Isolation and Sequencing of Muscarinic Acetylcholine Receptor Genes from Fishes

Abstract

In fish, adaptation to changes in light intensities is achieved in part by pigment granule movements within the retinal pigment epithelium. Based on studies using the cholinergic agonist carbachol it has been suggested that acetylcholine may play a role in light adaptive pigment granule dispersion. The effects of acetylcholine are mediated through the activation of either nicotinic or muscarinic receptors. Muscarinic receptors belong to the superfamily of receptors which initiate intracellular responses by interacting with G-proteins and are characterized by seven transmembrane domains. There are five known subtypes of muscarinic receptors (M1-M5). Pharmacological studies performed previously have hinted at the role of odd numbered muscarinic receptors (Mi, M3 and M5) in carbachol-induced pigment granule dispersion in retinal pigment epithelium in bluegill (Lepomis macrochirus), a model used in our lab to understand the signaling pathways involved in light adaptive pigment granule migration in retinal pigment epithelium. Due to the atypical affinity profiles exhibited by pharmacological agents for non-mammalian muscarinic receptors, it is necessary to molecularly characterize muscarinic receptors in bluegill in order to establish unambiguously the subtype identities of muscarinic receptors involved in light adaptive pigment granule movement. As a first step in that direction, a fragment of putative M5 gene from bluegill genomic DNA was isolated and amplified, using polymerase chain reaction employing primers based on the homologous regions among known putative M5 receptor genes from fish. The fragment is 1385 nucleotides in length and has an open reading frame encoding 461 amino acids. The deduced amino acid sequence showed higher identity to known M5 receptor proteins than to other subtypes of muscarinic receptors. A putative M2 receptor coding strand from fugu genomic database was also identified using search tools. This putative gene is 1500 nucleotides in length and encodes 500 amino acids. Comparison of the amino acid sequence with protein databases showed high identity with other M2 receptor proteins. Both the sequences, the bluegill putative M5 receptor fragment and fugu putative M2 receptor, have the critical amino acid motifs conserved across G-protein coupled receptors and the critical amino acids conserved among muscarinic receptors. These motifs have been shown by others to be required for ligand binding and G-protem coupling. Phylogenetic analyses of these putative receptors using both nucleotide and protein alignments and employing different methods grouped these receptors with their respective subtypes, thus confirming their subtype identity.