Activation of M1 muscarinic acetylcholine receptors (M1 mAChR) inhibits M-type potassium currents (I K(M)) and N-type calcium currents (I Ca) in mammalian sympathetic ganglia. Previous antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Gαq (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Gαq gene.
PTX-insensitive M1 mAChR inhibition ofI Ca was strongly reduced in Gαq −/− mouse SCG neurons and was fully restored by acute overexpression of Gαq. In contrast, M1mAChR inhibition of I K(M) persisted in Gαq−/− mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I K(M) was partly PTX-sensitive. Residual (PTX-insensitive)I K(M) inhibition was slightly reduced in Gαq −/− neurons, and the remaining response was then suppressed by anti-Gαq/11 antibodies.
Bradykinin (BK) also inhibits I K(M) in rat SCG neurons via a PTX-insensitive G-protein (Gq and/or G11; Jones et al., 1995). In mouse SCG neurons,I K(M) inhibition by BK was fully PTX-resistant. It was unchanged in Gαq −/− mice but was abolished by anti-Gαq/11 antibody.
We conclude that, in mouse SCG neurons (1) M1 mAChR inhibition of I Ca is mediated principally by Gq, (2) M1 mAChR inhibition ofI K(M) is mediated partly by Gq, more substantially by G11, and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I K(M) is mediated wholly by G11.
- M current
- calcium current
- superior cervical ganglion neuron
- knock-out mouse
- muscarinic receptor
- bradykinin receptor
The excitability of neurons is controlled by the convergent action of many different ion channels, including voltage-gated K+ and Ca2+ channels and ligand gated ion channels. N-Type Ca2+ channels have complex effects on cell excitability. They permit Ca2+ influx at presynaptic terminals, triggering exocytosis, thereby contributing to excitability. However, they also act to limit cell excitability by providing the Ca2+ necessary to open Ca2+-activated K+ channels, resulting in a prolonged afterhyperpolarization and action potential accommodation (Davies et al., 1996; Sah, 1996). The M-type potassium current (I K(M)) also acts to dampen cell excitability; it increases upon depolarization, hyperpolarizing the cell and keeping it clamped around rest and thus limiting action potential firing (Brown, 1988; Wang and McKinnon, 1995). Both the N-type Ca2+ channel andI K(M) can be inhibited by G-protein-coupled receptors, such as the muscarinic acetylcholine M1 receptor (M1 mAChR) (Marrion et al., 1989; Bernheim et al., 1992), and coincident inhibition of both currents results in large increases in cell excitability and action potential discharge (Jones and Adams, 1987;Brown, 1988, 1999).
In rat superior cervical ganglion (SCG), inhibition ofI Ca andI K(M) by agonists acting at the M1 mAChR appears to use very similar transduction mechanisms, and it has been suggested that they may share a common pathway (Hille, 1994). The first step in this pathway is the activation of a G-protein that is insensitive to pertussis toxin (PTX) (Brown et al., 1989; Bernheim et al., 1992). Previous experiments using the injection of antibodies directed against the common C terminus of Gαq and Gα11 (Caulfield et al., 1994) and the expression of separate antisense constructs specific to Gαq or Gα11(Delmas et al., 1998a; Haley et al., 1998a) suggested that the G-protein primarily involved is Gαq. However, because suppression by either method was incomplete, the participation of other G-protein(s) could not be discounted.
Bradykinin (BK) also inhibits I K(M) in rat SCG neurons through stimulation of B2 BK receptors (Jones et al., 1995). This effect was strongly attenuated by anti-Gαq/11 antibodies (Jones et al., 1995), but the specific G-protein involved has not been further identified. It may, in fact, differ from that required for mAChR-induced inhibition because BK-induced inhibition clearly involves the activation of phospholipase C, whereas M1 mAChR-induced inhibition appears not to (Cruzblanca et al., 1998; Haley et al., 1998b).
In an attempt to further define the role of Gαqin transducing the effects of M1 mAChR and B2 BK receptors, we have therefore investigated the inhibition of I Ca andI K(M) by agonists for these receptors in SCG neurons isolated from mice lacking Gαq(Offermanns et al., 1997a,b).
Parts of this work have been published previously in abstract form (Haley et al., 1998c).
