CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release

Channels (Austin). Jul-Aug 2010;4(4):266-77. doi: 10.4161/chan.4.4.12130. Epub 2010 Jul 18.

Abstract

It is well-established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca(2+) channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca(2+)-independent coupling of the human neuronal P/Q-type calcium channel (Ca(V)2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca(V)2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca(V)1.2, Ca(V)2.2 and Ca(V)2.3. The distinct kinetic properties of Ca(V)2.1 acquired by close association with Syt1 and t-SNAREs, suggests that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca(2+). To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted release mediated by Ca(V)2.1 is consistent with the model in which the VGCC plays a signaling role in triggering release, acting from within the exocytotic complex. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca(2+) entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.

MeSH terms

  • Animals
  • Botulinum Toxins / pharmacology
  • Botulinum Toxins, Type A / pharmacology
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Calcium Channels, N-Type / genetics
  • Calcium Channels, N-Type / metabolism*
  • Calcium Signaling* / drug effects
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Chelating Agents / pharmacology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Electric Capacitance
  • Evoked Potentials
  • Exocytosis* / drug effects
  • Female
  • Humans
  • Ion Channel Gating* / drug effects
  • Kinetics
  • Microinjections
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Oocytes
  • Patch-Clamp Techniques
  • R-SNARE Proteins / metabolism
  • Rabbits
  • Rats
  • Synaptic Transmission* / drug effects
  • Synaptosomal-Associated Protein 25 / metabolism
  • Synaptotagmin I / metabolism
  • Syntaxin 1 / metabolism
  • Xenopus laevis

Substances

  • CACNA1A protein, human
  • Calcium Channels
  • Calcium Channels, N-Type
  • Chelating Agents
  • Nerve Tissue Proteins
  • R-SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Synaptotagmin I
  • Syntaxin 1
  • voltage-dependent calcium channel (P-Q type)
  • Egtazic Acid
  • Botulinum Toxins
  • Botulinum Toxins, Type A
  • botulinum toxin type C
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid