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The Journal of Neuroscience, June 1, 2000, 20(11):3973-3979

Muscarinic Inhibition of Calcium Current and M Current in G_q-Deficient Mice

Jane E. Haley¹, Patrick Delmas¹, Stefan Offermanns², Fe C. Abogadie¹, Melvin I. Simon², Noel J. Buckley¹, and David A. Brown¹

¹ Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, London, WC1E 6BT, United Kingdom, and ² Division of Biology, California Institute of Technology, Pasadena, California 91125

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 G_q (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 G_q gene.

PTX-insensitive M₁ mAChR inhibition of I_Ca was strongly reduced in G_q / mouse SCG neurons and was fully restored by acute overexpression of G_q. In contrast, M₁ mAChR inhibition of I_K(M) persisted in G_q/ 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 G_q / neurons, and the remaining response was then suppressed by anti-G_q/11 antibodies.

Bradykinin (BK) also inhibits I_K(M) in rat SCG neurons via a PTX-insensitive G-protein (G_q 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 G_q / mice but was abolished by anti-G_q/11 antibody.

We conclude that, in mouse SCG neurons (1) M₁ mAChR inhibition of I_Ca is mediated principally by G_q, (2) M₁ mAChR inhibition of I_K(M) is mediated partly by G_q, 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.

Key words: M current; calcium current; G-protein; superior cervical ganglion neuron; knock-out mouse; muscarinic receptor; bradykinin receptor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 and I_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 of I_Ca and I_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 G_q and G₁₁ (Caulfield et al., 1994) and the expression of separate antisense constructs specific to G_q or G₁₁ (Delmas et al., 1998a; Haley et al., 1998a) suggested that the G-protein primarily involved is G_q. 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-G_q/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 G_q in transducing the effects of M₁ mAChR and B₂ BK receptors, we have therefore investigated the inhibition of I_Ca and I_K(M) by agonists for these receptors in SCG neurons isolated from mice lacking G_q (Offermanns et al., 1997a,b).

Parts of this work have been published previously in abstract form (Haley et al., 1998c).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

G_q-Deficient mice. Mice deficient in the G_q gene (G_q /) were generated by targeted disruption with a neomycin gene as described previously (Offermanns et al., 1997a). G_q-Deficient mice used in the experiments were obtained by mating either G_q knock-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 G_q 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 G_q 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, G_oA+B or G_q/11 antiserum (OC2 and CQ1 respectively, from G. Milligan) (Caulfield et al., 1994, Delmas et al., 1998a, 1999) or an expression plasmid encoding G_q 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 G_q-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 M) 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 G) were initially achieved and, after amphotericin-B permeabilization, access resistances were generally <25 M for I_K(M) recordings and <15 M for I_Ca recordings. 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.

I_K(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 the I_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).

I_Ca recording. Perforated patch recordings were conducted primarily as described previously (Delmas et al., 1999). Pipettes had a resistance of 2-3 M 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 and I_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 G_o or G_q/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 G_q after injection of the G_q plasmid was confirmed using the above protocol and preceding it with a 1 hr incubation with a 1:2000 dilution of a G_q antibody (IQB2) (Milligan et al., 1993).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

M₁ mAChR-induced inhibition of I_Ca and I_K(M) in mouse versus rat SCG

The mAChR agonist Oxo-M inhibited both I_Ca and I_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 for I_K(M) inhibition were 0.4 and 0.7 µM in rat and wild-type mouse, respectively. For M₁ mAChR inhibition of I_Ca, IC₅₀ were 800 nM and 1.1 µM in rat and mouse, respectively.

View larger version (21K): [in this window] [in a new window]   Figure 1.   Dose-response curves (mean ± SEM plus best-fit curve) for Oxo-M inhibition of ICa and IK(M) in rat and mouse SCG. ICa, Left, Oxo-M dose-response curves determined after treatment with PTX, thereby isolating the M1 mAChR-mediated component. n values for each concentration point range from 3 to 7. IK(M), Right, Oxo-M dose-response curve in wild-type mouse SCG (n = 9) is significantly different from that seen in rat SCG (n = 4; p < 0.001). The rat IK(M) dose-response curve data are taken from Haley et al. (1998a), and the rat ICa dose-response curve data are from Delmas et al. (1998a). Legend applies to both panels. All cells were recorded using the perforated patch method.

