SHORT COMMUNICATION A SULPHYDRYL REDUCING AGENT, DITfflOTHREITOL, MODIFIES AGONIST-NICOTINIC RECEPTOR INTERACTION IN AN IDENTIFIED INSECT NEURONE

Nicotinic acetycholine receptors are present at high density in the nervous system of insects (Sattelle, 1980). Studies on the structure of nicotinic acetylcholine receptors have shown a degree of homology between insect a-like subunits and a-subunits from other species (Bossy etal. 1988; Marshall etal. 1990; Sawruk etal. 1990a). The locust ar-subunit, when expressed in Xenopus oocytes, is able to form a functional receptor-channel that is apparently homo-oligomeric and mimics several properties of in vivo receptor pharmacology (Marshall et al. 1988,1990). It seems likely that the structure of native receptors will include other subunits; nona-subunits have been described in several species of insect (Hermans-Borgmeyer etal. 1986; Sawruk etal. 1990/?). Expression studies show that the a-subunit carries the binding site for acetylcholine and also for a number of other ligands, including nitromethylene insecticides (Leech et al. 1991). Sequencing ar-subunits from insects has shown the presence of conserved cysteine residues that could form an extracellular disulphide bond (Bossy et al. 1988; Marshall et al. 1990; Sawruk et al. 1990a). Compounds that reduce disulphide bonds, such as dithiothreitol (DTT), are known to decrease the sensitivity of nicotinic receptors to acetylcholine (Karlin and Bartels, 1966), suggesting that such a bond is located close to the anionic binding site of the neurotransmitter recognition site on the receptor-channel molecule. In this report we show that DTT partially blocks the response of the cockroach fast coxal depressor motor neurone (Df) to nicotine, thereby providing the first direct evidence that these cysteine residues play a role in the agonist binding site of insect nicotinic acetylcholine receptors (nAChRs). Adult male cockroaches (Periplaneta americana L.) were used throughout this investigat-on. The cell body of the fast coxal depressor motor neurone (Df) was located visually in an isolated, desheathed metathoracic ganglion and impaled with a microelectrode filled with 2moll" KC1 (electrode resistance 15-25MQ). The preparation was bathed at a constant Tate (2:7mlmin") with-saline of the