GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels

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 contractility. 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 gamma-aminobutyric acid (GABA)-induced inhibition of voltage-dependent calcium channels. This action of the transmitters was blocked by: (1) preincubation of the cells with pertussis toxin (a bacterial exotoxin catalysing ADP-ribosylation of G-proteins); or (2) intracellular administration of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of GTP to G-proteins. 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 neurones.

Fig. 1

Noradrenaline and OAG decrease action-potential duration and Ca current in chick DRG cells. a, c, Current-clamp recordings of DRG cell action potentials; b, d, voltage-clamp recordings of Ca currents. Traces were recorded before (1), during (2) and after (3) treatment with either 50 µM noradrenaline (a, b) or 60 µM OAG (c, d). Action potentials were evoked by direct current injection through the recording electrode. Ca currents were evoked by a +50 mV step depolarization from a holding potential of −60 mV. At this holding potential the predominant inward current is through non-inactivating Ca channels. Scale bars: a, 20 mV, 5 ms; b, 4 nA, 40 ms; c, 20 mV, 10 ms; d, 2 nA, 40 ms. Methods. Primary cultures of chick DRG cells were prepared as previously described,. Briefly, ganglia from 11–12 day-old embryos were dissociated in a Ca²⁺- and Mg²⁺-free Pucks solution, suspended in MEM (supplemented with nerve growth factor, 5% chick embryo extract, 10% horse serum, 2 mM glutamine, penicillin (50 U ml⁻¹) and streptomyocin (50 µg ml⁻¹), γ-irradiated (5,000 rads) and plated in 35-mm collagen-coated tissue culture dishes. Action potentials were recorded using an amplifier with an active bridge circuit allowing current injection through the recording microelectrodes (40–80 MΩ, filled with 2 M KCl). The bathing solution contained (mM): 132 NaCl, 2.5 KCl, 2.0 CaCl₂, 1.0 BaCl₂, 0.8 MgCl₂ and 25 HEPES (pH 7.4). Ca currents were recorded as described previously, using the whole-cell configuration of the patch-clamp technique. The pipette solution contained (mM): 150 CsCl, 5 bis(o-aminophenoxy)ethane-N,N,N′,N,-tetraacetic acid (BAPTA), 5 MgATP and 10 HEPES (pH 7.3); the external bathing solution contained 133 NaCl, 1 CaCl₂, 10 tetraethylam-monium, 0.3 µM tetrodotoxin and 25 HEPES (pH 7.3). Little or no rundown of the Ca current was observed during the first 10–15 min of recording. Noradrenaline, GABA and OAG (Sigma) were dissolved in the appropriate bathing solution and pressure ejected from blunt-tipped pipettes as previously described.

Fig. 2

Time- and temperature-dependent blockade by PTX of DRG cell responses to noradrenaline. a, Time dependence of the action of PTX. DRG cell cultures were incubated in MEM containing 140 ng ml⁻¹ PTX at 37 °C for 30, 50 and 70 min (circles). Control cultures (square) were incubated in MEM (without added PTX or (NH₄)₂SO₄) at 37 °C for 1 h. Recovery from PTX-treatment was tested at 48 h following washout of PTX (triangle). The results are expressed as the mean (±s.c.m.) percentage decrease in action-potential duration in response to 50 µM noradrenaline. In parentheses is indicated the fraction of cells tested that responded to noradrenaline. b, Temperature dependence of the action of PTX. Cultures were incubated in MEM containing 140 ng ml⁻¹ PTX for 4 h at 5, 23 and 37 °C (circles). Control cultures were incubated in MEM for 4 h at 5 °C (square). The fraction of cells responding to noradrenaline is also indicated in parentheses. The medium was HEPES-buffered and equilibrated with air when incubating at 5 and 23 °C. All results in a and b were obtained from cultures of the same plating.

Fig. 3

Blockade by GDP-β-S of DRG cell responses to noradrenaline. DRG cells were dialysed for 5 min with 100 (a), 250 (b) and 500 (c) µM GDP-β-S (trilithium salt, Boehringer Mannheim) by inclusion of the GDP analogue in the patch pipette solution, which allows diffusional exchange between the intracellular fluid contents and the pipette solution. The Ca current was evoked from a holding potential of −60 mV in response to depolarizing test pulses of 50–60 mV. The amplitude of the Ca current was monitored at 10-s intervals. The mean decrease in Ca current in response to noradrenaline (10 µM) is indicated for untreated control cells (unshaded columns) and cells dialysed with GDP-β-S (shaded columns). The responses to noradrenaline obtained from cells treated with GDP-β-S were compared with responses recorded from untreated cells in the same culture dish. All results shown were obtained from cultures of the same plating. Error bars indicate s.e.m. (number of cells is indicated in parentheses). GDP-β-S did not directly inhibit the Ca current, nor did it accelerate run-down of the current: the mean amplitude of the current recorded from 250 µM GDP-β-S-treated and untreated cells was similar after dialysis for 5 min (treated, 2.7 ± 0.3 nA, n = 10; untreated, 3.0 ± 0.4 nA, n = 11). Dialysis of DRG cells with 1.5 mM LiCl (n = 9) did not reduce the response to noradrenaline relative to the control response.