M₁ mAChR inhibition of I_Ca is reduced in G_q / 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 G_oA and a voltage- and PTX-insensitive M₁ mAChR pathway that mostly involves G_q (Delmas et al., 1998a). In mice, total mAChR inhibition of I_Ca was significantly (p < 0.002) reduced when G_q 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 (G_q +/+) mouse neurons and 29 ± 3% (n = 5) and 57 ± 3% (n = 5) inhibition in G_q / neurons. Inhibition in G_q / 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 G_oA and G_i in rat) (Caulfield et al., 1994; Delmas et al., 1999) was not altered when G_q was absent (wild type, 66 ± 3%, n = 8; G_q /, 62 ± 5%, n = 5).

Incubation with 1 µM PTX inactivates members of the G_o/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 G_q / mice. After PTX treatment, it became clear that the reduction in M₁ plus M₂ inhibition observed in the G_q / cells resulted from an almost complete loss of the voltage-insensitive M₁ mAChR-mediated inhibition (Fig. 2). To be sure that the loss of M₁ mAChR inhibition of I_Ca was attributable to the absence of G_q, we acutely overexpressed G_q in cultured SCG neurons from G_q / mice. Twenty-four hours after injection of a plasmid encoding for G_q, M₁ mAChR inhibition was fully restored (Fig. 2C). This reinstated inhibition was specific because NA inhibition was not rescued by G_q overexpression in PTX-treated cells (Fig. 2). These findings accord with previous data from this laboratory that M₁ mAChR inhibition of I_Ca in rat SCG is primarily mediated by G_q (Delmas et al., 1998a). It should be noted that the I_Ca density (normalized to cell capacitance) was significantly lower in G_q / 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 between I_Ca density in wild-type mouse and rat SCG (rat, 42 ± 2 pA/pF, n = 19).

View larger version (39K): [in this window] [in a new window]   Figure 2.   M1 mAChR inhibition of ICa is markedly reduced in Gq / mice. All experiments were performed on neurons pretreated with PTX. A, ICa waveforms in SCG neurons from wild-type (left) and Gq / mice (right) in the absence and presence of 10 µM Oxo-M, using the three-step voltage protocol illustrated above each waveform. The conditioning step to +90 mV did not reverse Oxo-M inhibition in either wild-type or Gq / neurons, confirming the voltage-independent nature of the PTX-insensitive inhibition. Calibration: 5 msec. B, Gq is expressed in a Gq / neuron after injection of a Gq expression plasmid (injected cell indicated by arrow). C, In Gq / neurons, Oxo-M inhibition of ICa is restored by exogenous expression of Gq. D, Summary of M1 mAChR and noradrenergic inhibitions of ICa. Note that NA inhibition cannot be restored in Gq / SCG neurons by expression of Gq. n ranges from 6 to 10 for Oxo-M data and from 4 to 7 for NA data.

M₁ mAChR inhibition of I_K(M) is not reduced in SCG from G_q / mouse

In rat SCG, inhibition of I_K(M) by mAChR agonists is mediated, at least in part, by G_q (Haley et al., 1998a), so one would predict a loss of inhibition in neurons lacking G_q. We were surprised, therefore, to discover that inhibition of I_K(M) by Oxo-M was not reduced in G_q / SCG compared with wild-type neurons (Fig. 3). Indeed, Oxo-M produced significantly more inhibition in G_q / neurons (p < 0.05), and the dose-response curve lay to the left of the wild-type dose-response (Fig. 3B). I_K(M) density (normalized to cell capacitance) was not changed in G_q / 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; G_q /, 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).

View larger version (22K): [in this window] [in a new window]   Figure 3.   M1 mAChR inhibition of IK(M) is not reduced in Gq / mice. A, IK(M) deactivation relaxation elicited by a 20 mV step for 1 sec from a holding potential of 25 mV. Waveforms (average of 6 traces) are from SCG neurons of wild-type (top) and Gq / (bottom) mice and are shown in the absence and presence of increasing concentrations of Oxo-M (micromolar). Dotted lines indicate 0 pA. Calibration: 250 pA. B, Mean ± SEM data (plus best-fit curves) for Oxo-M inhibition of IK(M) in wild-type and Gq / SCG neurons. Oxo-M dose-response curve in Gq / neurons (n = 8) was significantly different from wild type (n = 9; p < 0.05). IC50 and Hill slope values for inhibition in Gq / cells are 0.6 µM and 1.1, respectively. C, Oxo-M dose-response curves in wild-type and Gq / SCG neurons treated with PTX. IC50 and Hill slope values were 1.3 µM and 0.93, and 1.7 µM and 1.16 in wild-type and Gq / cells, respectively.

