Molecular mechanisms involved in regulation of nicotinic acetylcholine receptor genes by muscle activity.

In the nervous system, cellular electrical activity can cause long term changes in a cell via regulation of gene expression. The neuromuscular junction (NMJ) provides a convenient model for studying this regulation. At the NMJ, the nicotinic acetylcholine receptor (nAChR) genes located in the post-synaptic muscle cell are regulated by nerve induced muscle activity. These genes and their expression were used as markers to study this regulation. This study investigated the role of second messengers in this regulation and identified DNA sequences within the rat nAChR $\delta$-subunit's upstream regulatory region important for activity-dependent regulation. The effects of muscle activity on gene regulation were studied in both in vitro and in vivo systems. In vitro studies utilized muscle and non-muscle cell lines along with primary muscle cell cultures which were either stimulated to contract with electrodes or forced to remain inactive with the drug tetrodotoxin. In vivo experiments involved direct DNA injections into innervated (active) or denervated (inactive) muscle to identify elements within the $\delta$-subunit's upstream regulatory region important for activity-dependent regulation. Investigations into regulation by second messengers in rat muscle found that changes in intracellular calcium levels could regulate both embryonic-type and adult-type receptor genes. Phorbol ester induced protein kinase C, involved in regulating nAChR genes in chick muscle, was found to have no regulatory role with nAChR genes in rat muscle. However, regulation of these genes by changes in intracellular calcium was seen in both rat and chick muscle systems. Sequences within the $\delta$-subunit promoter from $-$1 to $-$47, relative to the transcription start site, were found to be critical for muscle specific expression and activity-dependent regulation. This region contains an E-box element (CACCTG), capable of binding myogenic factors, which was necessary but required additional upstream sequences to function as a muscle enhancer and confer activity-dependent regulation onto gene expression. Additionally, sequences located from $-$65 to $-$81 were required for high levels of expression in muscle, could act as a non-specific enhancer, and were also involved in conferring activity-dependent regulation onto gene expression.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104880/1/9610260.pdfDescription of 9610260.pdf : Restricted to UM users only