its modulation by BK. The continuous line marks the zero current. (C,) Effect of BK (IO nM) on Ica,” when applied after 3 min of GTPyS perfusion. Three overlapping traces are shown, before (Con), during (BK), and after (Wash) application of BK. (CZ) In the same cell shown in (C,), BK is applied again (IO nM) after 20 min of perfusion with GTPyS. Two overlapping traces are shown, one before (Con) and the other during (BK) BK application. The voltage-clamp protocols are described in Experimental Procedures.
Results Evidence that G Proteins Account for Both Types of Responses to BK Nonhydrolyzable analogs of GTP, such as GTPyS, slowly and irreversibly activate G proteins even in the absence of receptor stimulation (Gilman, 1987). Thus, the observation that GTPyS can irreversibly mimic and occlude a response to agonists would be consistent with the involvement of a G protein in that response. After establishing the whole-cell configuration with normal ionic conditions, the pipette was slowly perfused over a period of about 10 min with internal medium containing GTPyS (250 PM) and no GTP. Perfu-
sion with this nucleotide consistently (12 out of 12 cells tested) produced a slow outward current, followed by a long lasting inward current (Figure IB,). GTPyS may activate the same conductance activated by BK because both responseswere accompanied by a decrease of the cell’s input resistance and had similar reversal potentials (near -70 mV). Interestingly, the action of GTPyS, much like that of BK, is self-limited, because it is followed by a slow inward shift of the current. This responsewas never observed when GTP, rather than GTPyS, was perfused in the pipette (n = 44; data not shown). If GTPyS activates a G protein that is required for the increase of IK,BK,then it should not only mimic, but also occlude, the response to BK. We tested this idea in two different ways. First, BK was applied after perfusion of GTPyS and return of the holding current to near initial baseline level (Figure IBz). The response to BK was severely reduced in all cells tested under these conditions (mean = 13 pA; range = O-40 pA; n = 8 with GTPyS), as compared with the responses in the presence of GTP (mean = 102 pA; range = O-260 pA; n = 17). It is possible that long exposure of the cells to GTPyS could lead to an indirect effect on the BK pathway, such as desensitization of the response. Therefore, we also applied the transmitter to other cells at the peak of the outward current generated by GTPyS (Figure 1B3). Under these conditions, the stimulatory response to BK was entirely suppressed in all cells tested (n = 8). If cells were dialyzed for comparable durations with normal internal medium containing GTP, the response to BK remained robust (Figure IA). We used a similar approach to investigate the involvement of a G protein in the inhibition of ICa,” by BK. After isolation of Ic~,v, perfusion of the pipette with GTPyS elicited a slow decline of the peak Ic,,” (compare the peak currents in Figures ICI and 1C2). This phenomenon occurred consistently in all cells treated with GTPyS (n = 5). Application of BK during this phase produced a faster and largely irreversible inhibition of ICa,V(in 3 out of 3 cells tested); this would be consistent with promotion of GDP-GTPyS exchange by the receptor (Figure ICI). If BK was applied to GTPyS-treated cells after the peak Ica,” had stabilized at a depressed level, the neurotransmitter had no effect (n = 3; Figure IQ. In summary, these observations are consistent with the involvement of a G protein in both the activation of IK,BKand the inhibition of ICa,” by BK. Regulation of IK,BYand IQV by BK Is Insensitive to PTX Sensitivity to bacterial toxins, such as PTX, is a very useful criterion for indicating the broad class of G proteins potentially involved in a pathway. Since the susceptibility of IK,BKto PTX in NG108-15 cells was uncertain, we tested for its sensitivity to the toxin in the NG108-15 cells used for this work. A first group of cells was treated overnight with PTX (200 rig/ml). Previous
BK Modulation
of Potassium
and Calcium
Currents
111
50
w
0
A
f s 0 100 300 Peak Response f I PA I
SC0
BK
Figure 2. Pretreatment with PTX Does Not Block the Activation of ~.BK by BK (A, and AZ) Activation of IK,BK by BK (IO nM) in 2 cells, one that had been incubated overnight with PTX (A,) and the other with boiled PTX (AI). The slightly slower time course of the response in (A?) compared with (A,) was unrelated to the treatment. (B)Cumulativedistributionsofthevaluesofthe peak I~.~~evoked by BK in cells that were treated with PTX (squares) or boiled PTX (circles), or that received no treatment (triangles). Inset: response indices (the bars are proportional to the areas under each histogram) for each type of treatment. The PTX group was not significantly different from the two other groups. (C,-C,) Activation of IK,BK by BK and block of the effect of Leuenkephalin on Ica,v in the same cell pretreated with PTX. (C,) Action of BK under normal ionic conditions. The voltage-clamp protocol was as in (A,) and (A,). (C,) Action of Leu-enkephalin (Leu-Enk), after perfusing the pipette with cesium-EGTA solution and superfusing a high calcium-TTX solution in the bath (for about 5-6 min). Voltage-clamp protocols are described in the Experimental Procedures.
work has shown that this treatment was consistently capable of reducing the modulatory responses of Leuenkephalin, norepinephrine, and somatostatin in NG108-15 cells (Taussig et al., 1992). As controls, two additional groups of cells either received no treatment or were incubated with the same concentration of boiled PTX. The response to BK was measured in a sample of cells from each group. Examples of responses to BK after treatment with either active or boiled PTX are shown in Figures 2A1 and 2Az, respectively. Figure 2B summarizes activations of IK,IJK by BK. Cumulative histograms of responses show cells that were untreated (n = 7) ortreated with either PTX(n = IO) or boiled PTX (n = 7), as indicated. The corresponding response indices are shown in the inset. Direct inspection and statistical comparison of the distribution of the responses under the different conditions clearly indicate that treatment with PTX had no significant
effect on the regulation of II(,BK by BK (PTX versus boiled: p = 0.9; PTX versus no treatment: p = 0.8; Log-Rank Test). To demonstrate that the treatment of PTX was effective, we also measured the effect of Leu-enkephalin on these same cells. Inhibition of ICa,” by Leuenkephalin is a well-defined PTX-sensitive response (Hescheler et al., 1987; Taussig et al., 1992). After treatment with PTX, cells were examined under normal conditions for measuring IK,BK; a normal response to BK was evoked (mean = 207 pA; range = 60-440 pA; n = 4; Figure 2C,). The bath was then superfused with a solution containing high calcium and tetrodotoxin (T-TX), whereas the pipette and consequently the cell interior were perfused with a solution containing cesium and EGTA. Under these conditions, a voltagedependent calcium current that was insensitive to inhibition by Leu-enkephalin (Figure 2C2) could be readily isolated. Attenuation of the response to Leuenkephalin in cells with normal activation of I~,BK by BK was a consistent finding after treatment with PTX. On average, the inhibition of ICa,” by Leu-enkephalin was 9% (range = 2%-14%; n = 4) under these conditions. In contrast, inhibition of ICa,” by Leu-enkephalin was consistent with previously reported responses (Hescheler et al., 1987; Taussig et al., 1992) in the 1 untreated cell that was tested after exposure to BK and perfusion with the solutions for measuring Ic,,v. This also indicated that the block of the Leu-enkephalin response was not due to heterologous desensitization by BK. We also confirmed our previous finding (Taussig et al., 1992) that inhibition of ICa,” by BK is insensitive to PTX (4 cells responding out of 4; data not shown). Thus, any G proteins that may mediate activation of Ix,~xand inhibition of ICa,” by BK in NC10815 cells are insensitive to PTX.
An Antibody against the C Terminus of C,, and Cl,, Specifically Blocks the Activation of IK,B~ by BK In this series of experiments, we used raised against a peptide representing the terminus of both G,, and G,,, (Gutowski to test the hypothesis that members of this of G proteins are responsible for transducing fects of BK in NG108-15. Previous work that these antibodies are specific for G+, in immunoblots and immunoprecipitation)
antibodies common C et al., 1991) subfamily the efhas shown (as seen and that
they block the activation of PI-PLC by BK in membranes from NG108-15 cells (Gutowski et al., 1991). We first examined the effect of this antibody on the activation of IK,BK by BK. The response to application of BK was tested after 20-25 min of perfusion with either anti-G q,lla antibodies or preimmune IgG. When recording from a cell treated with antibodies to Gq,llu, the response to BK was clearly suppressed (Figure 3A,), compared with that observed from a cell treated with preimmune IgG (Figure 3A2). This was a consistent observation (cells with antibodies to G,,,,,: n = 21; with preimmune IgG: n = 17), as shown in Figure
Neuron 112
I3
A, +AB
COll 0
200
1CO Peak Response
Cl
1Oms
o\
d
I I PA I
D
.~
es,...
10 20 30 40 Mar ~nh~b,t~on , % ,
0
300
50
+AB
100% x A P
-
10s COll
0
A23187
100
300
Peak Response
Figure 3. The Anti-Gqfllo tion of IK,BKby BK
Antibody
Specifically
Blocks
500 I ( pA 1
the Activa-
(A,-B) Effect of the antibody on the activation of IK,BK induced by BK. (A, and AZ) Responses to BK (IO nM) in 2 cells: one (A,) after intrapipette perfusion with the purified IgC fraction containing the anti-Gs,,,,, antibodies CAB), and the other (AZ) after perfusion with preimmune IgC (Pi).(B) Cumulativedistributions of the peak IK,8K response to BK after perfusion with antibody (squares) or preimmune IgG (circles). Inset: response indices for each condition (the bars are proportional to areas under each histogram). The star indicates a highly significant statistical difference (p < 0.001; Log-Rank Test). (C-D) Effect of the antibody on the activation of a calciumdependent potassium current induced by the calcium ionophore A23187. (C, and C1) Responses to extracellular application of A23187 (20 NM) in 2 cells, one after perfusion with the purified IgG fraction containing the antibody (Cl), the other after perfusion with preimmune IgG (CJ. (D) Cumulative responsedistributions of the peak responses to the ionophore A23187 after application of antibody (squares) or control preimmune IgC (circles). Inset: response indices for each treatment. The AB group was not significantly different from the PI group. See Experimental Procedures for voltage-clamp protocols.
38, in which cumulative histograms, including data from all of the cells, are shown. The circles represent data from cells treated with preimmune IgG, whereas the squares represent data from cells treated with the anti-Gs,,,, antibodies. The corresponding response indices to BK under each of these conditions are illustrated in the inset. The difference between these two groups was highly significant (p < 0.001; Log-Rank test). To test whether the blocking action of the antibody was due to a direct effect on the channel itself, we mimicked the activation of IK,~kby BK, by extracellular application of the calcium ionophore, A23187 (Figures 3C, and 3C2). This compound acts by raising cyto-
Figure 4. The Anti-G,,,,, of Iti,v Induced by BK
Antibody
Does
Not Block
1Oms
the Inhibition
(A) Current traces recorded from a cell after perfusion with pre immune IgG (PI) fraction, before (Con) and during (BK) application of BK. The effect of BK was fully reversible upon washing. (B) Cumulative distributions of the peak inhibition of ICa,” induced by BK, after perfusion with anti-G,,,,, antibody (AB; squares) or preimmune IgG (PI; circles). Inset: response indices for each condition (the bars are proportional to areas under each histogram). The AB group was not significantly different from the PI group. (C,-C,) Effects of BK on IK,BKand Ic.,~ in the same cell, after perfusion with anti-Gs,,,. antibody (AB). (C,) Normal ionic conditions and after pipette perfusion with the antibody. (C2) After perfusing cesium-EGTA-antibody in the pipette and superfusing high calcium-T-TX solution in the bath. See Experimental Procedures for voltage-clamp protocols.
plasmic calcium levels directly, thus bypassing the activation of PI-PLC and the release of IPS produced by BK (Pressman, 1976). Application of A23187 (20 PM) produced a response resembling that evoked by BK (Figure 3C,), as shown by their similar time courses and reversal potentials. Similar responses were obtained previously by injection of either calcium or IP3 (Brown and Higashida, 1988a). When the cells were treated with the anti-G,,n, antibody, the ionophore evoked responses of similar amplitude to those from cells treated with preimmune IgG (compare Figures 3C, and 3C2). Cumulative histograms representing all of the responses to the ionophore are shown in Figure 3D, in which the circles depict responses after preimmune IgG (n = 7), and the squares refer to cells after treatment with the antibody (n = 7). The response indices (inset) obtained in the presence of antibody or preimmune IgG confirm that the anti-Gq,,lP antibody has no effect on this response (p = 0.7; Log-Rank Test). These data are consistent with anticipated action of the antibodies on G, and indicate that G, (or G,,) is required for the activation of LK,BKby BK. In
BK Modulation 113
of Potassium
and Calcium
Currents
principle, however, the data do not rule out the unlikelypossibilityof anonspecificeffectof theantibodies on the IP3 receptor or internal calcium stores. The
Antibody
against
Cq/lla
Does
Not
Block
Inhibition
of 1ca.v by BK
We next examined whether the anti-G,,ql, antibodies would affect a second modulatory effect of BK, the inhibition of ICa,V.Under appropriate ionic conditions, this current was readily isolated (Figure 4A). The response to the application of BK was tested after 20-25 min of perfusion in the pipette with either antiGq,lla or preimmune IgG. Inhibitory responses to BK were the same from cells that were treated either with anti-Gq,,,,, antibodies (Figure 4CJ or preimmune IgG (Figure 4A). Cumulative histograms (Figure 4B) demonstrate the consistency of these observations (n = n = 8 with preimmune IgG). The 11 with anti-G+,,; corresponding response indices are shown in the inset. The differences between the two groups were not significant (p = 0.8; Log-Rank Test). The clear lack of on the inhibitory effect of the anti-G,,,,, antibodies effect of BK on the calcium current suggests that a G protein different from Cq/,, mediates this action of the transmitter. A potential artifact in this experiment is that the antibodies could be inactivated by the cesium-EGTA solution present in the pipettes. This is an unlikely possibility because previous investigators have successfully used anti-G protein antibodies dissolved in cesium-EGTA solution to block transmitter modulation of ICa,V(see for example Lledo et al., 1992). In addition, we have examined the ability of these antibodies to immunoprecipitate C,, in vitro in the presence of the solutions used to measure either IK,BK or Ica,v. The antibodies effectively precipitated Gq,lla under both conditions (data not shown). It is also possible that the negative results with the modulation of Ica,v in vivo might simply be due to poor diffusion of antibodies into the cells under the conditions used to measure Ica,“. To test this idea, the pipette was perfused initially with the normal potassium-containing pipette solution and the anti-Gq,I,a antibody. As expected, the activation of IK,BKby BK was consistently suppressed (n = 4; mean = 32 pA; range = O-70 pA; Figure 4C,), which confirmed that the antibody had effectively blocked the endogenous C&. The pipette was then perfused with a cesium-EGTA solution containing the anti-C&, antibody, whereas the bath was simultaneously superfused with high calcium, TTXcontaining solution. After isolating ICa,v, BK was consistently able to elicit a normal inhibition of this current (Figure 4CJ. On the average, BK induced an 18% inhibition (range = 8%-32%; n = 4) of ICa,vunderthese conditions (two consecutive perfusions). This value compares favorably with the 25% inhibition (range = 12%-42%; n = 8) obtained after a single perfusion with the preimmune IgG (see above). The difference between these two groups was not statistically significant (p = 0.45; Log-Rank test). Although observations made in vitro (Gutowski et al.,
1991) and in vivo (Figure 4) indicate that the antiG q,lto antibody acts specifically on Gqjll, we further tested this specificity by examining its effect on a different G protein pathway, the GoA-mediated inhibition of Ica,v, by Leu-enkephalin (Taussig et al., 1992). In all 3 cells in which anti-Gqilla antibodies were perfused, Leu-enkephalin elicited normal inhibition of the ICa,v,which was identical to that obtained in 3 cells treated with preimmune IgG (data not shown). Discussion
In NG10815 cells, one can study the specific role of G proteins in two situations: the convergent actions of multiple neurotransmitters on the same channel (Taussig et al., 1992) and the divergent actions of the same neurotransmitter on multiple channels (this work). Previous work (Taussig et al., 1992) showed that distinct PTX-sensitive G proteins selectively coupled separategroups of transmitter receptors to inhibition of Ic~,~. Here, we investigated the opposite problem, the involvement of G proteins in mediating two separate actions of BK, activation of a potassium current and inhibition of Ica,v. First, the likely involvement of a G protein in both of these actions was indicated by treatment of cells with GTPyS, which initially mimicked and then occluded responses to BK. The slow actions of GTPyS in our experiments (compare Figures IA and IB,) parallel the slow activating effect of GTPyS on Gqilt in vitro (Hepler et al., 1993). We also attempted to block the actions of BK by perfusing the pipette with GDPBS. However, in normal ionic conditions, this nucleotide consistently evoked an outward current similar to that evoked by GTPyS. Although we do not have an explanation for this observation, this direct effect of GDPBS prevented its use as a blocker of the effects of BK (see also Rasenick et al., 1989). Our second finding, that the BK-mediated activation of IK,BKand inhibition of ICa,Vwere not blocked by PTX, is clearly demonstrated in Figure 2C under conditions in which PTX was able to suppress a well-known PTXsensitive response in the same cell. The action of BK on IK,~~ was previously reported to be PTX-sensitive in this same cell type (Higashida et al., 1986). The reason for this discrepancy is not clear. However, the original study was based on voltage, not conductance measurements, and the attenuation by PTX was small: perhaps nonspecificeffectsof thetoxin could account for that modest effect. Third, we tested whether the PTX-insensitive G protein, which presumably transducestheactionsofBK,mightbeG,orG,,.Thepipette used for recording was perfused with an antibody raised against the C terminus of Gq,tla. Unexpectedly, our data indicate that Gq,ll mediates activation of IK,~K but not inhibition of Ica,v by BK. Four lines of evidence indicate that inhibition of IK,~~ by the antibodies is due to a specific block of G,,,?: Perfusion with the antibody does not affect the resting electrical properties of the cell, nor the activation of a calcium-dependent potassium current obtained with
Neuron 114
Experimental Procedures K+
ca2+
Cultures NClOB-15 (Hamprecht
cells were grown et al., 1985).
