A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs.

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.