Dynamic association of the Ca2+ channel alpha1A subunit and SNAP-25 in round or neurite-emitting chromaffin cells.
Although the specific interaction between synaptic protein SNAP-25 and the alpha1A subunit of the Cav2.1 channels, which conduct P/Q-type Ca2+ currents, has been confirmed in in vitro-translated proteins and brain membrane studies, the question of how native proteins can establish this association in situ in developing neurons remains to be elucidated. Here we report data regarding this interaction in bovine chromaffin cells natively expressing both proteins. The two carboxyl-terminal splice variants of the alpha1A subunit identified in these cells share a synaptic protein interaction ('synprint') site within the II/III loop segment and are immunodetected by a specific antibody against bovine alpha1A protein. Moreover, both alpha1A isoforms form part of the P/Q-channels-SNARE complexes in situ because they are coimmunoprecipitated from solubilized chromaffin cell membranes by a monoclonal SNAP-25 antibody. The distribution of alpha1A and SNAP-25 was studied in round or transdifferentiated chromaffin cells using confocal microscopy and specific antibodies: the two proteins are colocalized at the cell body membrane in both natural cell types. However, during the first stages of the cell transdifferentiation process, SNAP-25 migrates alone out to the developing growth cone and what will become the nerve endings and varicosities of the mature neurites; alpha1A follows and colocalizes to SNAP-25 in the now mature processes. These observations lead us to propose that the association between SNAP-25 and alpha1A during neuritogenesis might promote not only the efficient coupling of the exocytotic machinery but also the correct insertion of P/Q-type channels at specialized active zones in presynaptic neuronal terminals.
Andrés-Mateos
Eva
E
Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.
Renart
Jaime
J
Cruces
Jesús
J
Solís-Garrido
Luisa M
LM
Serantes
Rocío
R
de Lucas-Cerrillo
Ana M
AM
Aldea
Marcos
M
García
Antonio G
AG
Montiel
Carmen
C
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008958:Models, Molecular; D016501:Neurites / Q000378:metabolism*; D050918:Phosphatidylethanolamine N-Methyltransferase / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment / Q000379:methods; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*
D002118:Calcium; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D004299:Dopamine beta-Hydroxylase; D050918:Phosphatidylethanolamine N-Methyltransferase; D011188:Potassium; D021122:Protein Subunits; D012333:RNA, Messenger; D050825:Synaptosomal-Associated Protein 25