and
differentiated
as described
Antibody
Figure
5. G Protein-Mediated
Pathways
Activated
by BK
Explanations for the symbols used: BK-R: BK receptor. Two recep tor subtypes might actually be involved. G PTX-ins: C protein insensitive to PTX. Other symbols are explained in the text.
A23187. Moreover, preimmune IgG, obtained from the same animal used to obtain the anti-G,,,,,antibodies, did not suppress activation of IK,BKby BK (Figure 3). Finally, the anti-Gqllla antibody did not suppress two other G protein-mediated modulatory pathways, the inhibition of ICa,vby either Leu-enkephalin or BK. Our data also strongly indicate that a PTX-insensitive G protein distinct from Gq,,, mediates the inhibition of 1Ca.Vby BK (Figure 5). The strongest argument for ruling out a role for Gq,,, is obtained from experiments in which the anti-G,/,,, antibody was demonstrated to be available inside the cell in sufficient concentrations to block IK,BK,but had no effect on inhibition of Ica,v by BK in the same cells. It should be emphasized that, in the present work, we have restricted our study to the inhibition of Ica,vthat is present in cells dialyzed with a high concentration of calcium buffer solution. We have not investigated the possibility that BK might produce calcium-induced inhibition of Ic~,v if solutions with low calcium-buffering capacity are used in the pipette (Brown and Higashida, 1988b; Kramer et al., 1991). If present, this pathway would be parallel to the G,,,,-independent pathway studied in the present paper and would be expected to be blocked by the anti-G,/,,, antibody. In conclusion, our work shows that G~II is required for the activation of a potassium conductance by BK (Figure 5). This action of G,/,, might appear simply to reflect the involvement of PI-PLC in generating the IPJcalcium signal that opens the potassium conductance. However, recent evidence from our laboratory shows that, parallel to the IPJcalcium pathway, BK and Gq,,, activate a membrane-delimited mechanism to open the potassium conductance (Harrington, S. G., and F. B., submitted). Moreover, if a G protein different from Gd,, mediates the inhibition of Ic=,v by BK(Figure5), then it is likelythat, much likethe inhibition of IG,V by GABAe in avian dorsal root cells (DiversbPierluissi and Dunlap, 1993), protein kinase C and, in general, activation of the PIP? metabolism do not mediate the inhibition of Ica,v by BK in NG108-15 cells. Although future work will attempt to identify the G protein that mediates the inhibition of IC+V by BK, the data presented here demonstrate that PTX-insensitive G protein pathways play a significant and complex role in regulating ion channels of the nervous system.
Generation Antibodies were raised against the C-terminal portion of G,, as described (Gutowski et al., 1991). The IgG fraction was purified from serum (Harlow and Lane, 1988) and gel filtered through a C-50 sephadex column in a final buffer containing 120 mM KCI and 20 mM HEPES (brought to pH 7.4 with KOH). Cesium replaced potassium in the antibody solution used in the experiments measuring calcium current. The preimmune IgG was obtained from preimmune serum from the same animal that was used to obtam the antl-G,,,,, antibody. Chemicals BK and Leu-enkephalin (Peninsula) were stored in dry aliquots at -25°C. The calcium ionophore, A23187 (Calbiochem), was dissolved in dimethylsulfoxide and stored in aliquots (20 mM) at +4%. Both compounds were reconstituted daily in extracellular solution (see below) at their final concentration. Vigorous sonication (2 min) was applied to the ionophore solution to facilitate itsdispersal in theaqueousmedium. BKand the ionophorewere applied by pressure through a blunt pipette positioned near the cell. All of the remaining chemicals were from Sigma, except ATP, GTP, and GTPyS, which were from Boehringer Mannheim. Solutions Extracellular solution for potassium responses contained 125 mM NaCI, 5.4 mM KCI, 1.8 mM CaCl?, 1.0 mM MgC&, 20 mM HEPES, and 5.0 mM glucose (brought to pH 7.4 with NaOH); for calcium currents contained 140 mM NaCI, 5.4 mM CsCI, 10.8 mM CaC12, 1 mM MgCI,, 10 mM HEPES, 10 mM glucose, and 0.0005 mM TTX (brought to pH 7.4 with NaOH). We used calcium, rather than barium, as the charge carrier to keep potential cellular metabolic pathways in a more physiological condition. Theextracellular solution was slowly superfused (I-2 ml/min) throughout the experiment using a peristaltic pump. This superfusion was arrested during the application of BK and maximally increased immediately after. The pipette solution to record potassium responses contained 115 mM KCI, 0.1 mM MgCl2,40 mM HEPES, 3 mM ATP, and 0.1 mM GTP (pH 7.3 with KOH); the pipette solution for calcium responses contained 112 mM CsCI, 1 mM MgCl?, 10 mM EGTA, and 30 mM HEPES (with CsOH). These solutions were used to fill the pipette and the perfusion apparatus at the beginning of the experiment and were also the basis for three types of test solutions: -Antibody solution: the IgG fraction containing the anti-Gq,,lo antibody was diluted l/IO with the pipette solution. -Preimmunesolution:theIgGfractionofthepreimmuneserum was similarly diluted. -GTPyS solution: the GTP in the pipette solution was replaced by 250 pM (or 500 PM in some experiments) GTP@ The preimmune IgG fraction, diluted l/IO, was also added in some experiments. The pipette solutions containing potassium and cesium generated junction potentials of 6 mV and 2 mV, respectively. These values have not been subtracted from thevalues reported in the figures and the text.