MATERIALS AND METHODS
Gαq -Deficient mice.Mice deficient in the Gαq gene (Gαq −/−) were generated by targeted disruption with a neomycin gene as described previously (Offermanns et al., 1997a). Gαq-Deficient mice used in the experiments were obtained by mating either Gαqknock-out males with heterozygous females or heterozygous males and females (to obtain wild-type and knock-out littermates). Mice were kept on a C57BL × 129/Sv background, and genotypes were confirmed by PCR on genomic DNA from tail snips of each mouse.
Cell culture. Sympathetic neurons were isolated from Gαq knock-out and wild-type mice that were at least 5 weeks old. Wild-type mice were either C57BL/6J (Harlan, Bicester, UK) or from litters also containing Gαq knock-out littermates; results obtained were the same from both these groups and so have been pooled. We also used ICR mouse (coding for outbred albino mouse; Harlan) to compare properties of SCG neurons derived from different strain of mouse. SCG were removed, and neurons were cultured using standard procedures as described previously for rat (Delmas et al., 1998b).
Microinjection. In a few experiments, GαoA+B or Gαq/11antiserum (OC2 and CQ1 respectively, from G. Milligan) (Caulfield et al., 1994, Delmas et al., 1998a, 1999) or an expression plasmid encoding Gαq were pressure-injected into the cytoplasm (antisera) or nucleus (plasmid) of SCG neurons 2 d in culture using a microinjector (Eppendorf, Hamburg, Germany). To allow identification of the injected neurons for recording, FITC-dextran (70,000 MW; Molecular Probes, Leiden, The Netherlands) was added in a final concentration of 0.2% (antisera) (Jones et al., 1995) or 0.5% (plasmid) (Haley et al., 1998a). Cells injected with the antisera were recorded at least 2 hr after injection, whereas those injected with the Gαq-encoding plasmid were recorded 24 hr later.
Electrophysiology. Currents were measured from SCG neurons cultured for 2–3 d, using the amphotericin-B perforated patch technique (Horn and Marty, 1988; Rae et al., 1991). Patch electrodes (2–5 MΩ) were filled by dipping the tip for 40 sec into the appropriate filtered internal solution, and the pipette was then back-filled with the internal solution containing 0.07–0.1 mg/ml amphotericin-B. High-resistance seals (>2 GΩ) were initially achieved and, after amphotericin-B permeabilization, access resistances were generally <25 MΩ for I K(M)recordings and <15 MΩ for I Carecordings. SCG neurons were perfused (5–10 ml/min) with an external solution consisting of (in mm): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, and tetrodotoxin 0.0005, pH 7.4, maintained at 32°C.
IK(M) recording. The internal solution comprised of (in mm): potassium acetate 80, KCl 30, HEPES 40, and MgCl2 3, adjusted to pH 7.3–7.4 with KOH (280 mOsm/l with K acetate). Cells were voltage-clamped using an Axoclamp-2A (switching frequencies 3–5 kHz, filter 0.1 kHz) from Axon Instruments (Foster City, CA).I K(M) was measured as a slowly developing inward deactivation relaxation after a 1 sec jump to a command potential of approximately −45 mV from a holding potential of approximately −25 mV (Haley et al., 1998a). Inhibition was measured as the fractional reduction in the amplitude of theI K(M) deactivation relaxation in response to either cumulatively increasing concentrations of oxotremorine methiodide (Oxo-M) (Research Biochemicals, Natick, MA) or a single application of 1 nm BK (Bachem, Torrance, CA).
ICa recording. Perforated patch recordings were conducted primarily as described previously (Delmas et al., 1999). Pipettes had a resistance of 2–3 MΩ when filled with the following solution (in mm): CsCl 30, caesium acetate 110, HEPES 10, and MgCl2 1, pH 7.2–7.3 with CsOH (300 mOsm/l). Cells were voltage-clamped using an Axopatch 200A amplifier (Axon Instruments). Series resistance and membrane capacitance were partially compensated (>80%). Current traces were low-pass filtered at 2–5 kHz using a four-pole Bessel filter. Leak and capacitance currents were subtracted digitally using the P/6 subtraction procedure of pClamp6 (Axon Instruments). I Ca was elicited from a holding potential of −70 mV with a three-voltage pulse protocol consisting of a test pulse to 0 mV applied before and after a conditioning depolarizing step to +90 mV.
For experiments with Bordetella PTX (Speywood, Maidenhead, Berkshire, UK), SCG neurons were incubated with 1 μg/ml PTX in the culture medium for at least 24 hr before recording. Data are expressed as mean ± SEM, and statistical analysis of dose–response curves used two-way ANOVA to compare treatments across all concentrations. If a significant effect of treatment was found overall, further analysis was performed using the two-way ANOVA to determine which treatment groups contributed to this significance. The bradykinin data were analyzed with one-way ANOVA, and if an overall effect of treatment was found, this was followed by Students–Newman–Keuls multiple comparison test. Analysis of I Ca andI K(M) current densities and neuron membrane potential was analyzed using Student's t test.p < 0.05 was considered significant.
Immunocytochemistry. Successful injection of Gαo or Gαq/11 antiserum was confirmed by staining. After recording, cells were fixed in acetone, washed, incubated with biotinylated Fab2 swine anti-rabbit antibody (the antiserum was raised in rabbit), and then incubated with avidin–biotin complex. The alkaline phosphatase substrate 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT) (Dako, Carpinteria, CA) was applied for 5 min, and the reaction was quenched with water. Neurons containing the injected antiserum were clearly distinguished as containing the dark purple BCIP/NBT product. Detection of expressed Gαqafter injection of the Gαq plasmid was confirmed using the above protocol and preceding it with a 1 hr incubation with a 1:2000 dilution of a Gαqantibody (IQB2) (Milligan et al., 1993).
M1 mAChR-induced inhibition ofICa and IK(M) in mouse versus rat SCG
The mAChR agonist Oxo-M inhibited bothI Ca andI K(M) (see also Hamilton et al., 1997) in SCG from mouse. However, the dose–response curves were shifted to the right in mouse compared with rat neurons (Fig.1). IC50 values forI K(M) inhibition were 0.4 and 0.7 μm in rat and wild-type mouse, respectively. For M1 mAChR inhibition ofI Ca, IC50 were 800 nm and 1.1 μm in rat and mouse, respectively.
M1 mAChR inhibition of ICais reduced in Gαq −/− mouse SCG neurons
In rat SCG, inhibition of I Ca by mAChR agonists results from the activation of two separate pathways: a voltage- and PTX-sensitive M4 mAChR pathway (M2 receptors in mice) (Shapiro et al., 1999) that requires GαoA and a voltage- and PTX-insensitive M1 mAChR pathway that mostly involves Gαq (Delmas et al., 1998a). In mice, total mAChR inhibition of I Ca was significantly (p < 0.002) reduced when Gαq was deleted. Thus, 1 and 10 μm Oxo-M resulted in 49 ± 4% (n = 8) and 68 ± 4% (n = 12) inhibition, respectively, in wild-type (Gαq+/+) mouse neurons and 29 ± 3% (n = 5) and 57 ± 3% (n = 5) inhibition in Gαq −/− neurons. Inhibition in Gαq −/− neurons was more voltage-dependent than in wild-type neurons, with facilitation ratio of 1.4 ± 0.2 (n = 10) and 0.94 ± 0.1 (n = 8), respectively (data not shown). Inhibition by 10 μm noradrenaline (NA) (which requires both GαoA and Gαi in rat) (Caulfield et al., 1994; Delmas et al., 1999) was not altered when Gαq was absent (wild type, 66 ± 3%,n = 8; Gαq −/−, 62 ± 5%, n = 5).
Incubation with 1 μm PTX inactivates members of the Gαo/i G-protein family, removes the PTX-sensitive mAChR inhibition, and isolates the M1 mAChR inhibition, as well as abolishes inhibition by NA (Schofield, 1991; Zhu and Ikeda, 1994). Inhibition by NA was, as expected, abolished in PTX-treated cells from both wild-type and Gαq −/− mice. After PTX treatment, it became clear that the reduction in M1 plus M2 inhibition observed in the Gαq −/− cells resulted from an almost complete loss of the voltage-insensitive M1mAChR-mediated inhibition (Fig. 2). To be sure that the loss of M1 mAChR inhibition ofI Ca was attributable to the absence of Gαq, we acutely overexpressed Gαq in cultured SCG neurons from Gαq −/− mice. Twenty-four hours after injection of a plasmid encoding for Gαq, M1 mAChR inhibition was fully restored (Fig.2 C). This reinstated inhibition was specific because NA inhibition was not rescued by Gαqoverexpression in PTX-treated cells (Fig. 2). These findings accord with previous data from this laboratory that M1mAChR inhibition of I Ca in rat SCG is primarily mediated by Gαq (Delmas et al., 1998a). It should be noted that theI Ca density (normalized to cell capacitance) was significantly lower in Gαq−/− neurons (27 ± 2 pA/pF, n = 10) compared with wild-type mouse SCG (38 ± 2 pA/pF, n = 12,p < 0.01). There was no difference betweenI Ca density in wild-type mouse and rat SCG (rat, 42 ± 2 pA/pF, n = 19).
