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1. Using perforated-patch recordings, we have examined the part played by endogenous G-protein subunits in the alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ currents in sympathetic neurones. 2. Two components of ICa inhibition by noradrenaline were recorded: a prominent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTX-resistant pathway. 3. PTX-sensitive inhibition was reduced by microinjection of antibodies against either GalphaoA,B or Galphai1,2. The voltage-dependent fraction of inhibition was reduced by anti-Galphao but not by anti-Galphai antibody. 4. Antisense depletion of GalphaoA led to a marked reduction of noradrenaline-induced inhibition and voltage dependence. By contrast, Galphai depletion attenuated noradrenergic modulation without affecting the voltage dependence. 5. Expression of the betagamma-binding agents beta-adrenergic receptor kinase 1 (C-terminus, betaARK1C-ter) or Galphai1 with a Cys3 to Ser mutation partially prevented noradrenergic inhibition while alpha-transducin abolished it. Residual inhibition was mostly voltage independent in cells expressing betaARK1C-ter but was strongly reversed by depolarization in Galphai1 Cys3Ser-expressing cells. 6. Expression of the PTX-resistant Galphai1 Cys351Ile mutant in cells treated with PTX restored alpha2-adrenoceptor inhibition. This restored inhibition was weakly reversed by depolarization. Both the degree and voltage dependence of inhibition were correlated with the level of expression of the Galphai1 Cys351Ile subunit. 7. Our findings identify betagamma dimers associated with GalphaoA and Galphai as mediators of the PTX-sensitive alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ channels. Different betagamma combinations may account for the differential voltage-dependent effects of Go and Gi on ICa.


Journal article


J Physiol

Publication Date





23 - 36


Animals, Antibodies, Blocking, Calcium Channels, Cells, Cultured, Electric Stimulation, Electrophysiology, GTP-Binding Protein alpha Subunits, Gi-Go, Heterotrimeric GTP-Binding Proteins, Immunohistochemistry, Membrane Potentials, Neurons, Norepinephrine, Oligonucleotides, Antisense, Patch-Clamp Techniques, Pertussis Toxin, Plasmids, Rats, Receptors, Adrenergic, alpha-2, Sympathetic Nervous System, Virulence Factors, Bordetella