Pharmacological properties and regulation of the rat neuronal nicotinic acetylcholine receptor alpha-3/beta-4 subtype stably expressed in HEK 293 cells

The neuronal nicotinic acetylcholine receptor (nAChR) family consists receptor subtypes defined by the different combinations of {dollar}alpha{dollar} and {dollar}beta{dollar} subunits of which they are composed. In most cases, these receptors are thought to consist of two {dollar}alpha{dollar} and three {dollar}beta{dollar} subunits which form a ligand-gated cation channel that passes Na{dollar}sp+,{dollar} K{dollar}sp+,{dollar} and in some cases Ca{dollar}sp{lcub}++{rcub}.{dollar} Molecular biological techniques have revealed eight {dollar}alpha{dollar} subunit genes {dollar}(alpha2{lcub}-{rcub}alpha9){dollar} and three {dollar}beta{dollar} subunit genes {dollar}(beta2{lcub}-{rcub}beta4){dollar} in vertebrate nervous systems. Our laboratory has generated a library of stably transfected HEK 293 cell lines, each expressing one combination of {dollar}alpha{dollar} and {dollar}beta{dollar} subunits. The studies for this thesis research were carried out using a transfected cell line called KX{dollar}alpha3beta{dollar}4R2, which expresses the {dollar}alpha3/beta4{dollar} nicotinic receptor subtype.; The {dollar}alpha3{dollar} and {dollar}beta4{dollar} subunits are highly expressed in vertebrate autonomic ganglia, making the {dollar}alpha3/beta4{dollar} subtype a leading candidate for a receptor that mediates ganglionic transmission. The objectives of this research were to examine the basic pharmacology and the regulation by chronic nicotine of the rat {dollar}alpha3/beta4{dollar} subtype expressed in KX{dollar}alpha3beta4{dollar}R2 cells.; The mRNA for both the {dollar}alpha{dollar}3 and {dollar}beta{dollar}4 subunits is highly expressed in KX{dollar}alpha3beta4{dollar}R2 cells. The density of {dollar}alpha3/beta4{dollar} binding sites is very high, measuring 8,900 fmols/mg protein by ({dollar}sp3{dollar}H) ({dollar}pm{dollar})epibatidine ( ({dollar}sp3{dollar}H) ({dollar}pm{dollar})EB) binding. The K{dollar}rmsb{lcub}d{rcub}{dollar} for ({dollar}sp3{dollar}H) ({dollar}pm{dollar})EB was approximately 300 pM. Nicotinic drugs competed for all of the (3H) ({dollar}pm{dollar})EB binding sites in KX{dollar}alpha3beta4{dollar}R2 cells with the rank order of affinities: epibatidine {dollar}>{dollar} anatoxin {dollar}>{dollar} A85380 {dollar}>{dollar} cytisine {dollar}>{dollar} nicotine {dollar}>{dollar} acetylcholine {dollar}>{dollar} carbamylcholine {dollar}>{dollar} d-tubocurarine {dollar}>{dollar} dihydro-{dollar}beta{dollar}-erythoidine.; Measurements of {dollar}rmsp{lcub}86{rcub}Rbsp+{dollar} efflux were used to assess nicotinic receptor function. The order of affinities for nicotinic agonists in stimulating {dollar}rmsp{lcub}86{rcub}Rbsp+{dollar} efflux from KX{dollar}alpha3beta4{dollar}R2 cells was epibatidine {dollar}>{dollar} cytisine {dollar}geq{dollar} nicotine {dollar}>{dollar} acetylcholine {dollar}>{dollar} carbamylcholine. The nicotinic antagonists mecamylamine, hexamethonium and d-tubocurarine inhibited nicotine stimulated {dollar}rmsp{lcub}86{rcub}Rbsp+{dollar} efflux in a noncompetitive manner, while dihydro-{dollar}beta{dollar}-erythoidine acted as a competitive inhibitor.; When KX{dollar}alpha3beta4{dollar}R2 cells were chronically exposed to nicotine, the density of {dollar}alpha3/beta4{dollar} receptors increased two to five fold in a time and dose related manner. A concentration dependent decrease in function of {dollar}alpha3/beta4{dollar} receptors was also noted in cells that were chronically exposed to nicotine.; These results indicate that the KX{dollar}alpha3beta4{dollar}R2 cell line is a good model in which to study the pharmacology and regulation of the rat {dollar}alpha3/beta4{dollar} nAChR subtype and that it provides a useful tool for comparison to other nAChR subtypes.