Identification of a novel Vamp1 splice variant in the cochlear nucleus

Abstract

Cochlear nucleus neurons propagate auditory impulses to higher brain stem centers at rapid firing rates with high fidelity. Intrinsic to synaptic transmission are the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins engaged in vesicle fusion, release and recycling. Herein we report a novel splice variant of the SNARE protein Vamp1 (vesicle-associated membrane protein 1) within the cochlear nucleus. We previously demonstrated, through serial analysis of gene expression and microarray studies, that Vamp1 is differentially expressed among the subdivisions of the rat cochlear nucleus. The 3′ end of this transcript, however, was poorly characterized and we could not initially confirm our findings. In this study, we designed RT-PCR primers using conserved 5′ regions and the mouse 3′ domain to validate the expression of Vamp1. Several species of Vamp1 were subsequently amplified from a rat brain cDNA library including a full length clone of Vamp1as and a novel splice variant we termed Vamp1nv. Using regional brain libraries Vamp1nv showed expression in the medulla and lack of expression in the cortex, cerebellum and thalamus. Expression of Vamp1nv was further confirmed and characterized by RT-PCR and real-time PCR in each of the cochlear nucleus subdivisions. The predicted protein sequence for Vamp1nv demonstrates a unique modification of the carboxy-terminal end of the protein as compared to known variants. This includes the appearance of two intra-vesicular serine residues with high predicted potential as kinase phosphorylation sites. Such splice variants of Vamp1 may alter the kinetics of SNARE complex formation and vesicle release and impart unique features to expressing neurons. This may be important for central auditory function and contribute to the distinct physiological properties observed in auditory neurons.