Campbell, Veronica Ann; (1995) Components of neuronal calcium channels and their interaction with GTP-binding proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Immunocytochemistry utilising antipeptide antibodies and confocal microscopy was used in this study to reveal the localisation of components of voltage-dependent calcium channels in cultured dorsal root ganglion neurones. The αl subunits of the dihydropyridine-, ω-Conotoxin GVIA-, and ω-Agatoxin-IVA-sensitive calcium channels were found to have different localisations within the soma and neurite processes of dorsal root ganglion neurones. The calcium channel B-subunits were found to have an intracellular localisation, while the α2/δ component was found to be wholly exofacial. Immunoreactivity associated with the a subunits of the GTP-binding proteins GO and Gi was found to occur on the cytoplasmic surface of the somal membrane and neurite processes in these cells. Antisense oligonucleotides were used to transiently suppress the expression of calcium channel B-subunits or a-subunits of the GTP-binding proteins Go and Gi. The antisense oligonucleotides, complementary to a conserved sequence on the mRNA of calcium channel B- subunits or Go/Gi a-subunits, were microinjected into the cytoplasm of dorsal root ganglion neurones and the effect on levels of the target proteins was monitored using confocal microscopy. The calcium channel B-subunit antisense oligonucleotide produced a maximal 90% decrease in B- subunit levels 110 hours after microinjection of the oligonucleotide. In contrast, the GTP-binding protein a subunit antisense oligonucleotides produced an approximate 80 % reduction in levels of Gai and Gao 24 hours after microinjection. The dihydropyridine calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of GTP hydrolysis in rat frontal cortex membranes, the maximal stimulation of GTPase activity being observed with 10 nM (-)-BayK 8644. Affinity purified anti-GTP-binding protein antibodies, raised against the a subunit of either GO or Gi, revealed that the stimulation of GTPase by (-)-BayK 8644 was due exclusively to activation of the intrinsic GTPase activity of the GAO subtype of GTP-binding protein. In contrast, the GABABergic stimulation of GTP hydrolysis was found to be coupled to both the Gao and Gai GTP-binding protein subtypes in this preparation. An antipeptide antiserum which recognises neuronal calcium channel B-subunits abolished the (-)-BayK 8644-stimulated GTP hydrolysis of GAO and also caused a 30 % reduction in the dihydropyridine binding capacity of the cortical membranes. Furthermore, a peptide which mimics the B-subunit binding domain on the calcium channel a1 subunit, also abolished (-)-BayK 8644- mediated stimulation of GTPase. The findings from this study suggest that dihydropyridine agonist binding is coupled to an increase in GTPase activity. The calcium channel B-subunit appears to be the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Gao. This provides a possible mechanism by which calcium channels can act to limit the duration of a GTP-binding protein modulatory signal.

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