Association of CaV1.3 L-type calcium channels with Shank

J Neurosci. 2005 Feb 2;25(5):1037-49. doi: 10.1523/JNEUROSCI.4554-04.2005.

Abstract

Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. Both CaV1.2 and CaV1.3 subunits contain class I PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-binding consensus at their C termini. In yeast two-hybrid screen of rat brain cDNA library with the C-terminal bait of CaV1.3a (long C-terminal splice variant) L-type Ca2+ channel subunit, we isolated multiple clones of postsynaptic adaptor protein Shank. We demonstrated a specific association of CaV1.3a C termini, but not of CaV1.2 C termini, with Shank PDZ domain in vitro. We further demonstrated that the proline-rich region present in C termini of CaV1.3a subunit binds to Shank Src homology 3 domain. We established that CaV1.3a and Shank localized to postsynaptic locations in cultured rat hippocampal neurons. By expressing epitope-tagged recombinant CaV1.3 subunits in rat hippocampal neuronal cultures, we demonstrated that the presence of Shank-binding motifs in CaV1.3a sequence is both necessary and sufficient for synaptic clustering of CaV1.3 L-type Ca2+ channels. In experiments with dominant-negative peptides and dihydropyridine-resistant CaV1.3a mutants, we demonstrated an importance of Shank-binding motif in CaV1.3a sequence for phosphorylated cAMP response element-binding protein (pCREB) signaling in cultured hippocampal neurons. Our results directly link CaV1.3 neuronal L-type Ca2+ channels to macromolecular signaling complex formed by Shank and other modular adaptor proteins at postsynaptic density and provide novel information about the role played by CaV1.3 L-type Ca2+ channels in pCREB signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Adaptor Proteins, Signal Transducing / physiology
  • Animals
  • Calcium Channels / chemistry*
  • Calcium Channels / physiology
  • Calcium Channels, L-Type / chemistry*
  • Calcium Signaling
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Hippocampus / embryology
  • Hippocampus / metabolism*
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / physiology
  • Neurons / metabolism*
  • Oocytes
  • Protein Binding
  • Protein Interaction Mapping
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Two-Hybrid System Techniques
  • Xenopus laevis

Substances

  • Adaptor Proteins, Signal Transducing
  • Calcium Channels
  • Calcium Channels, L-Type
  • Cyclic AMP Response Element-Binding Protein
  • Nerve Tissue Proteins
  • Recombinant Fusion Proteins
  • Shank1 protein, rat
  • Shank3 protein, rat
  • Cacna1d protein, rat