M₁ mAChR inhibition of I_K(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 G_q-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 G_q / mice (p < 0.0001) (Fig. 4). In fact, the effect of PTX was greater in G_q-deficient neurons, and the dose-response curve for the PTX-insensitive component of inhibition showed a significant (p < 0.02) rightward shift in G_q / neurons. Thus, at 0.3 µM Oxo-M, the mean inhibition of I_K(M) was reduced from 15 ± 3 to 6 ± 4%. Hence, it appeared that G_q contributed a component of PTX-insensitive inhibition at low agonist concentrations but that, at high agonist concentrations, activation of another PTX-insensitive G-protein predominated.

View larger version (33K): [in this window] [in a new window]   Figure 4.   PTX-sensitive G-proteins and G11 mediate Oxo-M inhibition of IK(M) in Gq / neurons. A, Representative waveforms (1 sec step from 25 to 45 mV) recorded from Gq / neurons treated with PTX and injected with either GoA+B antibody or Gq/11 antibody. Whereas cumulative concentrations of Oxo-M (micromolar) still produce inhibition in the GoA+B antibody-injected cell, there was very little inhibition in presence of the Gq/11 antibody. Dotted lines indicate 0 pA. Calibration: 250 pA. B, Mean ± SEM data (plus best-fit curves) for Oxo-M inhibition of IK(M) in wild-type and Gq / neurons. PTX treatment significantly reduced Oxo-M inhibition in both wild-type (n = 8; p < 0.01) and Gq / neurons (n = 10; p < 0.0001). Injection of a GoA+B antibody had no effect on PTX-treated cells from either wild-type (n = 7) or Gq / (n = 7) mice and served as an injection control. Injection of a Gq/11 antibody reduced the remaining PTX-insensitive inhibition in both the wild-type (n = 4; p < 0.0001 compared with GoA+B antibody-injected cells) and Gq / (n = 5; p < 0.0001 compared with GoA+B antibody-injected cells) neurons. Legend applies to both panels. Insets demonstrate the presence of the injected antibodies in SCG neurons (injected cells indicated by arrow): left, GoA+B antibody in wild-type neuron; right, Gq/11 antibody in Gq / neuron.

One possibility is that this residual PTX-insensitive inhibition might have been mediated by G₁₁, because expression of constitutively activated G₁₁ 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 G_q/11 into the cell cytosol of PTX-treated neurons, using antibodies against G_o as a control (Caulfield et al., 1994). Anti-G_q/11 strongly reduced PTX-insensitive inhibition in neurons from wild-type mice and virtually annulled the residual inhibition in neurons from G_q / mice (p < 0.0001). Thus, the residual PTX-insensitive inhibition in neurons from G_q / mice results from activation of G₁₁ because the antibody is specific to G_q and G₁₁, and G_q is absent.

Inhibition of I_K(M) by BK is mediated by G₁₁ in G_q / 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.

View larger version (28K): [in this window] [in a new window]   Figure 5.   G11 mediates BK inhibition of IK(M) in Gq / neurons. A, Representative waveforms (1 sec step from 25 to 45 mV) recorded from Gq / neurons treated with PTX and injected with GoA+B (left) or Gq/11 (right) antibody. BK (1 nM) inhibited IK(M) in GoA+B-injected neurons but not when the Gq/11 antibody was injected. The dotted lines represent 0 pA. Calibration: 250 pA. B, Mean ± SEM data for inhibition by 1 nM BK in wild-type and Gq / neurons. Neither PTX treatment nor injection of the GoA+B antibody significantly altered inhibition by BK in either wild-type or Gq / neurons. Injection of the Gq/11 antibody reduced BK inhibition in both wild-type and Gq / cells (p < 0.01 for both compared with PTX plus GoA+B antibody).