M1 mAChR inhibition of IK(M)is not reduced in SCG from Gαq −/− mouse
In rat SCG, inhibition of I K(M)by mAChR agonists is mediated, at least in part, by Gαq (Haley et al., 1998a), so one would predict a loss of inhibition in neurons lacking Gαq. We were surprised, therefore, to discover that inhibition ofI K(M) by Oxo-M was not reduced in Gαq −/− SCG compared with wild-type neurons (Fig. 3). Indeed, Oxo-M produced significantly more inhibition in Gαq −/− neurons (p < 0.05), and the dose–response curve lay to the left of the wild-type dose–response (Fig.3 B). I K(M) density (normalized to cell capacitance) was not changed in Gαq −/− mouse SCG compared with wild type, and neither was the resting membrane potential (wild type, 2.5 ± 0.5 pA/pF, n = 8; −58.0 ± 1.3 mV,n = 9; Gαq −/−, 3.3 ± 1.1 pA/pF, n = 9; −56.3 ± 1.3 mV,n = 11), and these did not differ from rat SCG (2.4 ± 0.3 pA/pF, n = 10; −59.9 ± 1.3 mV).
M1 mAChR inhibition of IK(M)partly involves PTX-sensitive G-proteins in mouse SCG
One factor contributing to the persistence of mAChR-induced inhibition of I K(M) in neurons from Gαq-deficient mice became apparent when we tested the effect of PTX. In contrast to previous observations on rat SCG neurons (Brown et al., 1989; Bernheim et al., 1992; Haley et al., 1998a), PTX treatment significantly reduced inhibition by Oxo-M in neurons from both wild-type mice (p < 0.01) and Gαq −/− mice (p < 0.0001) (Fig. 4). In fact, the effect of PTX was greater in Gαq-deficient neurons, and the dose-response curve for the PTX-insensitive component of inhibition showed a significant (p < 0.02) rightward shift in Gαq −/− neurons. Thus, at 0.3 μm Oxo-M, the mean inhibition ofI K(M) was reduced from 15 ± 3 to 6 ± 4%. Hence, it appeared that Gαqcontributed a component of PTX-insensitive inhibition at low agonist concentrations but that, at high agonist concentrations, activation of another PTX-insensitive G-protein predominated.
One possibility is that this residual PTX-insensitive inhibition might have been mediated by G11, because expression of constitutively activated Gα11 is also capable of inhibiting I K(M) in rat SCG neurons (Haley et al., 1998a). To test this, we injected antibodies directed against the C terminus of Gαq/11 into the cell cytosol of PTX-treated neurons, using antibodies against Gαo as a control (Caulfield et al., 1994). Anti-Gαq/11 strongly reduced PTX-insensitive inhibition in neurons from wild-type mice and virtually annulled the residual inhibition in neurons from Gαq −/− mice (p < 0.0001). Thus, the residual PTX-insensitive inhibition in neurons from Gαq −/− mice results from activation of Gα11 because the antibody is specific to Gαq and Gα11, and Gαq is absent.
Inhibition of IK(M) by BK is mediated by Gα11 in Gαq −/− neurons
For these experiments, a single application of a low concentration (1 nm) of BK was made to each neuron tested because slow recovery from inhibition precluded repeated applications to the same neuron and desensitization precluded applications of incremental concentrations (Jones et al., 1995; Cruzblanca et al., 1998). At 1 nm, BK inhibited I K(M) in wild-type mouse SCG neurons by 25 ± 4% (n = 8). This was not significantly different from that in rat SCG neurons (29 ± 7%; n = 8; data not shown). Unlike mAChR-induced inhibition, the effect of BK on mouse neurons was not reduced by PTX (Fig. 5). This accords with previous observations on rat neurons (Jones et al., 1995). No inhibition of I Ca by 1 nm BK could be detected.
No significant reduction of BK-induced inhibition ofI K(M) was observed in Gαq −/− neurons. However, injection of an antibody against Gαq/11 substantially (p < 0.05 vs anti-Goantibody) reduced I K(M) inhibition in wild-type mouse neurons (as in rat neurons; Jones et al., 1995) and abolished inhibition in neurons from Gαq −/− mice (p < 0.01) (Fig. 5 B).
