Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. II. The rundown and inward rectification of agonist-elicited whole-cell currents and identification of receptor subunits by in situ hybridization

Abstract

Our previous study demonstrated for the first time that nicotinic currents evoked in rat hippocampal neurons could be grouped into four categories (types IA, IB, II and III) according to their functional and pharmacological characteristics. In the second part of our continuing studies, the structural and functional diversity of nicotinic receptors expressed in hippocampal neurons was further explored. Type IA, the predominant and alpha-bungarotoxin-sensitive current, but not type II, the alpha-bungarotoxin-insensitive current, showed rundown in the peak amplitude during the whole-cell recording. The rundown of type IA currents could be prevented when the ATP-regenerating compound phosphocreatine, alone or in combination with ATP and creatine phosphokinase, was added to the internal recording solution. The addition to the internal solution of either the microfilament-stabilizing agent phalloidin (5 microM) or the microtubule-stabilizing agent taxol (50 microM) did not alter or prevent rundown in type IA currents. Type IA and type II currents showed inward rectification. The inward rectification of type IA currents was dependent on the presence of intracellular Mg++, whereas that of type II currents was independent of Mg++. When Mg++ was present in the internal pipette solution, the inward rectification of type IA currents was sustained throughout the recording time. However, when nominally Mg(++)-free internal solution was used, the inward rectification decreased with recording time in type IA currents, but not in type II currents, as a consequence of removal of intracellular Mg++. In situ hybridization demonstrated the presence of alpha 7-, alpha 4- and beta 2-nicotinic acetylcholine receptor subunit mRNAs in cultured hippocampal neurons. The distribution among the neurons of the mRNAs for alpha 7- and alpha 4-nicotinic acetylcholine receptor subunits, correlated with the frequency with which type IA and type II currents, respectively, could be evoked in these neurons. The present results provide evidence for 1) the presence of intracellular high-energy phosphate-dependent processes linked with the nicotinic acetylcholine receptor subserving type IA currents, 2) a requirement of intracellular Mg++ for the inward rectification of type IA currents and 3) a correlation between the distribution of nAChR subunits and the different probabilities of eliciting distinct types of nicotinic currents in hippocampal neurons.