Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons^*

Zhiping P. Pang¹², Wei Xu¹, Peng Cao, , and Thomas C. Südhof³

From the Department of Molecular and Cellular Physiology and
the Howard Hughes Medical Institute, Stanford University, Palo Alto, California 94304-5543

ABSTRACT Back to Top

Abstract: Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca²⁺ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca²⁺ binding to an unidentified secondary Ca²⁺ sensor. To test whether CaM could be the enigmatic Ca²⁺ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of 70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca²⁺ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca²⁺/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.

Key Words: Calcium • Calmodulin • Calcium Calmodulin-dependent Protein Kinase (CaMK) • Cell Adhesion • Exocytosis • Neurobiology • Neuroscience • Synapses

Received for publication June 1, 2010. Revision received July 23, 2010.

FOOTNOTES Back to Top

² To whom correspondence may be addressed: Dept. of Molecular and Cellular Physiology, Stanford University, 1050 Arastradero Rd., Palo Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: zpang{at}stanford.edu.

³ To whom correspondence may be addressed: Dept. of Molecular and Cellular Physiology, Stanford University, 1050 Arastradero Rd., Palo Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: tcs1{at}stanford.edu.

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