No significant reduction of BK-induced inhibition of I_K(M) was observed in G_q / neurons. However, injection of an antibody against G_q/11 substantially (p < 0.05 vs anti-G_o antibody) reduced I_K(M) inhibition in wild-type mouse neurons (as in rat neurons; Jones et al., 1995) and abolished inhibition in neurons from G_q / mice (p < 0.01) (Fig. 5B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Using mice lacking G_q, we have demonstrated that M₁ mAChR inhibition of I_Ca requires G_q because regulation was essentially lost in G_q / neurons (Fig. 2). This was not a result of secondary effects on transduction mechanisms arising from an absence of G_q during development because inhibition could be fully restored by acute overexpression of G_q in G_q / 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 G_q 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 G_q is the primary G-protein involved in M₁ mAChR-induced inhibition of I_Ca but is not involved in the inhibition produced by activating M_2/4 mAChRs or ₂ adrenergic receptors. It is especially worth noting (particularly in connection with our observations on I_K(M); see below) that G₁₁ appears not to be able to substitute for G_q in G_q / 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 G₁₁ can inhibit I_Ca just like G_q (unpublished observations; Delmas et al., 1998a).

In contrast [and surprisingly, in view of previous observations in rat SCG neurons using G_q antisense (Haley et al., 1998a)], mAChR-induced inhibition of I_K(M) was not reduced in neurons from G_q / 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 G_q because the dose-response curve for Oxo-M-induced M current inhibition was shifted significantly to the right in G_q / mice. Nevertheless, it was clear that a considerable proportion of the inhibition must have been mediated by another PTX-insensitive G-protein. In G_q-deficient neurons, this G-protein was identifiable as G₁₁, because the residual PTX-insensitive inhibition was virtually annulled using an antibody directed against the unique but common C terminus to G_q and G₁₁. Thus, in the G_q-deficient mice, we conclude that mAChR-induced M current inhibition is maintained by G₁₁ 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 G₁₁. 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 inhibit I_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 G₁₁, in previous experiments on rat neurons, anti-G_q 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 G_q / mouse neurons. However, although this might also suggest the involvement of another PTX-insensitive G-protein, antisense to G₁₁ had no effect on the inhibitory effect of Oxo-M in rat neurons. Hence, it was concluded that the limited effect of G_q 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 G₁₁ can sustain mAChR-induced M current inhibition in G_q-deficient mice, we have no direct evidence from the present experiments that G₁₁ mediates any part of the response of normal (wild-type) mouse neurons to Oxo-M; the reduced inhibition seen in the presence of the G_q/11 antibody could equally well have been attributable to antagonism of either endogenous G_q or G₁₁. The large contribution of G₁₁ in G_q / 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 G₁₁ 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 G_q. 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 of I_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 M₂ receptors 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 G_q or G₁₁ (or both) but could not identify which. Because in the present experiments inhibition was unchanged in neurons from G_q / mice and then annulled by anti-G_q/11 antibody, we infer that inhibition in normal SCG neurons is probably mediated exclusively by G₁₁ (although with the caveat regarding possible adaptive responses after G_q deletion expressed above). Lack of any involvement of G_q would accord with the fact that, unlike mAChR stimulation, BK did not inhibit I_Ca in the mouse ganglion cells.

The reason for this apparent selectivity of BK receptors for G₁₁ in mouse neurons (and possibly in rat neurons) is unclear. Although it is usually assumed that BK can activate G_q, we are unaware of any experiments that unequivocally distinguish effects mediated by G_q from those that might equally be mediated by G₁₁. 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 G_q, whereas Wilk-Blaszczak et al. (1994b) identified G₁₃ as the G-protein responsible for the PTX-insensitive component of I_Ca inhibition in neuroblastoma hybrid cells. [In previous experiments, these authors (Wilk-Blaszczak et al., 1994a) had identified G_q/11 as mediating the phospholipase C-driven activation of I_K(Ca) in these cells but, because this was from anti-G_q/11 antibody infusion, it could have resulted from activation of G_q,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 K_M⁺ channels in SCG neurons (Fig. 6). Thus, in the mouse ganglion, M₁ mAChR inhibition of I_Ca appears to be mediated exclusively (or almost so) by G_q, whereas inhibition of I_K(M) also involves both G₁₁ and an additional unidentified PTX-sensitive G-protein. BK does not inhibit I_Ca, and its inhibition of I_K(M) is probably mediated exclusively by G₁₁. 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 K_M⁺ channels might equally imply that, contrary to previous inferences (Hille, 1994), the modulation of these two channels may also involve different transduction pathways.

View larger version (23K): [in this window] [in a new window]   Figure 6.   Diagram to summarize projected G-protein involvement for M1 mAChR- and B2 BK receptor-induced inhibition of ICa and IK(M) channels in rat SCG neurons and in wild-type and Gq-deficient mouse SCG neurons.

