Dynamic association of the Ca²⁺ channel α_1A subunit and SNAP‐25 in round or neurite‐emitting chromaffin cells

Abstract

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.

Calcium channel subtypes differentially regulate fusion pore stability and expansion

“…In addition to the evidences yielded by the functional studies aforementioned, molecular techniques have also indicated that P/Q Ca 2+ channels are tightly coupled to the exocytotic machinery in chromaffin cells. Two P/Q channel α 1 subunit isoforms have been identified in bovine chromaffin cells, which share a synprint site and interact with SNAP‐25 in situ (Andrés‐Mateos et al. 2005).…”

The Immediately Releasable Pool of Mouse Chromaffin Cell Vesicles Is Coupled to P/Q-Type Calcium Channels via the Synaptic Protein Interaction Site

“…Therefore, it is possible that a similar type of interaction between dense core vesicles and Ca 2+ channels may occur for the IRP in chromaffin cells. Previous results that show the feasibility of this hypothesis include the identification of synprint in different splice variants of the P/Q α 1A subunit in bovine chromaffin cells; the co-immunoprecipitation of P/Q channels and the SNARE complex with a monoclonal antibody against SNAP-25; and the co-localization of α 1A and SNAP-25 at the membrane of intact chromaffin cells [20].…”

Bovine CACNA1A gene and comparative analysis of the CAG repeats associated to human spinocerebellar ataxia type-6

“…In fact, the immunodetection of both variants of the protein in bovine chromaffin cells has been previously reported by our group (Andres-Mateos et al, 2005). In the short variant exon 47 codes only for the stop codon (TAG).…”