Electrical Recordings The whole-cell version of patch clamp was used (Hamill et al., 1981). Bullet-shaped, wide-mouthed pipettes (0.5-1.5 Mn) connected to an Axopatch-IB (Axon Instruments, Foster City, CA) amplifier were employed. No series resistance compensation was applied. A 386-based IBM PC-compatible computer (Lucky Computer, Dallas, TX), connected to a TL-1 interface (Axon Instruments) and using Axon Instruments software, was employed for the generation of the voltage-clamp commands and for storing the calcium current records. The experiments measuring
BK Modulation 115
potassium back and
of Potassium
currents analysis.
were
and Calcium
recorded
Currents
on digital
tape,
for later play-
Perfusion of CTPyS and Antibodies A simplified version of the intrapipette perfusion technique was used (Tang et al., 1990). A fine polyethylene tubing (PE 10; ClayAdams, Parsippan, NJ) was gently heated in a stream of hot air and then manually pulled to produce tipswith an internal diameter around 100 urn. This tip was positioned inside the recording pipette within 200-100 urn of the pipette mouth. The other end of the tubing, outside the holder (#PC-S3-30; Wright, Guilford, CT), was immersed in a 1 ml Eppendorf tube containing pipette solution (perfusion line). At the beginning of the experiment (with pipette solution in the perfusion line and pipette), negative pressure inside the holder was created with the suction line and used to establish a seal on the cell and subsequently to break the patch. The external end of the perfusion line was then manually placed in a second Eppendorf tube, which contained one of the test solutions (see above). An air bubble was initially included in the perfusion line to insulate the pipette from the external portion of the line electrically during these manipulations. Finally, gentle suction was used to perfuse the test solution (80 ~1) over a period of IO-15 min. Analysis of the Data Our analysis of thedatacan bedivided in three phases: measurement of the response to BK, graphical representation of the data obtained, and statistical comparison between groups of data. First, activation of IK,BKby BK: the cells were held either at -40 or -50 mV and subjected to 2 s steps to -70 mV every IO s to measure the cell’s conductance. A more positive holding potential was not used because under our conditions (prolonged perfusion), it appears to produce irreversible deterioration of the cell. The response to BK was directly measured as the peak current at -40 mV. Only cells that exhibited a positive or null holding current at -40 mV at the end of the experiment were included in the analysis. To analyze the modulation of Ic..v, cells were held at -50 mV. Every 10 s, a prepulse (2 s; -90 mV) was applied, followed by a 5 ms interval and a test pulse (0 mV; 100 ms). The peak inhibition by neurotransmitters was expressed as a percentage of baseline current, after analog correction of the capacitive current and digital subtraction of capacitive and leakage currents. Only ICa,v that declined less than 5% over the duration of the experiment was analyzed. Second, the analysis of the data was complicated by the fact that cells showed a substantial variability of the size of the IK,BK activation by BK. To analyze the actions of BK, the procedure described by McGehee et al. (1992) was used. Briefly, the data were plotted as cumulative histograms. The ordinate of each point represents the percentage of responses that are larger than its value. The area underlying a particular histogram is called the response index. This yields a central value for the responses under a particular set of conditions and also contains information about their scatter. Bars proportional to the response indices obtained under different conditions allow direct comparison between groups of responses. Error bars were not used since they were inappropriate. Third, in the present work, we were faced with two statistical difficulties. First, IK,BKdesensitizes so greatly after the first application that we were forced to comparedata taken from different cells. Second, many cells did not respond to BK at all, which produced highly asymmetrical distributions of the data. The statistical comparison between groups of data was obtained using the Log-Rank test. This test is particularly powerful for the comparison of this type of data, because it is very sensitive to the tail of L-shaped distributions. The Statistical Analysis System, which contains the Log-Rank Test, was used on the local VAX 8800 computer.
Received
July 22, 1993; revised
October
25, 1993.
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Cilman, A. G. (1987). C proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615-649. Grandt, R., Greiner, C., Zubin, P., and Jakobs, K. H. (1986). Bradykinin stimulates GTP hydrolysis in NCIOB15 membranes by a high-affinity, pertussis toxin-insensitive GTPase. FEBS Lett. 796, 279-283. Gutowski, S., Smrcka, A., Nowak, L., Wu, D., Simon, M., and Sternweis, P. C. (1991). Antibodies to the as subfamily of guanine nucleotide-binding regulatory protein a subunits attenuate activation of phosphatidylinositol 4,5bisphosphate hydrolysis by hormones. J. Biol. Chem. 266, 20519-20524. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch. 397, 85-100. Hamprecht, B., Glaser, T., Reiser, G., Bayer, E., and Propst, F. (1985). Culture and characteristics of hormone-responsive neuroblastoma x glioma hybrid cells. In Methods in Enzymology (Orlando, Florida: Academic Press, Inc.), pp. 316-347. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual (Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory). Hepler, J. R., and Cilman, them. Sci. 17, 383-387.
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Acknowledgments We are indebted up the perfusion
custom-modified version of the Log-Rank software, A. S. French for the graphic software, P. Middleton, M. A. Harrington, M. L. Burleson, and S. Siegelbaum for comments on the manuscript, and K. Edwards for expert and patient secretarial assistance. This work was supported by USPHS grant GM31954 to P. S., USPHS grants CM47721 and NS25186 to F. B., and a Klingenstein Fellowship to F. 8.; F. B. is an Established Investigator of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.
to C. Hartzell for generous system, C. Oxford and
assistance in setting D. McCehee for the
Higashida, H., Streaty, R. A., Klee, W., and Nirenberg, M. (1986). Bradykinin-activated transmembrane signals are coupled via N,,
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Rasenick, M. M., Hughes, J. M., and Wang, N. (1989). Guanosine5’-0-thiodiphosphate functions as a partial agonist for the receptor-independent stimulation of neural adenylate cyclase. Brain Res. 488, 105-173. Robbins, J., Trouslard, J., Marsh, S. J., and Brown, D. A. (1992). Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells. J. Physiol. 457,159185. Robbins, J., Marsh, S. J., and Brown, D.A. (1993). On the mechanism of M-current inhibition by muscarinic ml receptors in DNAtransfected rodent neuroblastoma x gliomacells. J. Physiol. 469, 153-178. Ross, E. M. (1989). Signal sorting and amplification protein-coupled receptors. Neuron 3, 141-152. Schafer, S., Behe, P., and Meves, current in NC10815 neuroblastoma ers Arch. 418, 581-591. Simon, M. I., Strathmann, of G proteins in signal
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H. (1991). Inhibition of the M x glioma hybrid cells. Pfltig-
M. P., and Gautam, N. (1991). Diversity transduction. Science 252, 802-808.
Smrcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991). Regulation of polyphosphoinositide-specific phospholipase C activity by purified C,. Science 257, 804-807. Sternweis, connection.
P. C.., and Pang, I.-H. (1990). The Trends Neurosci. 73, 122-126.
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Sternweis, P. C., and Smrcka, A. V. (1992). Regulation of phospholipase C by G proteins. Trends Biochem. Sci. 77, 502-505. Tang, J. M., Wang, Perfusing pipettes.
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Taussig, R., Sanchez, S., Rifo, M., Cilman, A. G., and Belardetti, F. (1992). Inhibition of thew-conotoxin-sensitive calcium current by distinct G proteins. Neuron 8, 799-809. Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991). Activation of the PI isozyme of phospholipase C by a subunits of the G, class of G proteins. Nature 3.50, 516-518. Yano, K., Higashida, H., Inoue, R., and Nozawa,Y. (1984). Bradykinin-induced rapid breakdown of phosphatidylinositol 4,5bisphosphate in neuroblastoma x glioma hybrid NC10815cells. J. Biol. Chem. 259, 10201-10207.