	FOOTNOTES

Received Nov. 9, 1999; revised Feb. 28, 2000; accepted March 17, 2000.

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 G_o and G_q/11 antisera.

Correspondence should be addressed to Prof. David Brown, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. E-mail: d.a.brown{at}ucl.ac.uk.

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.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Ashkenazy A, Peralta EG, Winslow JW, Ramachandran J, Capon D (1989) Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 56:487-493[ISI][Medline].
• Bernheim L, Mathie A, Hille B (1992) Characterization of muscarinic receptor subtypes inhibiting Ca²⁺ current and M current in rat sympathetic neurons. Proc Natl Acad Sci USA 89:9544-9548[Abstract/Free Full Text].
• Brown DA (1988) M currents. In: Ion channels, Vol 1 (Narahashi T, ed), pp 55-99. New York: Plenum.
• Brown DA (1999) M-Current: from discovery to single channel currents. In: Slow synaptic responses and modulation (Kuba K, ed), pp 15-26. Berlin: Springer.
• Brown DA, Marrion NV, Smart TG (1989) On the transduction mechanism for muscarine-induced inhibition of M-current in rat sympathetic neurons. J Physiol (Lond) 413:469-488[Abstract/Free Full Text].
• Caulfield MP, Jones S, Vallis Y, Buckley NJ, Kim G-D, Milligan G, Brown DA (1994) Muscarinic M-current inhibition via G_q/11 and -adrenoceptor inhibition of Ca²⁺ current via G_o in rat sympathetic neurones. J Physiol (Lond) 477:415-422[ISI][Medline].
• Cruzblanca H, Koh D-S, Hille B (1998) Bradykinin inhibits M current via phospholipase C and Ca²⁺ release from IP₃-sensitive Ca²⁺ stores in rat sympathetic neurons. Proc Natl Acad Sci USA 95:7151-7156[Abstract/Free Full Text].
• Davies PJ, Ireland DR, McLachlan EM (1996) Sources of Ca²⁺ for different Ca²⁺-activated K⁺ conductances in neurones of the rat superior cervical ganglion. J Physiol (Lond) 495:353-366[ISI][Medline].
• Delmas P, Abogadie FC, Dayrell M, Haley JE, Milligan G, Caulfield MP, Brown DA, Buckley NJ (1998a) G-proteins and G-protein subunits mediating cholinergic inhibition of N-type calcium currents in sympathetic neurons. Eur J Neurosci 10:1654-1666[ISI][Medline].
• Delmas P, Brown DA, Dayrell M, Abogadie FC, Caulfield MP, Buckley NJ (1998b) On the role of endogenous G-protein subunits in N-type Ca²⁺ current inhibition by neurotransmitters in rat sympathetic neurones. J Physiol (Lond) 506:319-329[Abstract/Free Full Text].
• Delmas P, Abogadie FC, Milligan G, Buckley NJ, Brown DA (1999) dimers derived from G_o and G_i proteins contribute different components of adrenergic inhibition of Ca²⁺ channels in rat sympathetic neurones. J Physiol (Lond) 518:23-36[Abstract/Free Full Text].
• Fernandez-Fernandez JM, Wanaverbecq N, Halley P, Caulfield MP, Brown DA (1999) A role for M₂ receptors in the muscarinic activation of G protein-gated K⁺ (GIRK) channels expressed in isolated rat sympathetic neurones. J Physiol (Lond) 515:631-637[Abstract/Free Full Text].
• Gudermann T, Kalkbrenner F, Schultz G (1996) Diversity and selectivity of receptor-G protein interaction. Annu Rev Pharmacol Toxicol 36:429-459[ISI][Medline].
• Haley JE, Abogadie FC, Delmas P, Dayrell M, Vallis Y, Milligan G, Caulfield MP, Brown DA, Buckley NJ (1998a) The  subunit of G_q contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons. J Neurosci 18:4521-4531[Abstract/Free Full Text].
• Haley JE, Abogadie FC, Dayrell M, Buckley NJ, Brown DA (1998b) Antisense against phospholipase C-4 reduces inhibition of M-current (I_K(M)) by bradykinin, but not by the muscarinic agonist oxotremorine-M (Oxo-M) in rat sympathetic ganglion (SCG) neurones. J Physiol (Lond) 515P:131P.
