Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

Abstract

In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases~2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor a subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of a-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (-17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation ofthe corresponding mRNAs.The acetylcholine receptor (AcChoR) is an oligomeric protein composed of five transmembrane subunits with an a213y8 structure (reviewed in ref.

A 5'-flanking region of the chicken acetylcholine receptor alpha-subunit gene confers tissue specificity and developmental control of expression in transfected cells.

“…Finally, muscle electrical activity represses the expression of the AChR (15), and this effect can be studied in vitro, e.g., by using tetrodotoxin to block the spontaneous electrical activity of cultured myotubes (39). Such changes in AChR expression have been correlated with variations of the mRNA levels for its a-subunit (24); similar results have been obtained after muscle denervation in vivo (24,28).…”

“…Characterization of 5' end of chicken AChR a-subunit gene. Several laboratories, including ours, had previously isolated and identified genomic clones encoding the chicken AChR a-subunit (2,24) on the basis of its high degree of sequence conservation with its counterparts in other species (5,12,33,34). To map the first exon, the length of mRNA between the sequence corresponding to the exon named P2 in the human gene (24,33) and the mRNA capsite was assessed by primer extension with reverse transcriptase, using poly(A)+ RNA * Corresponding author.…”

Protein Kinase C and Calcium/Calmodulin-activated Protein Kinase II (CaMK II) Suppress Nicotinic Acetylcholine Receptor Gene Expression in Mammalian Muscle

“…In contrast, when denervated muscle is electrically stimulated, intracellular calcium concentrations are elevated (4), and receptor expression is suppressed (6 -8). Similarly, receptor expression is suppressed in TTX-treated myotubes when they are exposed to calcium-elevating drugs (2,3,9,10). Although muscle depolarization and increases in intracellular calcium can initiate the process of nAChR suppression in extrajunctional nuclei, the signal transduction pathways involved in these processes remain controversial.…”

Protein synthesis is required for the denervation‐triggered activation of acetylcholine receptor genes

“…Recent analysis of the mechanisms underlying the increased receptor synthesis rate has revealed that denervation also results in a remarkable increase in mRNAs coding for the individual receptor subunits [4][5][6][7][8][9][10][11]. The increased message concentrations in turn are at least in part caused by enhanced transcription rates of receptor genes [1,12]; in the chicken a denervation signal reaches the genome and activates the t~-, 6-, and 7-subunit genes within 12 h after nerve section [12].…”

“…For example, it is not known if denervation results in the appearance of an activating factor or in the loss of an inhibitor. Treatment of primary muscle cells with the sodium channel blocker tetrodotoxin results in an increase of AChR protein and ct-subunit mRNA, and has been employed to analyze the effects of membrane electrical activity on receptor gene expression [5,11,13]. Using this in vitro model of denervation, Duclert et al [14] have shown that inhibition of protein synthesis blocks the tetrodotoxin-induced rise in a-subunit message level We have investigated the metabolism of the AChR subunit messages in denervated chick skeletal muscle.…”