Nicotinic acetylcholine receptors are pentameric, transmembrane, ligand-gated ion channels critical for neuromuscular signal transmission. Prior to innervation, the genes encoding these receptors are expressed in nuclei throughout the muscle fiber. Muscle innervation leads to a dramatic decrease in expression of these genes in extrasynaptic nuclei. This reduction in gene expression can be reversed by muscle denervation. The effects of denervation on receptor gene expression can be blocked by electrical stimulation of muscle using extracellular electrodes. The molecular mechanisms by which muscle electrical activity leads to altered patterns of nicotinic acetylcholine receptor gene expression are not well understood. Using an in vitro electrical stimulation paradigm to induce muscle activity, we have been able to mimic the effect of innervation on extrasynaptic acetylcholine receptor gene expression. We have found that a 93-bp region of 5'-flanking DNA, spanning nucleotides -150 to -57 relative to the transcription start site of the gamma subunit gene, is required for the suppression of gene expression in response to muscle activity. Sequences downstream of this region are transcriptionally active but are not responsive to muscle activity. However, these downstream sequences become responsive to muscle activity when placed under the control of the gamma subunit muscle-specific enhancer.
A 1.45 kb DNA sequence encoding the rat alpha 6 GABAA receptor subunit (nucleotides 33-1483) was cloned from a Sprague-Dawley rat brain cDNA library by PCR amplification. Dideoxy sequencing of two individual clones revealed that the nucleotide sequence differed at only one basepair (T480-->G) from that published previously. This difference altered the deduced amino acid sequence, producing a conservative amino acid substitution (His121-->Gln). A Gln residue is present at the same location in the bovine alpha 6 subunit. Restriction endonuclease analysis of the total PCR product demonstrated that this variant of the rat alpha 6 subunit was the only allele found in this particular rat brain library, the original allele was not present. These results were further verified by RNAse protection assays performed with RNA isolated from individual rat cerebella. alpha 6, beta 1, and gamma 2S subunits were transiently expressed in L929 cells for electrophysiological analysis. Whole-cell recordings obtained from the cells demonstrated that GABAA receptor channels with the expected GABA and benzodiazepine pharmacology were produced. Excised outside out single channel recordings from the same cells revealed that GABA elicited brief duration openings to a 33 pS main conductance level and to at least one smaller (approximately 21 pS) subconductance level. Thus this allelic variant of rat alpha 6 subunit could assemble with other subunits to form a functional GABAA receptor channel with similar properties to the original allelic form.
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