A calcitonin gene-related peptide-induced signaling pathway directs the synaptic expression of collagen-tail subunit (ColQ) of acetylcholinesterase in muscle

In the vertebrate neuromuscular junction (nmj), the asymmetric acetylcholinesterase is anchored to synaptic basal lamina through a collagen-tailed subunit (ColQ). There are two types of ColQ mRNAs, namely ColQ-1 and ColQ-1a which are different in their first exons. ColQ-1 mRNA is mainly expressed in slow-twitch muscle throughout the entire muscle fiber while ColQ-1a mRNA is expressed in fast-twitch muscle with a synapse-specific manner. The differential expressions of ColQ-1 and ColQ-1a mRNAs in different muscles are regulated by two different promoters, namely pColQ-1 and pColQ-1a (Lee. et al., 2004). However, the molecular mechanism in regulating the ColQ synaptic expression in muscle is still not clear. The level of ColQ mRNAs in rat muscles dropped dramatically after denervation, which suggested that nerve-derived factors and nerve-induced electrical activity might play a regulatory role(s). The ColQ promoters tagged with a luciferase reporter gene (pColQ-1-Luc and pColQ-1a-Luc) are used as tools to map out the gene regulations in muscle. The promoter constructs were shown functionally intact muscle cell (Lee. et al., 2004). Calcitonin gene-related peptide (CGRP), a nerve-derived factor, induced a cAMP-dependent signaling pathway in muscle, which phosphorylated cAMP-responsive regulatory element binding protein (CREB) and up-regulated the mRNA and promoter activity of ColQ-1a but not ColQ-1. Application of cAMP potentiated the CREB-mediated ColQ-1a expression. Two cAMP responsive elements were identified in ColQ-1a promoter. In addition, the endogenous level of CREB is much higher in fast muscle (e.g. tibialis) than in slow muscle (e.g. soleus). These results indicated that CGRP-mediated cAMP-dependent pathway may act as one of crucial regulatory mechanisms to drive the synaptic expression of ColQ-1a in fast muscle. ColQ-1 and ColQ-1a promoter activities were suppressed by the application of tetrodotoxin, a sodium channel blocker disrupting the muscular activity. Moreover, ColQ mRNA and promoter expressions were induced by A23187 which is a calcium ionophore elevating intracellular calcium level. These results suggested that the nerve-induced muscular activity could induce ColQ gene expression. Various signaling molecules such as Calcium/Calmudulin-Dependent Protein Kinase II (CaMK II) and calcineurin are regulated by intracellular calcium, and their roles in mediating the muscular activity-induced ColQ gene expression would be investigated. Over-expressing the active form of CaMKII could induce ColQ promoter activities. KN62, a CaMKII specific blocker, attenuated the A23187-induced ColQ promoter activities. These results suggested that ColQ gene expression could possibly be regulated by muscular activity via CaMK II mediated signaling pathway