Exotoxin-insensitive G proteins mediate synaptically evoked muscarinic sodium current in rabbit sympathetic neurones.
1. The involvement of G proteins in the transduction pathway that links muscarinic receptors to the low-threshold voltage-dependent sodium current (INa,M) was studied in neurones from intact sympathetic prevertebral ganglia using the whole-cell configuration of the patch-clamp technique. Experiments were performed in the presence of the nicotinic receptor antagonists hexamethonium (50 microM) and d-tubocurarine (50 microM). 2. INa,M was activated by either bath-applying muscarinic agonists or stimulating the preganglionic splanchnic nerves. Synaptically and agonist-mediated INa,M did not display significant run-down or changes in their properties in cells tested, irrespective of whether the pipette solutions contained GTP. 3. Dialysis of sympathetic neurones with GDP beta S (500-750 microM) decreased the amplitude of INa,M by approximately 65% compared with control neurones within 30 min. 4. In the absence of muscarinic receptor stimulation, intracellular dialysis with GTP gamma S (500 microM) for 10 min slowly and slightly (20-25%) activated INa,M. GTP gamma S dialysis markedly slowed down the decay of INa,M after its transient activation with carbachol pulses (10-20 s) or nerve stimulation (3-5 s). The INa,M activation became fully irreversible 2.9 min after the start of GTP gamma S dialysis. Dialysing cells with the G protein activator AIF4-led to a rapid but transient activation of INa,M. 5. Synaptically and agonist-evoked INa,M were not affected in ganglia treated with 0.5-1 microgram ml-1 pertussis toxin (PTX) for 7-24 h at 37 degrees C. Control experiments showed that this treatment severely reduced the PTX-sensitive inhibition of N-type calcium currents induced by carbachol (CCh) and noradrenaline. Application of NEM (N-ethylmaleimide) for 2 min depressed the INa,M evoked in response to bath-applied CCh by only 27%. 6. Incubating ganglia with 5-10 micrograms ml-1 of cholera toxin for 7 h had no effect on the carbachol-induced INa,M but greatly potentiated (approximately 250%) the synaptically evoked INa,M, presumably via a presynaptic mechanism. 7. These results show that the coupling between muscarinic receptors and NaM channels is mediated by pertussis toxin- and cholera toxin-insensitive G proteins, possibly of the Gq/11 or G12 class.
Delmas
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Laboratoire de Neurobiologie LNB4, CNRS, Marseille, France.
Gola
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D000818:Animals; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D004594:Electrophysiology; D005098:Exotoxins / Q000494:pharmacology*; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D005728:Ganglia, Sympathetic / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D018721:Muscarinic Agonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013564:Sympathetic Nervous System / Q000187:drug effects / Q000502:physiology*; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D005098:Exotoxins; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D015222:Sodium Channels; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D005459:Fluorides