Using mice lacking Gαq, we have demonstrated that M1 mAChR inhibition ofI Ca requires Gαq because regulation was essentially lost in Gαq −/− neurons (Fig. 2). This was not a result of secondary effects on transduction mechanisms arising from an absence of Gαq during development because inhibition could be fully restored by acute overexpression of Gαq in Gαq −/− neurons (Fig. 2); thus, the remaining components of the inhibitory pathway were still present and functional in these mutant cells. In contrast, neither the inhibition produced by NA nor the PTX-sensitive (M2) component of mAChR inhibition (Shapiro et al., 1999) were reduced after deletion of the Gαq genes. Hence, these results in mouse neurons are in complete accord with previous conclusions from observations on rat SCG neurons using G-protein antibody injections and antisense depletion (Caulfield et al., 1994; Delmas et al., 1998a) that Gαq is the primary G-protein involved in M1 mAChR-induced inhibition ofI Ca but is not involved in the inhibition produced by activating M2/4 mAChRs or α2 adrenergic receptors. It is especially worth noting (particularly in connection with our observations onI K(M); see below) that Gα11 appears not to be able to substitute for Gαq in Gαq −/− mice, although it is able to interact with M1 mAChRs (Offermanns et al., 1994; Gudermann et al., 1996) and although expression of a constitutively active form of Gα11 can inhibitI Ca just like Gαq (unpublished observations; Delmas et al., 1998a).
In contrast [and surprisingly, in view of previous observations in rat SCG neurons using Gαq antisense (Haley et al., 1998a)], mAChR-induced inhibition ofI K(M) was not reduced in neurons from Gαq −/− mice. One contributory reason for this seemed to be that I K(M)inhibition after mAChR stimulation was partly sensitive to PTX. When this component was eliminated, it appeared that some part of the inhibition (particularly at low agonist concentrations) was mediated by Gαq because the dose–response curve for Oxo-M-induced M current inhibition was shifted significantly to the right in Gαq −/− mice. Nevertheless, it was clear that a considerable proportion of the inhibition must have been mediated by another PTX-insensitive G-protein. In Gαq-deficient neurons, this G-protein was identifiable as Gα11, because the residual PTX-insensitive inhibition was virtually annulled using an antibody directed against the unique but common C terminus to Gαq and Gα11. Thus, in the Gαq-deficient mice, we conclude that mAChR-induced M current inhibition is maintained by Gα11 and by an increased contribution of the (unidentified) PTX-sensitive pathway.
Thus, the present experiments reveal two clear differences from the inferences drawn from previous work on mAChR-induced M current inhibition in rat SCG neurons: the additional involvement of a PTX-sensitive G-protein and the greater potential involvement of Gα11. One possible explanation for the apparent involvement of a PTX-sensitive G-protein is that, in mouse ganglion cells, stimulation of M4 or M2 receptors might also inhibitI K(M). However, this seems unlikely, because Hamilton et al. (1997) found that mAChR-induced M current inhibition was completely annulled in mice deficient in the M1 receptor gene, implying that, as in rat neurons (Marrion et al., 1989; Bernheim et al., 1992), muscarinic inhibition of I K(M) was mediated entirely by M1 mAChRs. Although M1 receptors are usually considered to couple exclusively to PTX-insensitive G-proteins, there have been occasional reports of at least partial PTX-sensitive responses after expression of cloned M1 receptors (Stein et al., 1988;Ashkenazy et al., 1989). Because this PTX-sensitive pathway seems unique to mouse neurons and may have limited general significance, we have not so far made any serious attempt to identify the species of G-protein involved.