• Haley JE, Delmas P, Offermanns S, Abogadie FC, Buckley NJ, Simon MI, Brown DA (1998c) Muscarinic M1 inhibition of calcium current, but not M-type potassium current, is reduced in Gq knockout mice. Naunyn Schmiedebergs Arch Phamacol 358:R646.
• Hamilton SE, Loose MD, Qi M, Levey AI, Hille B, McKnight GS, Idzerda RL, Nathanson NM (1997) Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice. Proc Natl Acad Sci USA 94:13311-13316[Abstract/Free Full Text].
• Hille B (1994) Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci 17:531-536[ISI][Medline].
• Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92:145-159[Abstract/Free Full Text].
• Jones S, Brown DA, Milligan G, Willer E, Buckley NJ, Caulfield MP (1995) Bradykinin excites rat sympathetic neurons by inhibition of M current through a mechanism involving B₂ receptors and G_q/11. Neuron 14:399-405[ISI][Medline].
• Jones SW, Adams PR (1987) The M-current and other potassium currents of vertebrate neurons. In: Neuromodulation (Kaczmarek LK, Levitan IB, eds), pp 159-186. New York: Oxford UP.
• Marrion NV, Smart TG, Marsh SJ, Brown DA (1989) Muscarinic suppression of the M-current in the rat sympathetic ganglion is mediated by receptors of the M₁-subtype. Br J Pharmacol 98:557-573[ISI][Medline].
• Milligan G, Mullaney I, McCallum JF (1993) Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins G_q and G₁₁: absence of G₁₁ in human platelets and haemopoietically derived cell lines. Biochem Biophys Acta 1179:208-212[Medline].
• Offermanns S, Wieland T, Homann D, Sandmann J, Bombien E, Spicher K, Schultz G, Jakobs KH (1994) Transfected muscarinic actelycholine receptors selectively couple to G_i-type G proteins and G_q/11. Mol Pharmacol 45:890-898[Abstract].
• Offermanns S, Hashimoto K, Watanabe M, Sun W, Kurihara H, Thompson RF, Inoue Y, Kano M, Simon MI (1997a) Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Gq. Proc Natl Acad Sci USA 94:14089-14094[Abstract/Free Full Text].
• Offermanns S, Toombs CF, Hu Y-H, Simon MI (1997b) Defective platelet activation in G_q-deficient mice. Nature 389:183-186[Medline].
• Rae J, Cooper K, Gates P, Watsky M (1991) Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37:15-26[ISI][Medline].
• Ricupero DA, Polgar P, Taylor L, Sowell MO, Gao Y, Bradwin G, Mortensen RM (1997) Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein _q subunit. Biochem J 327:803-809.
• Sah P (1996) Ca²⁺-activated K⁺ currents in neurones: types, physiological roles and modulation. Trends Neurosci 19:150-154[ISI][Medline].
• Schofield GG (1991) Norepinephrine inhibits a Ca²⁺ current in rat sympathetic neurons via a G-protein. Eur J Pharmacol 207:195-207[ISI][Medline].
• Shapiro MS, Loose MD, Hamilton SE, Nathanson NM, Gomeza J, Wess J, Hille B (1999) Assignement of muscarinic receptor subtypes mediating G-protein modulation of Ca²⁺ channels by using knockout mice. Proc Natl Acad Sci USA 96:10899-10904[Abstract/Free Full Text].
• Stein R, Pinkas-Kramarski R, Sokolovsky M (1988) Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover. EMBO J 7:3031-3035[ISI][Medline].
• Wang H-S, McKinnon D (1995) Potassium currents in rat prevertebral and paravertebral sympathetic neurones: control of firing properties. J Physiol (Lond) 485:319-335[ISI][Medline].
• Wilk-Blaszczak MA, Gutowski S, Sternweis PC, Belardetti F (1994a) Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways. Neuron 12:109-116[ISI][Medline].
• Wilk-Blaszczak MA, Singer WD, Gutowski S, Sternweis PC, Belardetti F (1994b) The G protein G₁₃ mediates inhibition of voltage-dependent calcium current by bradykinin. Neuron 13:1215-1224[ISI][Medline].
• Zhu Y, Ikeda SR (1994) Modulation of Ca²⁺-channel currents by protein kinase C in adult rat sympathetic neurons. J Neurophysiol 72:1549-1560[Abstract/Free Full Text].

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