Abstract
The modulation of voltage-dependent calcium channels by hormones and neurotransmitters has important implications for the control of many Ca2+-dependent cellular functions including exocytosis and contractility1–7. We made use of electrophysiological techniques, including whole-cell patch-clamp recordings from dorsal root ganglion (DRG) neurones, to demonstrate a role for GTP-binding proteins (G-proteins) as signal transducers in the noradrenaline- and γ-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels8–11. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins12); or (2) intracellular administration of guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins13. Our findings provide the first direct demonstration of the G-protein-mediated inhibition of voltage-dependent calcium channels by neurotransmitters. This mode of transmitter action may explain the ability of noradrenaline and GABA to presynaptically inhibit Ca2+-dependent neurosecretion from DRG sensory neurones4,5.
Recordings were obtained from primary cultures of embryonic chick DRG cells (see Fig. 1). When bathed in solutions containing 1 mM Ba2+ and 2 mM Ca2+, these cells generate action potentials with a prominent calcium-dependent plateau phase. The plateau results from a regenerative inward current carried by Ba2+ and Ca2+ ions and is blocked by cobalt14. Previous studies have demonstrated that noradrenaline and GABA decrease the duration of these action potentials by inhibiting the depolarization-induced calcium current8,9. In the present study, a saturating concentration of noradrenaline (50 µM) reduced the action-potential duration in 75% of the cells tested by an average of 43 ± 4.7% (Fig. 1a, Table 1). The inhibitory action of noradrenaline was also observed as a decrease in the Ca current recorded from voltage-clamped DRG cells (Fig. 1b). Similar decreases in action-potential duration and Ca current were also observed during application of GABA (Table 1).
Table 1.
Noradrenaline | GABA | |||
---|---|---|---|---|
Cells responding |
Mean decrease in action- potential duration (%) |
Cells responding |
Mean decrease in action- potential duration (%) |
|
Control | 15/20 | 43 ± 4.7 | 16/19 | 44 ± 4.6 |
PTX-treated | 2/22 | 24 ± 5.0 | 3/16 | 36 ± 2.5 |
Vehicle-treated | 9/10 | 45 ± 6.0 | — | — |
DRG cell responses to 50 µM noradrenaline and 50 µM GABA were recorded as a decrease in action-potential duration measured at 1/2 peak spike amplitude. Only reversible and repeatable responses were included in the results. Under the recording conditions used, measurements of decreases in action-potential duration of <10% were considered unreliable and were therefore scored as no effect. PTX was stored at 4 °C as a stock suspension (1.4 mg ml−1) in saturated (NH4)2SO4. The stock suspension was diluted 1:1,000 in 10 mM sodium phosphate buffer (pH 7.2) containing 50 mM NaCl and 0.04% heat-inactivated bovine serum albumin. Freshly diluted PTX was then diluted 10-fold in MEM containing 0.1% glutamine (no horse serum, nerve growth factor or penicillin-streptomyocin added) to give a final dilution factor of 1:10,000 containing 140 ng ml−1 PTX. DRG cell cultures were incubated in 2 ml of 140 ng ml−1 PTX at 37 °C for 4–8 h. Control cultures were incubated at 37 °C for 4–8 h in MEM containing sodium phosphate buffer but with no added PTX or (NH4)2SO4. Vehicle-treated cultures were incubated at 37 °C for 4–8 h in MEM containing saturated (NH4)2SO4 diluted 1:10,000. All results were obtained from cultures of the same plating. Results are expressed as mean ±s.e.m. We did not observe any direct effects of PTX on the electrical properties of the neurones. The mean resting membrane potential, action-potential duration and action-potential amplitude of PTX-treated cells did not differ significantly from that of untreated cells. Furthermore, the mean amplitude of the Ca current recorded from voltage-clamped cells was similar for both PTX-treated and untreated cells.
Pertussis toxin (PTX) blocks G-protein-mediated responses to hormones and neurotransmitters in many cell types12. Specifically, PTX catalyses ADP-ribosylation of G-proteins, thereby preventing agonist-induced dissociation of the proteins into active subunits15,16. When applied to DRG cells, PTX inhibits the transmitter-induced decrease in action-potential duration (Table 1). Following exposure to PTX (140 ng ml−1), only 9 and 19% of the cells responded to noradrenaline and GABA, respectively. Note that the mean percentage decrease in action-potential duration for those PTX-treated cells which did respond to noradrenaline and GABA was also reduced relative to control. The responses recorded from PTX-treated cells were very slow in onset: the maximal decrease in action-potential duration was observed only after continued application of noradrenaline or GABA for 1–2 min. In contrast, noradrenaline and GABA responses recorded from cells untreated with PTX were rapid in onset, reaching a maximal decrease in action-potential duration after only 20–30 s of continual application.
