Acute desensitization of nociceptin/orphanin FQ inhibition of voltage-gated calcium channels in freshly dissociated hippocampal neurons.

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like receptor, has been shown to inhibit high-voltage-gated calcium channels (VGCCs) in acutely dissociated rat hippocampal pyramidal cells [Knoflach, F., Reinscheid, R.K., Civelli, O. & Kemp, J.A. (1996), J. Neurosci., 16, 6657]. In this study, it was further demonstrated that N/OFQ inhibition of calcium channel current was blocked by its specific antagonist PGN, [Phe1-psi(CH2-NH)-Gly2]nociceptin (1-13)-NH2, and the EC50 of the N/OFQ inhibition was approximately 10 nM, indicating that this effect was really mediated via the opioid receptor-like receptor. The N/OFQ inhibition of the calcium channel current was significantly reduced, as the maximal inhibition decreased from 36 to 23%, by 1-min pretreatment of freshly dissociated hippocampal neurons with the same peptide. The inhibition completely recovered from this acute desensitization in less than 20 min. The N/OFQ inhibition was also greatly attenuated by pretreatment of the neurons with the GABAB (gamma-aminobutyric acid) agonist baclofen while the baclofen inhibition of the calcium channel current was significantly reduced by N/OFQ pretreatment, revealing the agonist-induced desensitization was heterologous in nature. This desensitization was blocked by pretreating the neurons with the sodium channel blocker, tetrodotoxin (TTX), or by removing the extracellular calcium, which indicates the necessity of membrane depolarization and extracellular calcium influx in the process. Furthermore, pretreatment of the neurons with the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), attenuated the N/OFQ inhibition of the calcium channel current whereas the cAMP-dependent kinase A activator, forskolin, showed no effect, suggesting the probable involvement of PKC in the N/OFQ-induced desensitization.

The amino terminus with a conserved glutamic acid of G protein-coupled receptor kinases is indispensable for their ability to phosphorylate photoactivated rhodopsin.

To investigate functions of the consensus amino terminus of G protein-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.