Vol. 12, 109-116,
January,
1994, Copyright
0 1994 by Cell Press
Bradykinin Modulates Potassium and Calcium Currents in Neuroblastoma Hybrid Cells via Different Pertussis Toxin-Insensitive Pathways M. A. Wilk-Blaszczak, S. Gutowski, and F. Belardetti Department of Pharmacology University of Texas Southwestern at Dallas Dallas, Texas 75235
P. C. Sternweis,
Medical
Center
Summary In NGlOB-15 cells, bradykinin (BK)activatesapotassium current (Ix,Bx) and inhibits the voltage-dependent calcium current (I&. BK also stimulatesa phosphatidylinositol-specific phospholipase C (PI-PLC). The subsequent release of inositol 1,4,5-trisphosphate and increase in intracellular calcium contribute to IK,BK,through activation of a calcium-dependent potassium current. In membranes from these cells, stimulation of PI-PLC by BK is mediated by C, and/or GI1, two homologous, pertussis toxin-insensitive G proteins. Here, we have investigated the role of G@,, in the electrical responses to BK. GTPTS mimicked and occluded both actions of BK, and both effects were insensitive to pertussis toxin. Perfusion of an anti-G@,,. antibody into the pipette suppressed IK,BK, but not the inhibition of Ica,” by BK. Thus, BK couples to Ix,~x via GM,,, but coupling to IcI,v is most likely via a different, pertussis toxin-insensitive G protein. Introduction CTP-binding proteins (G proteins) are membraneassociated proteins that couple receptors for neurotransmitters and hormones to effector proteins. They have been implicated in the regulation of ion channel activities by many neurotransmitters (Brown, 1990; Sternweis and Pang, 1990; Hille, 1992). Although the putative G proteins in many of these pathways have been characterized with respect to their sensitivity to the bacterial toxins, pertussis toxin (PTX) and cholera toxin, the identification of the specific G proteins utilized in vivo has been rare. This is especially true for the systems that are insensitive to PTX. The receptor-sensitive G proteins are heterotrimers of three unique subunits (Gilman, 1987; Ross, 1989; Birnbaumer, 1990). The a subunit contains the guanine nucleotide-binding site and, when present, the ADP ribosylation sites for the bacterial toxins. When receptors bind agonists, they can activate G proteins by catalyzing the exchange of GTP for GDP bound to the a subunit of the G protein. This activation also promotes dissociation of the a from its by subunits, thus generating two potential regulatory molecules. The a subunit is generally thought to provide the specificity for coupling to the effector, although recently defined actions of the 8~ subunits indicate that the
picture may be more complex (Sternweis and Smrcka, 1992; Birnbaumer, 1992; tiiiguez-Lluhi et al., 1993). Several types of novel a subunits that lack sites for ADP ribosylation by PTX have recently been identified (Simon et al., 1991; Hepler and Gilman, 1992). Two of these, the highly homologous G,, and GIla, have been shown to regulate phosphatidylinositol-specific phospholipase C (PI-PLC) activity in vitro (Smrcka et al., 1991; Gutowski et al., 1991; Taylor et al., 1991) and mobilization of calcium in vivo (LaMorte et al., 1993). The importance of G, or G,, in mediating the PTXinsensitive regulation of several ion channels has not been established. In the neuroblastoma-glioma hybrid cell line, NG108-15 (Hamprecht et al., 1985), bradykinin (BK) transiently activates a potassium current (IK,BK;Brown and Higashida, 1988a, 1988b) and inhibits the voltagedependent calcium current (Ica,“; Brown and Higashida, 1988b). In the same cells, BK stimulates PI-PLC activity (Yano et al., 1984) via the G,,,, proteins (Gutowski et al., 1991). PI-PLC generates inositol 1,4,5trisphosphate (IP3, which in turn raises intracellular calcium. This BK-triggered calcium signal is thought to activate a calcium-dependent potassium current, which would account for, or at least contribute to, theobserved IK,BKin thesecells (Brown and Higashida, 1988a, 1988b, 1988c). The PTX sensitivity of potassium channel regulation by BK in NG108-15 cells is controversial. One group has reported that the inhibitory responses elicited by BK, as well as stimulation of GTPase activity by BK, are sensitive to PTX; this led to the proposal that G, mediates the response to BK (Higashida et al., 1986). However, other groups have claimed that the stimulation of GTPase by BK (Grandt et al., 1986) and the pathway that mediates Ica,v inhibition by BK (Taussig et al., 1992) are insensitive to PTX. Furthermore, in NG108-15 cells (Gutowski et al., 1991) and in a related cell line (Jackson et al., 1987), activation of PI-PLC by BK was PTX insensitive. A third action of BK is to produce long lasting increases of excitability in NG108-15 cells. This is thought to be mediated by inhibition of the potassium M-current (Brown and Higashida, 1988b; Robbins et al., 1992). Under our experimental conditions, this action was modest and highly variable (Schafer et al., 1991; see also Discussion in Robbins et al., 1993). In addition, the modulation of the M-current may have also been obscured by the simultaneous activation of an inward current, not previously described (see Figure 2Q. This effect of BK was not studied further. In the present work, we have used whole-cell recording and perfusion with a specific antibody (Gutowski et al., 1991) to investigate the role of G,,,, in mediating the activation of IK,BKand the inhibition of I ca.v by BK.
B
B
30s
4
BK
Cl
c2
+
GTP’)‘S
,... .,. COll
Wash 1Oms
Figure cludes
1. The Perfusion with GTPyS the Responses to BK
First Mimics
and Then
Oc-
(A) Rapid activation of IK,BKby BK (IO nM) in a cell dialyzed with GTP (0.1 mM) for more than 10 min. (B,-B,) Slow activation of IK,~K in 2 cells. The pipette was perfused with CTPyS (0.25 mM), and the perfusion was started 20 s after the beginning of the record (left). In the first cell shown (B,-BZ), BK was applied after 24 min of perfusion with GTPyS. In the second cell (B,), BK was applied at the peak of the GTPyS effect. The arrows (A-B3 mark the application of BK. The continuous lines mark the zero current. (C,-C2) Effect of the pipette perfusion with GTPyS (0.5 mM) on the peak ICa,” and on its modulation by BK. The continuous line marks the zero current. (C,) Effect of BK (IO nM) on Ica,” when applied after 3 min of GTPyS perfusion. Three overlapping traces are shown, before (Con), during (BK), and after (Wash) application of BK. (CZ) In the same cell shown in (C,), BK is applied again (IO nM) after 20 min of perfusion with GTPyS. Two overlapping traces are shown, one before (Con) and the other during (BK) BK application. The voltage-clamp protocols are described in Experimental Procedures.
Results Evidence that G Proteins Account for Both Types of Responses to BK Nonhydrolyzable analogs of GTP, such as GTPyS, slowly and irreversibly activate G proteins even in the absence of receptor stimulation (Gilman, 1987). Thus, the observation that GTPyS can irreversibly mimic and occlude a response to agonists would be consistent with the involvement of a G protein in that response. After establishing the whole-cell configuration with normal ionic conditions, the pipette was slowly perfused over a period of about 10 min with internal medium containing GTPyS (250 PM) and no GTP. Perfu-
sion with this nucleotide consistently (12 out of 12 cells tested) produced a slow outward current, followed by a long lasting inward current (Figure IB,). GTPyS may activate the same conductance activated by BK because both responseswere accompanied by a decrease of the cell’s input resistance and had similar reversal potentials (near -70 mV). Interestingly, the action of GTPyS, much like that of BK, is self-limited, because it is followed by a slow inward shift of the current. This responsewas never observed when GTP, rather than GTPyS, was perfused in the pipette (n = 44; data not shown). If GTPyS activates a G protein that is required for the increase of IK,BK,then it should not only mimic, but also occlude, the response to BK. We tested this idea in two different ways. First, BK was applied after perfusion of GTPyS and return of the holding current to near initial baseline level (Figure IBz). The response to BK was severely reduced in all cells tested under these conditions (mean = 13 pA; range = O-40 pA; n = 8 with GTPyS), as compared with the responses in the presence of GTP (mean = 102 pA; range = O-260 pA; n = 17). It is possible that long exposure of the cells to GTPyS could lead to an indirect effect on the BK pathway, such as desensitization of the response. Therefore, we also applied the transmitter to other cells at the peak of the outward current generated by GTPyS (Figure 1B3). Under these conditions, the stimulatory response to BK was entirely suppressed in all cells tested (n = 8). If cells were dialyzed for comparable durations with normal internal medium containing GTP, the response to BK remained robust (Figure IA). We used a similar approach to investigate the involvement of a G protein in the inhibition of ICa,” by BK. After isolation of Ic~,v, perfusion of the pipette with GTPyS elicited a slow decline of the peak Ic,,” (compare the peak currents in Figures ICI and 1C2). This phenomenon occurred consistently in all cells treated with GTPyS (n = 5). Application of BK during this phase produced a faster and largely irreversible inhibition of ICa,V(in 3 out of 3 cells tested); this would be consistent with promotion of GDP-GTPyS exchange by the receptor (Figure ICI). If BK was applied to GTPyS-treated cells after the peak Ica,” had stabilized at a depressed level, the neurotransmitter had no effect (n = 3; Figure IQ. In summary, these observations are consistent with the involvement of a G protein in both the activation of IK,BKand the inhibition of ICa,” by BK. Regulation of IK,BYand IQV by BK Is Insensitive to PTX Sensitivity to bacterial toxins, such as PTX, is a very useful criterion for indicating the broad class of G proteins potentially involved in a pathway. Since the susceptibility of IK,BKto PTX in NG108-15 cells was uncertain, we tested for its sensitivity to the toxin in the NG108-15 cells used for this work. A first group of cells was treated overnight with PTX (200 rig/ml). Previous
BK Modulation
of Potassium
and Calcium
Currents
111
50
w
0
A
f s 0 100 300 Peak Response f I PA I
SC0
BK
Figure 2. Pretreatment with PTX Does Not Block the Activation of ~.BK by BK (A, and AZ) Activation of IK,BK by BK (IO nM) in 2 cells, one that had been incubated overnight with PTX (A,) and the other with boiled PTX (AI). The slightly slower time course of the response in (A?) compared with (A,) was unrelated to the treatment. (B)Cumulativedistributionsofthevaluesofthe peak I~.~~evoked by BK in cells that were treated with PTX (squares) or boiled PTX (circles), or that received no treatment (triangles). Inset: response indices (the bars are proportional to the areas under each histogram) for each type of treatment. The PTX group was not significantly different from the two other groups. (C,-C,) Activation of IK,BK by BK and block of the effect of Leuenkephalin on Ica,v in the same cell pretreated with PTX. (C,) Action of BK under normal ionic conditions. The voltage-clamp protocol was as in (A,) and (A,). (C,) Action of Leu-enkephalin (Leu-Enk), after perfusing the pipette with cesium-EGTA solution and superfusing a high calcium-TTX solution in the bath (for about 5-6 min). Voltage-clamp protocols are described in the Experimental Procedures.
work has shown that this treatment was consistently capable of reducing the modulatory responses of Leuenkephalin, norepinephrine, and somatostatin in NG108-15 cells (Taussig et al., 1992). As controls, two additional groups of cells either received no treatment or were incubated with the same concentration of boiled PTX. The response to BK was measured in a sample of cells from each group. Examples of responses to BK after treatment with either active or boiled PTX are shown in Figures 2A1 and 2Az, respectively. Figure 2B summarizes activations of IK,IJK by BK. Cumulative histograms of responses show cells that were untreated (n = 7) ortreated with either PTX(n = IO) or boiled PTX (n = 7), as indicated. The corresponding response indices are shown in the inset. Direct inspection and statistical comparison of the distribution of the responses under the different conditions clearly indicate that treatment with PTX had no significant
effect on the regulation of II(,BK by BK (PTX versus boiled: p = 0.9; PTX versus no treatment: p = 0.8; Log-Rank Test). To demonstrate that the treatment of PTX was effective, we also measured the effect of Leu-enkephalin on these same cells. Inhibition of ICa,” by Leuenkephalin is a well-defined PTX-sensitive response (Hescheler et al., 1987; Taussig et al., 1992). After treatment with PTX, cells were examined under normal conditions for measuring IK,BK; a normal response to BK was evoked (mean = 207 pA; range = 60-440 pA; n = 4; Figure 2C,). The bath was then superfused with a solution containing high calcium and tetrodotoxin (T-TX), whereas the pipette and consequently the cell interior were perfused with a solution containing cesium and EGTA. Under these conditions, a voltagedependent calcium current that was insensitive to inhibition by Leu-enkephalin (Figure 2C2) could be readily isolated. Attenuation of the response to Leuenkephalin in cells with normal activation of I~,BK by BK was a consistent finding after treatment with PTX. On average, the inhibition of ICa,” by Leu-enkephalin was 9% (range = 2%-14%; n = 4) under these conditions. In contrast, inhibition of ICa,” by Leu-enkephalin was consistent with previously reported responses (Hescheler et al., 1987; Taussig et al., 1992) in the 1 untreated cell that was tested after exposure to BK and perfusion with the solutions for measuring Ic,,v. This also indicated that the block of the Leu-enkephalin response was not due to heterologous desensitization by BK. We also confirmed our previous finding (Taussig et al., 1992) that inhibition of ICa,” by BK is insensitive to PTX (4 cells responding out of 4; data not shown). Thus, any G proteins that may mediate activation of Ix,~xand inhibition of ICa,” by BK in NC10815 cells are insensitive to PTX.
An Antibody against the C Terminus of C,, and Cl,, Specifically Blocks the Activation of IK,B~ by BK In this series of experiments, we used raised against a peptide representing the terminus of both G,, and G,,, (Gutowski to test the hypothesis that members of this of G proteins are responsible for transducing fects of BK in NG108-15. Previous work that these antibodies are specific for G+, in immunoblots and immunoprecipitation)
antibodies common C et al., 1991) subfamily the efhas shown (as seen and that
they block the activation of PI-PLC by BK in membranes from NG108-15 cells (Gutowski et al., 1991). We first examined the effect of this antibody on the activation of IK,BK by BK. The response to application of BK was tested after 20-25 min of perfusion with either anti-G q,lla antibodies or preimmune IgG. When recording from a cell treated with antibodies to Gq,llu, the response to BK was clearly suppressed (Figure 3A,), compared with that observed from a cell treated with preimmune IgG (Figure 3A2). This was a consistent observation (cells with antibodies to G,,,,,: n = 21; with preimmune IgG: n = 17), as shown in Figure
Neuron 112
I3
A, +AB
COll 0
200
1CO Peak Response
Cl
1Oms
o\
d
I I PA I
D
.~
es,...
10 20 30 40 Mar ~nh~b,t~on , % ,
0
300
50
+AB
100% x A P
-
10s COll
0
A23187
100
300
Peak Response
Figure 3. The Anti-Gqfllo tion of IK,BKby BK
Antibody
Specifically
Blocks
500 I ( pA 1
the Activa-
(A,-B) Effect of the antibody on the activation of IK,BK induced by BK. (A, and AZ) Responses to BK (IO nM) in 2 cells: one (A,) after intrapipette perfusion with the purified IgC fraction containing the anti-Gs,,,,, antibodies CAB), and the other (AZ) after perfusion with preimmune IgC (Pi).(B) Cumulativedistributions of the peak IK,8K response to BK after perfusion with antibody (squares) or preimmune IgG (circles). Inset: response indices for each condition (the bars are proportional to areas under each histogram). The star indicates a highly significant statistical difference (p < 0.001; Log-Rank Test). (C-D) Effect of the antibody on the activation of a calciumdependent potassium current induced by the calcium ionophore A23187. (C, and C1) Responses to extracellular application of A23187 (20 NM) in 2 cells, one after perfusion with the purified IgG fraction containing the antibody (Cl), the other after perfusion with preimmune IgG (CJ. (D) Cumulative responsedistributions of the peak responses to the ionophore A23187 after application of antibody (squares) or control preimmune IgC (circles). Inset: response indices for each treatment. The AB group was not significantly different from the PI group. See Experimental Procedures for voltage-clamp protocols.
38, in which cumulative histograms, including data from all of the cells, are shown. The circles represent data from cells treated with preimmune IgG, whereas the squares represent data from cells treated with the anti-Gs,,,, antibodies. The corresponding response indices to BK under each of these conditions are illustrated in the inset. The difference between these two groups was highly significant (p < 0.001; Log-Rank test). To test whether the blocking action of the antibody was due to a direct effect on the channel itself, we mimicked the activation of IK,~kby BK, by extracellular application of the calcium ionophore, A23187 (Figures 3C, and 3C2). This compound acts by raising cyto-
Figure 4. The Anti-G,,,,, of Iti,v Induced by BK
Antibody
Does
Not Block
1Oms
the Inhibition
(A) Current traces recorded from a cell after perfusion with pre immune IgG (PI) fraction, before (Con) and during (BK) application of BK. The effect of BK was fully reversible upon washing. (B) Cumulative distributions of the peak inhibition of ICa,” induced by BK, after perfusion with anti-G,,,,, antibody (AB; squares) or preimmune IgG (PI; circles). Inset: response indices for each condition (the bars are proportional to areas under each histogram). The AB group was not significantly different from the PI group. (C,-C,) Effects of BK on IK,BKand Ic.,~ in the same cell, after perfusion with anti-Gs,,,. antibody (AB). (C,) Normal ionic conditions and after pipette perfusion with the antibody. (C2) After perfusing cesium-EGTA-antibody in the pipette and superfusing high calcium-T-TX solution in the bath. See Experimental Procedures for voltage-clamp protocols.