Recordings from voltage-clamped DRG cells demonstrated that, as expected, PTX also blocked the transmitter-induced decrease in Ca current. Before PTX treatment, noradrenaline (10 µM) reduced the Ca current by 35 ± 6.0% in seven of 8 cells tested. After treatment with 140 ng ml−1 PTX, the fraction of cells responding to noradrenaline was reduced to two of eight, and the average decrease in Ca current was only 13 ± 2.4%. The action of PTX seems to be selective for receptor-mediated alterations in Ca channel function: PTX did not block responses to the diacylglycerol analogue 1,2-oleoyl acetylglycerol (OAG), an activator of protein kinase C that mimics the effects of noradrenaline and GABA on DRG cells17. A saturating concentration of OAG (60 µM) decreased the action-potential duration by an average of 37 ± 2.9% in 74% of the cells tested (n = 19) and decreased the Ca current by 38 ± 5.3% in five of five cells tested (Fig. 1c, d). In cultures treated with PTX (140 ng ml−1), the responses to OAG were not attenuated.
ADP-ribosylation of G-proteins by PTX requires prior internalization and activation of the toxin12. The effects of PTX, therefore, occur with some delay. When PTX was tested on DRG cells, we found its action to be slow in onset and prolonged in duration (Fig. 2a). A progressive decrease in the number of cells responding to noradrenaline was observed after treatment with PTX for 30, 50 and 70 min. A progressive decrease in the magnitude of the response to noradrenaline was also observed. A lag time of ~30 min preceded the inhibitory actions of PTX. To test for recovery, cultures were exposed to 140 ng ml−1 PTX for 4h, washed repeatedly with minimal essential medium (MEM) and re-incubated in culture medium. Even after 48 h of recovery, a total blockade of transmitter action was observed (Fig. 2a).
As expected for a process of internalization, the action of PTX was temperature-dependent. Incubation of cultures in MEM containing 140 ng ml−1 PTX for 4 h at 5 °C did not diminish the response to noradrenaline (Fig. 2b). When cultures were incubated in PTX for 4 h at 23 °C, the number of cells responding to noradrenaline was reduced, and the magnitude of the response decreased relative to the control response. With an increase in incubation temperature to 37 °C, a total blockade of the action of noradrenaline was observed.
The blockade of noradrenaline and GABA responses by PTX suggests that G-proteins mediate the inhibitory actions of transmitters on neuronal Ca channels. To substantiate such a role for G-proteins, DRG cells were voltage-clamped and loaded with GDP-β-S by the whole-cell recording variation of the patch-clamp technique18. GDP-β-S competes with GTP for the guanine nucleotide binding site on G-proteins, thereby blocking GTP-dependent activation of the proteins by hormones and neurotransmitters19–21. Intracellular dialysis of DRG cells with GDP-β-S (100–500 µM) blocked the noradrenaline-induced decrease in Ca current in a dose-dependent manner (Fig. 3). The blockade of noradrenaline responses was observed using concentrations of GDP-β-S similar to that reported to block adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets20. Additional experiments demonstrated that GDP-β-S does not interfere with the action of OAG on DRG cells. Before exposure to GDP-β-S, OAG reduced the Ca current by 38 ± 5.3% (n = 5), whereas after treatment with 250 µM GDP-β-S, OAG decreased the Ca current by 35 ± 3.0% (n = 5).
The noradrenaline and GABA receptors mediating inhibition of DRG cell Ca channels are similar to α-2 adrenergic22 and GABA-B23 receptors, two receptor subtypes known to be negatively coupled to adenylate cyclase24,25. The actions of GDP-β-S and PTX on chick DRG cells may therefore result from their ability to block noradrenaline and GABA receptor-mediated activation of the G-protein (Ni) promoting transmitter inhibition of adenylate cyclase15,20,26,27. In this manner, noradrenaline and GABA would inhibit Ca channel function by lowering intracellular concentrations of cyclic AMP. To test this hypothesis, we dialysed DRG cells with solutions containing 5 mM cAMP and 250 µM 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). As previously reported28, cAMP did not attenuate the noradrenaline-induced inhibition of the Ca current (N = 5 cells). These findings indicate that in DRG cells the PTX substrate mediating transmitter inhibition is a G-protein structurally related to Ni, but not directly coupled to adenylate cyclase. One possibility is that this DRG cell G-protein corresponds to the PTX-sensitive N0 α-protein of relative molecular mass 39,000 isolated from bovine brain29,30.
Previous studies support a role for G-proteins in the receptor-mediated activation of membrane phospholipases31–35. It remains to be determined whether a similar G-protein-mediated stimulation of phospholipase activity underlies the inhibitory actions of noradrenaline and GABA on neuronal Ca channels. The best evidence implicating phospholipases in the inhibition of neuronal Ca channels is the demonstration that the protein kinase C activator OAG36 blocks the DRG cell Ca current in a manner similar to that of noradrenaline and GABA17. Alternatively, G-proteins may directly couple transmitter receptors to the Ca channel. Although these points remain to be resolved, our findings clearly indicate that cellular processes controlling Ca homeostasis are influenced by alterations in the structure and function of G-proteins. In terms of neuronal function, therefore, G-proteins are likely to serve as important intermediaries in processes governing receptor-mediated inhibition or facilitation of Ca2+-dependent neurosecretion.
Acknowledgments
We thank Dr Ron Sekura for the gift of PTX and Drs Paul Brehm and Michael Goy for their advice. This work was supported by grants to K.D. from the Klingenstein Foundation and the American Heart Association.
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