The fast actions of the neurotransmitter acetylcholine (ACh) are mediated by nicotinic acetylcholine receptors (nAChRs). These pentameric receptors are members of the cysteine-loop family of ionotropic receptors and contain an integral ion channel which becomes transiently permeable to cations following binding of the neurotransmitter. ACh binds to residues in the extracellular N-terminal region and four membrane spanning regions (M1-4) of each subunit form the ion channel. M2 makes a major contribution to the channel lining, though residues at the extracellular end of M1 are also exposed to the channel lumen. A generalized subunit stoichiometry (a)2(non-a)3 is observed for muscle and certain neuronal nAChRs. a subunits are defined by the presence of two adjacent cysteine residues which contribute to the Tyr-x-Cys-Cys motif of loop C of the ACh binding site. Previous studies have identified several nAChR subunit genes in C. elegans:
deg-3 [a] (Treinin and Chalfie, 1995); Ce21 [a] (Ballivet et al., 1996);
acr-2 [non-a] (Squire et al., 1995);
unc-38 [a];
lev-1 [non-a] and
unc-29 [non-a] (Fleming et al., 1997) and
acr-3 [non-a] (Baylis et al., 1997). In the present study we have used Reverse Transcriptase-Polymerase Chain Reaction to demonstrate the transcription of 8 novel candidate nicotinic acetylcholine receptor (nAChR) a subunit genes in Caenorhabditis elegans. Hence this is now the largest known family of nAChR a subunit genes in a single species. We have identified 4 groups of a subunits: DEG-3-like; Ce21-like; UNC-38-like and ZC504.2-like. Five C. elegans nAChR a subunits contain a modification in loop C of the ACh binding site, in which the normally conserved Tyr-x-Cys-Cys motif, is replaced by Tyr-x-x-Cys-Cys. Variation is also found in the channel lining M2 regions, including the replacement in 4 subunits of the highly conserved 9! Leu residue by Val and most notably, the replacement in all ZC504.2-like subunits of the highly conserved -1! Glu residue by His. Molecular dynamics simulations have been used to generate homo-pentameric M2 helix bundle-models for selected a subunits and possible functional implications examined. The calculated electrostatic potential energy profile for the M2 region of ZC504.2 is radically altered due to the presence of His at the -1! position, indicating a major perturbation of nAChR channel cation permeability in the presence of this subunit type. Ballivet, M., Alliod, C., Bertrand, S., and Bertrand, D. (1996) J.Mol.Biol. 258, 261-269 Baylis, H.A., Matsuda, K., Squire, M.D., Fleming, J.T., Harvey, R., Darlison, M.G., Barnard, E.A., and Sattelle, D.B. (1997) Receptors and Channels 5 149-158 Fleming, J.T., Squire, M.D., Barnes, T.M., Tornoe, C.T., Matsuda, K., Sulston, J.E., Barnard, E.A., Sattelle, D.B., and Lewis, J.T. (1997) J.Neurosci. 17 5843-5857 Squire, M.D., Baylis, H.A., Fleming, J.T., Barnard, E.A., and Sattelle, D.B. (1995) Receptors and Channels 3 107-115 Treinin, M. and Chalfie, M. (1995) Neuron 14 871-877