Ligandgated Channels

Ligand-gated channels (LGCs) are integral membrane proteins specialized in the trans-duction of neurotransmitter flux into electrical signal. Upon agonist binding, LGCs undergo fast conformational transitions which, as demonstrated by patch-clamp recordings, must be in the sub-millisecond time scale (22). When observed at the whole cell level, LGCs response times are slowed down to a few milliseconds owing to neurotransmitter diffusion, integration of the activity occurring over thousands of receptors, and the cell time constant. Typical postsynaptic currents can be as large as a several hundred picoamperes and firing of the action potential by the postsynaptic cell depends on summation of many excitatory and/or inhibitory presynaptic signals. It is worth recalling that a neuron can receive as many as 50,000 synaptic endings. The amplitude as well as the timing of each synaptic event can thus become critical. Potentiation or inhibition of a family of LGCs by a diffusible messenger can profoundly modify the activity of neurons and as a result the information processing of the neuronal networks in which they participate.

Following the initial work carried out on the acetylcholine receptor (23-25), a large number of LGCs have been identified as transmembrane proteins resulting from the assembly of different subunits. In each family, various isoforms (or subtypes) of the receptors have been identified and it has been shown that the different subunits can assemble and form a wide diversity of hetero-oligomers. These receptors display distinct pharmacological profiles with a high specificity for agonists, antagonists, and kinetic responses (26,27). Some of the subunit DNA sequences have been cloned. The currently accepted tertiary structure of a LGC subunit (24,25,28,29) assumes that a large hydro-philic segment faces the synaptic cleft and comprises the neurotransmitter binding site. Two to four hydrophobic segments, depending on the receptor type, have been identified as transmembrane-spanning domains. One of them appears to line the ionic pore. Some of the intracellular loops can display sites of regulation by phosphorylation/dephospho-

Fig. 1. Schematic representation of the structure of the nAChR. The left panel represents the transmembrane organization of the protein. The ligand-binding site (here ACh) and protein segments spanning the membrane are represented by filled structures. The second transmembrane segments (TM2), which border the aqueous ionic pore, are symbolized by the dark cylinders. The right panel represents the pentameric disposition of a nAChR with two a and three p subunits. Main and complementary parts, or residues, of the ACh-binding site are symbolized (A—E).

Fig. 1. Schematic representation of the structure of the nAChR. The left panel represents the transmembrane organization of the protein. The ligand-binding site (here ACh) and protein segments spanning the membrane are represented by filled structures. The second transmembrane segments (TM2), which border the aqueous ionic pore, are symbolized by the dark cylinders. The right panel represents the pentameric disposition of a nAChR with two a and three p subunits. Main and complementary parts, or residues, of the ACh-binding site are symbolized (A—E).

rylation mechanisms (30-33). Many features of the tertiary and quaternary structures of the Torpedo nicotinic acetylcholine receptor (nAChR) were studied by electron microscopy at 9 Â resolution (34,35), and similar features are thought to be present in neuronal nAChRs and other LGCs such as GABAa (36), glycine (37,38) or serotonin 5HT3 receptors (39). The four transmembrane segment scheme illustrated in Fig. 1 represents the currently accepted tertiary structure of the nAChRs and other members of this ionotropic receptors family.

The nAChR is currently the best characterized LGC. Up to the present time, 11 genes coding for putative subunits of the neuronal nAChR have been identified in vertebrates (24,25,40-44), whereas fewer closely related genes have been identified in invertebrates (25,45,46). Sequence analysis of the proteins suggest that all of these subunits derive from a common ancestor molecule by gene duplication and progressive spontaneous mutagenesis (25,47).

Compelling biochemical and electrophysiological evidences suggest that neuronal nAChRs result, as for the muscle receptor, from the assembly of five subunits, which are arranged with an axial pseudosymetry (29,34,48,49). Although single subunit genes (a7-a9) yield functional receptors when expressed in Xenopus oocytes or cell lines (50-55), all of the other subunits must co-assemble and produce functional nAChRs only when expressed under adequate combinations of at least two subunits, one a and one p (25,56). It is worth noting that the homomeric forms of nAChRs (a7-a9) are highly sensitive to the snake toxin a-bungarotoxin (a-Bgt) as is the muscle receptor.

Recent experiments combining molecular biology with both biochemistry and elec-trophysiology have brought new insights into the acetylcholine (ACh) binding domain. One conclusion of these experiments is that the ACh-binding site lies at the interface between two adjacent subunits (25,57-60). The main component is formed by the a subunit while the complementary component being contributed by the adjacent subunit, which is either a non-a or another segment of an a subunit, as in the case of the homomeric nAChRs.

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