Nicotinic Receptor Subtypes in the Developing Chick Brain: Appearance of a Species Containing the a4, b2, and a5 Gene Products

Increasing evidence suggests nicotinic receptors regulate developmental events in the nervous system. We used [H]epibatidine and I-a-bungarotoxin, together with subunit-specific monoclonal antibodies, to distinguish and quantify nicotinic receptor subtypes in developing chick brain. The results show that more than three fourths of the epibatidine-binding receptors at both early and late embryonic stages contain a4 and b2 subunits, representing receptors previously distinguished by high affinity nicotine binding. A fraction of these also contain the a5 gene product, which is consistent with studies on transfected cells showing that the a4, b2, and a5 gene products coassemble to produce epibatidine-binding receptors. A small portion of the receptors contain a3 and b4 subunits, assembled in part with either a4 or b2 subunits. The most abundant nicotinic receptors, however, at both early and late embryonic stages are those having high affinity for a-bungarotoxin rather than epibatidine. Most contain a7 subunits, whereas about half contain a8 subunits as well. The sharpest developmental increase between embryonic days 8 and 17/18 occurs with receptors containing a5 subunits, whereas receptors containing a3 or b4 subunits undergo no specific increase. The three major receptor species (containing a4 and b2 but not a5 subunits; a7 subunits; or a7 and a8 subunits) each increase '3fold during the same period. The results indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in neuronal nicotinic receptors and draw attention to the role of a5 subunits in late development. AChRs in the vertebrate nervous system are ligand-gated ion channels that depolarize neurons by permitting cations to flow across the plasma membrane. Like their counterparts in vertebrate skeletal muscle, the receptors are thought to be pentameric transmembrane proteins encoded by one or more members of a multigene family (Karlin and Akabas, 1996). It is clear that neuronal AChRs can participate in a variety of functions. Presynaptically, the receptors can modulate neurotransmitter release, whereas postsynaptically, they can generate synaptic currents from both synaptic and perisynaptic locations (Role and Berg, 1996; Zhang et al., 1996; Wonnacott, 1997). Some classes of AChRs have a high relative permeability to calcium and can influence calcium-dependent events in neurons, including activation of second messenger cascades. Neuronal AChRs may play important developmental roles as well. The receptors are expressed early during embryogenesis, as is the enzyme responsible for synthesis of ACh (Zoli et al., 1995; Role and Berg, 1996). AChRs can be found on the tips of growing neurites in cell culture and, when activated, can influence the pattern of neurite growth. Presynaptic AChRs also can enhance neurotransmitter release at newly formed neuromuscular synapses in culture (Fu and Liu, 1996), and they may contribute to the early stages of synap-