Association of CaV1.3 L-type calcium channels with Shank.
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.
Zhang
Hua
H
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
Maximov
Anton
A
Fu
Yu
Y
Xu
Fang
F
Tang
Tie-Shan
TS
Tkatch
Tatiana
T
Surmeier
D James
DJ
Bezprozvanny
Ilya
I
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000502:physiology; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry* / Q000502:physiology; D020746:Calcium Channels, L-Type / Q000737:chemistry*; D020013:Calcium Signaling; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D006624:Hippocampus / Q000196:embryology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C120531:Shank1 protein, rat; C120532:Shank3 protein, rat; C093712:Cacna1d protein, rat