plasmic calcium levels directly, thus bypassing the activation of PI-PLC and the release of IPS produced by BK (Pressman, 1976). Application of A23187 (20 PM) produced a response resembling that evoked by BK (Figure 3C,), as shown by their similar time courses and reversal potentials. Similar responses were obtained previously by injection of either calcium or IP3 (Brown and Higashida, 1988a). When the cells were treated with the anti-G,,n, antibody, the ionophore evoked responses of similar amplitude to those from cells treated with preimmune IgG (compare Figures 3C, and 3C2). Cumulative histograms representing all of the responses to the ionophore are shown in Figure 3D, in which the circles depict responses after preimmune IgG (n = 7), and the squares refer to cells after treatment with the antibody (n = 7). The response indices (inset) obtained in the presence of antibody or preimmune IgG confirm that the anti-Gq,,lP antibody has no effect on this response (p = 0.7; Log-Rank Test). These data are consistent with anticipated action of the antibodies on G, and indicate that G, (or G,,) is required for the activation of LK,BKby BK. In
BK Modulation 113
of Potassium
and Calcium
Currents
principle, however, the data do not rule out the unlikelypossibilityof anonspecificeffectof theantibodies on the IP3 receptor or internal calcium stores. The
Antibody
against
Cq/lla
Does
Not
Block
Inhibition
of 1ca.v by BK
We next examined whether the anti-G,,ql, antibodies would affect a second modulatory effect of BK, the inhibition of ICa,V.Under appropriate ionic conditions, this current was readily isolated (Figure 4A). The response to the application of BK was tested after 20-25 min of perfusion in the pipette with either antiGq,lla or preimmune IgG. Inhibitory responses to BK were the same from cells that were treated either with anti-Gq,,,,, antibodies (Figure 4CJ or preimmune IgG (Figure 4A). Cumulative histograms (Figure 4B) demonstrate the consistency of these observations (n = n = 8 with preimmune IgG). The 11 with anti-G+,,; corresponding response indices are shown in the inset. The differences between the two groups were not significant (p = 0.8; Log-Rank Test). The clear lack of on the inhibitory effect of the anti-G,,,,, antibodies effect of BK on the calcium current suggests that a G protein different from Cq/,, mediates this action of the transmitter. A potential artifact in this experiment is that the antibodies could be inactivated by the cesium-EGTA solution present in the pipettes. This is an unlikely possibility because previous investigators have successfully used anti-G protein antibodies dissolved in cesium-EGTA solution to block transmitter modulation of ICa,V(see for example Lledo et al., 1992). In addition, we have examined the ability of these antibodies to immunoprecipitate C,, in vitro in the presence of the solutions used to measure either IK,BK or Ica,v. The antibodies effectively precipitated Gq,lla under both conditions (data not shown). It is also possible that the negative results with the modulation of Ica,v in vivo might simply be due to poor diffusion of antibodies into the cells under the conditions used to measure Ica,“. To test this idea, the pipette was perfused initially with the normal potassium-containing pipette solution and the anti-Gq,I,a antibody. As expected, the activation of IK,BKby BK was consistently suppressed (n = 4; mean = 32 pA; range = O-70 pA; Figure 4C,), which confirmed that the antibody had effectively blocked the endogenous C&. The pipette was then perfused with a cesium-EGTA solution containing the anti-C&, antibody, whereas the bath was simultaneously superfused with high calcium, TTXcontaining solution. After isolating ICa,v, BK was consistently able to elicit a normal inhibition of this current (Figure 4CJ. On the average, BK induced an 18% inhibition (range = 8%-32%; n = 4) of ICa,vunderthese conditions (two consecutive perfusions). This value compares favorably with the 25% inhibition (range = 12%-42%; n = 8) obtained after a single perfusion with the preimmune IgG (see above). The difference between these two groups was not statistically significant (p = 0.45; Log-Rank test). Although observations made in vitro (Gutowski et al.,
1991) and in vivo (Figure 4) indicate that the antiG q,lto antibody acts specifically on Gqjll, we further tested this specificity by examining its effect on a different G protein pathway, the GoA-mediated inhibition of Ica,v, by Leu-enkephalin (Taussig et al., 1992). In all 3 cells in which anti-Gqilla antibodies were perfused, Leu-enkephalin elicited normal inhibition of the ICa,v,which was identical to that obtained in 3 cells treated with preimmune IgG (data not shown). Discussion
In NG10815 cells, one can study the specific role of G proteins in two situations: the convergent actions of multiple neurotransmitters on the same channel (Taussig et al., 1992) and the divergent actions of the same neurotransmitter on multiple channels (this work). Previous work (Taussig et al., 1992) showed that distinct PTX-sensitive G proteins selectively coupled separategroups of transmitter receptors to inhibition of Ic~,~. Here, we investigated the opposite problem, the involvement of G proteins in mediating two separate actions of BK, activation of a potassium current and inhibition of Ica,v. First, the likely involvement of a G protein in both of these actions was indicated by treatment of cells with GTPyS, which initially mimicked and then occluded responses to BK. The slow actions of GTPyS in our experiments (compare Figures IA and IB,) parallel the slow activating effect of GTPyS on Gqilt in vitro (Hepler et al., 1993). We also attempted to block the actions of BK by perfusing the pipette with GDPBS. However, in normal ionic conditions, this nucleotide consistently evoked an outward current similar to that evoked by GTPyS. Although we do not have an explanation for this observation, this direct effect of GDPBS prevented its use as a blocker of the effects of BK (see also Rasenick et al., 1989). Our second finding, that the BK-mediated activation of IK,BKand inhibition of ICa,Vwere not blocked by PTX, is clearly demonstrated in Figure 2C under conditions in which PTX was able to suppress a well-known PTXsensitive response in the same cell. The action of BK on IK,~~ was previously reported to be PTX-sensitive in this same cell type (Higashida et al., 1986). The reason for this discrepancy is not clear. However, the original study was based on voltage, not conductance measurements, and the attenuation by PTX was small: perhaps nonspecificeffectsof thetoxin could account for that modest effect. Third, we tested whether the PTX-insensitive G protein, which presumably transducestheactionsofBK,mightbeG,orG,,.Thepipette used for recording was perfused with an antibody raised against the C terminus of Gq,tla. Unexpectedly, our data indicate that Gq,ll mediates activation of IK,~K but not inhibition of Ica,v by BK. Four lines of evidence indicate that inhibition of IK,~~ by the antibodies is due to a specific block of G,,,?: Perfusion with the antibody does not affect the resting electrical properties of the cell, nor the activation of a calcium-dependent potassium current obtained with
Neuron 114
Experimental Procedures K+
ca2+
Cultures NClOB-15 (Hamprecht
cells were grown et al., 1985).
and
differentiated
as described
Antibody
Figure
5. G Protein-Mediated
Pathways
Activated
by BK
Explanations for the symbols used: BK-R: BK receptor. Two recep tor subtypes might actually be involved. G PTX-ins: C protein insensitive to PTX. Other symbols are explained in the text.
A23187. Moreover, preimmune IgG, obtained from the same animal used to obtain the anti-G,,,,,antibodies, did not suppress activation of IK,BKby BK (Figure 3). Finally, the anti-Gqllla antibody did not suppress two other G protein-mediated modulatory pathways, the inhibition of ICa,vby either Leu-enkephalin or BK. Our data also strongly indicate that a PTX-insensitive G protein distinct from Gq,,, mediates the inhibition of 1Ca.Vby BK (Figure 5). The strongest argument for ruling out a role for Gq,,, is obtained from experiments in which the anti-G,/,,, antibody was demonstrated to be available inside the cell in sufficient concentrations to block IK,BK,but had no effect on inhibition of Ica,v by BK in the same cells. It should be emphasized that, in the present work, we have restricted our study to the inhibition of Ica,vthat is present in cells dialyzed with a high concentration of calcium buffer solution. We have not investigated the possibility that BK might produce calcium-induced inhibition of Ic~,v if solutions with low calcium-buffering capacity are used in the pipette (Brown and Higashida, 1988b; Kramer et al., 1991). If present, this pathway would be parallel to the G,,,,-independent pathway studied in the present paper and would be expected to be blocked by the anti-G,/,,, antibody. In conclusion, our work shows that G~II is required for the activation of a potassium conductance by BK (Figure 5). This action of G,/,, might appear simply to reflect the involvement of PI-PLC in generating the IPJcalcium signal that opens the potassium conductance. However, recent evidence from our laboratory shows that, parallel to the IPJcalcium pathway, BK and Gq,,, activate a membrane-delimited mechanism to open the potassium conductance (Harrington, S. G., and F. B., submitted). Moreover, if a G protein different from Gd,, mediates the inhibition of Ic=,v by BK(Figure5), then it is likelythat, much likethe inhibition of IG,V by GABAe in avian dorsal root cells (DiversbPierluissi and Dunlap, 1993), protein kinase C and, in general, activation of the PIP? metabolism do not mediate the inhibition of Ica,v by BK in NG108-15 cells. Although future work will attempt to identify the G protein that mediates the inhibition of IC+V by BK, the data presented here demonstrate that PTX-insensitive G protein pathways play a significant and complex role in regulating ion channels of the nervous system.
Generation Antibodies were raised against the C-terminal portion of G,, as described (Gutowski et al., 1991). The IgG fraction was purified from serum (Harlow and Lane, 1988) and gel filtered through a C-50 sephadex column in a final buffer containing 120 mM KCI and 20 mM HEPES (brought to pH 7.4 with KOH). Cesium replaced potassium in the antibody solution used in the experiments measuring calcium current. The preimmune IgG was obtained from preimmune serum from the same animal that was used to obtam the antl-G,,,,, antibody. Chemicals BK and Leu-enkephalin (Peninsula) were stored in dry aliquots at -25°C. The calcium ionophore, A23187 (Calbiochem), was dissolved in dimethylsulfoxide and stored in aliquots (20 mM) at +4%. Both compounds were reconstituted daily in extracellular solution (see below) at their final concentration. Vigorous sonication (2 min) was applied to the ionophore solution to facilitate itsdispersal in theaqueousmedium. BKand the ionophorewere applied by pressure through a blunt pipette positioned near the cell. All of the remaining chemicals were from Sigma, except ATP, GTP, and GTPyS, which were from Boehringer Mannheim. Solutions Extracellular solution for potassium responses contained 125 mM NaCI, 5.4 mM KCI, 1.8 mM CaCl?, 1.0 mM MgC&, 20 mM HEPES, and 5.0 mM glucose (brought to pH 7.4 with NaOH); for calcium currents contained 140 mM NaCI, 5.4 mM CsCI, 10.8 mM CaC12, 1 mM MgCI,, 10 mM HEPES, 10 mM glucose, and 0.0005 mM TTX (brought to pH 7.4 with NaOH). We used calcium, rather than barium, as the charge carrier to keep potential cellular metabolic pathways in a more physiological condition. Theextracellular solution was slowly superfused (I-2 ml/min) throughout the experiment using a peristaltic pump. This superfusion was arrested during the application of BK and maximally increased immediately after. The pipette solution to record potassium responses contained 115 mM KCI, 0.1 mM MgCl2,40 mM HEPES, 3 mM ATP, and 0.1 mM GTP (pH 7.3 with KOH); the pipette solution for calcium responses contained 112 mM CsCI, 1 mM MgCl?, 10 mM EGTA, and 30 mM HEPES (with CsOH). These solutions were used to fill the pipette and the perfusion apparatus at the beginning of the experiment and were also the basis for three types of test solutions: -Antibody solution: the IgG fraction containing the anti-Gq,,lo antibody was diluted l/IO with the pipette solution. -Preimmunesolution:theIgGfractionofthepreimmuneserum was similarly diluted. -GTPyS solution: the GTP in the pipette solution was replaced by 250 pM (or 500 PM in some experiments) GTP@ The preimmune IgG fraction, diluted l/IO, was also added in some experiments. The pipette solutions containing potassium and cesium generated junction potentials of 6 mV and 2 mV, respectively. These values have not been subtracted from thevalues reported in the figures and the text.
Electrical Recordings The whole-cell version of patch clamp was used (Hamill et al., 1981). Bullet-shaped, wide-mouthed pipettes (0.5-1.5 Mn) connected to an Axopatch-IB (Axon Instruments, Foster City, CA) amplifier were employed. No series resistance compensation was applied. A 386-based IBM PC-compatible computer (Lucky Computer, Dallas, TX), connected to a TL-1 interface (Axon Instruments) and using Axon Instruments software, was employed for the generation of the voltage-clamp commands and for storing the calcium current records. The experiments measuring
BK Modulation 115
potassium back and
of Potassium
currents analysis.
were
and Calcium
recorded
Currents
on digital
tape,
for later play-
Perfusion of CTPyS and Antibodies A simplified version of the intrapipette perfusion technique was used (Tang et al., 1990). A fine polyethylene tubing (PE 10; ClayAdams, Parsippan, NJ) was gently heated in a stream of hot air and then manually pulled to produce tipswith an internal diameter around 100 urn. This tip was positioned inside the recording pipette within 200-100 urn of the pipette mouth. The other end of the tubing, outside the holder (#PC-S3-30; Wright, Guilford, CT), was immersed in a 1 ml Eppendorf tube containing pipette solution (perfusion line). At the beginning of the experiment (with pipette solution in the perfusion line and pipette), negative pressure inside the holder was created with the suction line and used to establish a seal on the cell and subsequently to break the patch. The external end of the perfusion line was then manually placed in a second Eppendorf tube, which contained one of the test solutions (see above). An air bubble was initially included in the perfusion line to insulate the pipette from the external portion of the line electrically during these manipulations. Finally, gentle suction was used to perfuse the test solution (80 ~1) over a period of IO-15 min. Analysis of the Data Our analysis of thedatacan bedivided in three phases: measurement of the response to BK, graphical representation of the data obtained, and statistical comparison between groups of data. First, activation of IK,BKby BK: the cells were held either at -40 or -50 mV and subjected to 2 s steps to -70 mV every IO s to measure the cell’s conductance. A more positive holding potential was not used because under our conditions (prolonged perfusion), it appears to produce irreversible deterioration of the cell. The response to BK was directly measured as the peak current at -40 mV. Only cells that exhibited a positive or null holding current at -40 mV at the end of the experiment were included in the analysis. To analyze the modulation of Ic..v, cells were held at -50 mV. Every 10 s, a prepulse (2 s; -90 mV) was applied, followed by a 5 ms interval and a test pulse (0 mV; 100 ms). The peak inhibition by neurotransmitters was expressed as a percentage of baseline current, after analog correction of the capacitive current and digital subtraction of capacitive and leakage currents. Only ICa,v that declined less than 5% over the duration of the experiment was analyzed. Second, the analysis of the data was complicated by the fact that cells showed a substantial variability of the size of the IK,BK activation by BK. To analyze the actions of BK, the procedure described by McGehee et al. (1992) was used. Briefly, the data were plotted as cumulative histograms. The ordinate of each point represents the percentage of responses that are larger than its value. The area underlying a particular histogram is called the response index. This yields a central value for the responses under a particular set of conditions and also contains information about their scatter. Bars proportional to the response indices obtained under different conditions allow direct comparison between groups of responses. Error bars were not used since they were inappropriate. Third, in the present work, we were faced with two statistical difficulties. First, IK,BKdesensitizes so greatly after the first application that we were forced to comparedata taken from different cells. Second, many cells did not respond to BK at all, which produced highly asymmetrical distributions of the data. The statistical comparison between groups of data was obtained using the Log-Rank test. This test is particularly powerful for the comparison of this type of data, because it is very sensitive to the tail of L-shaped distributions. The Statistical Analysis System, which contains the Log-Rank Test, was used on the local VAX 8800 computer.
Received
July 22, 1993; revised
October
25, 1993.
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Acknowledgments We are indebted up the perfusion
custom-modified version of the Log-Rank software, A. S. French for the graphic software, P. Middleton, M. A. Harrington, M. L. Burleson, and S. Siegelbaum for comments on the manuscript, and K. Edwards for expert and patient secretarial assistance. This work was supported by USPHS grant GM31954 to P. S., USPHS grants CM47721 and NS25186 to F. B., and a Klingenstein Fellowship to F. 8.; F. B. is an Established Investigator of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.
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