Concanavalin A prevents acetylcholine receptor redistribution in Xenopus nerve-muscle cultures

Abstract

During neuromuscular junction formation ACh receptors accumulate at the nerve-contact region. It has been shown that this is at least partly due to lateral migration of existing receptors in the membrane (Anderson et al., 1977). Randomly diffusing ACh receptor molecules in the membrane may be trapped at the nerve-contact region to form a high receptor density area. If this were the major mechanism, cross-linking ACh receptors by tetravalent concanavalin A (Con A) should immobilize receptors and prevent nerve-induced receptor accumulation. We examined the effect of Con A on nerve-induced receptor accumulation and on the mobility of ACh receptors in cultured Xenopus muscle cells. ACh receptors were stained with tetramethyl rhodamine conjugated alpha-bungarotoxin. The cells were then treated briefly with Con A, and neural tube cells were added to these cultures. The mobility of ACh receptors was measured by the fluorescence photobleaching recovery method. The Con A treatment prevented rapid diffusion of ACh receptors as well as nerve-induced receptor accumulation. Functional synapse formation was not inhibited by this treatment. In contrast, divalent succinyl Con A did not affect the mobility of ACh receptors nor prevent nerve-induced ACh receptor accumulation. When the Con A concentration was varied, the blocking effect on the nerve-induced receptor accumulation changed in parallel with the mobile fraction of receptors. Newly inserted ACh receptors after the Con A treatment were found to be mobile and to accumulate at the nerve-contact region. In these cultures, new receptors accumulated around old, immobilized receptors in some areas along the nerve contact. This observation suggests that new receptors were inserted elsewhere and migrated to the nerve-contact region surrounding immobilized old ones. In addition to the accumulation of receptors, the nerve disperses preexisting receptor clusters prior to induction of high-density regions along the contact area, and, at this early stage, denervation disperses nerve-induced receptor clusters in Xenopus cultures (Kuromi and Kidokoro, 1984a, b). When cultures were treated with Con A, neither of these events occurred, suggesting that these are also diffusion-mediated.

Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin

“…At what point during neuromuscular synaptogenesis do these clusters of Ca2+ channels form? For the case of AChRs, it is known that, during synaptogenesis, preexisting mobile AChRs in neighboring extrajunctional regions accumulate, and become immobilized, at the site of nerve-muscle contact, thereby contributing to the buildup of the high-density of AChRs in the newly forming postsynaptic membrane (Anderson and Cohen, 1977;Ziskind-Conhaim et al, 1984;Kidokoro et al, 1986). Do similar mechanisms contribute to the accumulation of Ca2+ channels at developing active zones?…”

Intracellular and surface acetylcholine receptors during the normal development of a frog skeletal muscle

“…At the onset of synaptogenesis in Xenopus myotomal muscle (Anderson and Cohen, 1977;Kuromi et al, 1985;Kidokoro et al, 1986), rat muscle (Ziskind-Conhaim et al, 1984), and chick muscle (Role et al, 1985), mobile AChRs in neighboring regions of the surface membrane enter and become immobilized in the newly forming postsynaptic membrane, thereby contributing to the build-up of the high AChR density. In rat and chick muscle (Kelly and Zacks, 1969;Nakai, 1968; see also Englander and Rubin, 1987), but not in Xenopus myotomal muscle (Kullberg et al, 1977), this is followed by an accumulation ofmuscle nuclei immediately below the postsynaptic membrane.…”

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures

“…The neural agent(s) that initiates these remarkable changes in AChR distribution has not yet been identified, but it is known that it can act in the absence of activation of AChRs, as well as in the absence of muscle and neuronal action potentials Rubin et al, 1980;Davey and Cohen, 1986). The response of the muscle cell to the triggering event includes a process of receptor redistribution whereby preexisting mobile AChRs in neighboring regions of the surface membrane aggregate along the path of neurite-muscle contact where they become immobilized Stya and Axelrod, 1984;Ziskind-Conhaim et al, 1984;Kidokoro et al, 1986). An increased incorporation of newly inserted AChRs can also contribute to the build-up of a high density of AChRs at sites of contact (Role et al, 1985).…”