title: Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca²+ channel peptides.
abstract: Modulation of presynaptic voltage-dependent Ca2+ channels is a major means of controlling neurotransmitter release. The CaV2.2Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I-II loop. The NT and I-II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV2 channel activity. Here, we investigate the effects of an amino terminal (CaV2.2[45-55]) 'NT peptide' and a I-II loop alpha interaction domain (CaV2.2[377-393]) 'AID peptide' on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV2.2 amino terminal and I-II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation.
authors.affiliation: School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AJ, UK.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bucci
authors.forename: Sumiko
authors.initials: S
authors.lastname: Mochida
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Stephens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020864:Calcium Channels, N-Type / Q000138:chemical synthesis / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology*; D008969:Molecular Sequence Data; D009433:Neural Inhibition / Q000502:physiology*; D010455:Peptides / Q000138:chemical synthesis / Q000502:physiology*; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D009435:Synaptic Transmission / Q000502:physiology*
chemical_list: C503279:Cacna1b protein, rat; D020864:Calcium Channels, N-Type; D010455:Peptides; D019204:GTP-Binding Proteins