Regarding the involvement of Gα11, in previous experiments on rat neurons, anti-Gαq antisense produced a rightward shift of the dose–response curve for inhibition of I K(M) by Oxo-M (Haley et al., 1998a), not dissimilar to that seen in Gαq−/− mouse neurons. However, although this might also suggest the involvement of another PTX-insensitive G-protein, antisense to Gα11 had no effect on the inhibitory effect of Oxo-M in rat neurons. Hence, it was concluded that the limited effect of Gαq antisense probably resulted from incomplete protein suppression rather than to the additional effect of another G-protein. Nevertheless, it is necessary to point out that, although Gα11 can sustain mAChR-induced M current inhibition in Gαq-deficient mice, we have no direct evidence from the present experiments that Gα11 mediates any part of the response of normal (wild-type) mouse neurons to Oxo-M; the reduced inhibition seen in the presence of the Gαq/11 antibody could equally well have been attributable to antagonism of either endogenous Gαq or Gα11. The large contribution of Gα11 in Gαq −/− neurons might then be an adaptive change, perhaps resulting from a redistribution of G-proteins in the plasma membrane, because there is no evidence for compensatory overexpression of Gα11 in the nervous system of these mice (Offermanns et al., 1997b). Hence, the present observations do not necessarily negate our previous conclusion that mAChR-induced M current inhibition in normal rat neurons results primarily from activation of Gαq. In this context, it should be noted that previous observations have revealed differences between the coupling of muscarinic receptors to ion channels in mouse and rat SCG neurons. Thus, in the mouse, inhibition ofI Ca is mediated by M2 receptors (Shapiro et al., 1999) whereas the corresponding response in rat SCG neurons is mediated by M4 receptors (Bernheim et al., 1992); indeed, although rat neurons possess functional M2receptors capable of activating PTX-sensitive G-proteins, they appear not to inhibit I Ca(Fernandez-Fernandez et al., 1999).
The effects of BK on I K(M) in mouse ganglion cells seem to match those on rat SCG neurons much more closely. Thus, 1 nm BK produced the same amount of inhibition in both, and neither showed any sensitivity to PTX (unlike muscarinic inhibition). Previous experiments on rat neurons using G-protein antibodies (Jones et al., 1995) strongly suggested that inhibition was mediated by either Gαq or Gα11 (or both) but could not identify which. Because in the present experiments inhibition was unchanged in neurons from Gαq −/− mice and then annulled by anti-Gαq/11 antibody, we infer that inhibition in normal SCG neurons is probably mediated exclusively by Gα11 (although with the caveat regarding possible adaptive responses after Gαq deletion expressed above). Lack of any involvement of Gαq would accord with the fact that, unlike mAChR stimulation, BK did not inhibitI Ca in the mouse ganglion cells.
The reason for this apparent selectivity of BK receptors for Gα11 in mouse neurons (and possibly in rat neurons) is unclear. Although it is usually assumed that BK can activate Gαq, we are unaware of any experiments that unequivocally distinguish effects mediated by Gαq from those that might equally be mediated by Gα11. On the contrary, Ricupero et al. (1997) have reported that BK-induced stimulation of phospholipase C was enhanced, rather than inhibited, in mouse embryonic stem cells lacking Gαq, whereas Wilk-Blaszczak et al. (1994b)identified G13 as the G-protein responsible for the PTX-insensitive component of I Cainhibition in neuroblastoma hybrid cells. [In previous experiments, these authors (Wilk-Blaszczak et al., 1994a) had identified Gq/11 as mediating the phospholipase C-driven activation of I K(Ca)in these cells but, because this was from anti-Gq/11 antibody infusion, it could have resulted from activation of Gq,G11, or both.]
In conclusion, the present results point to a surprising degree of divergence at the G-protein level in the coupling of M1 mAChR and B2 BK receptors to Ca2+ and KM + channels in SCG neurons (Fig. 6). Thus, in the mouse ganglion, M1 mAChR inhibition ofI Ca appears to be mediated exclusively (or almost so) by Gαq, whereas inhibition ofI K(M) also involves both Gα11 and an additional unidentified PTX-sensitive G-protein. BK does not inhibitI Ca, and its inhibition ofI K(M) is probably mediated exclusively by Gα11. This divergence at the level of G-protein coupling may go some way toward explaining the apparent divergence in the subsequent steps to M current inhibition after activation of M1 mAChRs or B2 BK receptors, namely, that BK-induced inhibition appears to involve activation of a phospholipase C, whereas M1 muscarinic inhibition does not (Cruzblanca et al., 1998; Haley et al., 1998b). The difference in G-protein coupling from M1 mAChRs to Ca2+ channels and KM + channels might equally imply that, contrary to previous inferences (Hille, 1994), the modulation of these two channels may also involve different transduction pathways.
This work was supported by the Wellcome Trust and the UK Medical Research Council. We thank Mariza Dayrell, Brenda Browning, and Misbah Malik-Hall for tissue culture expertise and Prof. Graeme Milligan (Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK) for the gift of Gαo and Gαq/11 antisera.
Correspondence should be addressed to Prof. David Brown, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. E-mail:.
Dr. Offermanns's present address: Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69–73, 14195 Berlin, Germany.
Dr. Buckley's present address: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.