Local accumulation of acetylcholine receptors is neither necessary nor sufficient to induce cluster formation

Abstract

Acetylcholine receptors (AChRs) accumulate at developing neuromuscular junctions in part via lateral migration of diffusely expressed receptors. Using a model system - cultured Xenopus muscle cells exposed to electric fields - we have shown that AChRs, concentrated at the cathode-facing cell pole, continue to aggregate there after the field is terminated (Stollberg and Fraser, 1988). These observations are consistent with the possibility that the field-induced increase in receptor concentration triggers the aggregation event. Only 2 other molecular events could initiate the electric field-induced receptor aggregation: (1) a local increase in the density of some other molecules, or (2) a voltage-sensitive mechanism. Treatment of muscle cell cultures with neuraminidase changes the cell surface charge and has been reported to reverse the direction of electromigration for AChRs and concanavalin A binding sites (Orida and Poo, 1978). Using digitally analyzed fluorescence videomicroscopy, we find that AChRs in neuraminidase-treated cultures accumulate at both cell poles in an electric field. After termination of the field, the AChR continues to aggregate at the cathode-facing pole, as in cells not treated with neuraminidase. However, receptor density decreases at the anode-facing pole, indicating that elevated AChR density does not initiate receptor aggregation. Cells pretreated with neuraminidase and trypsin (which blocks receptor aggregation) display reversed receptor distributions compared to untreated controls, indicating that electromigration has indeed been reversed. The rate at which neuraminidase- and trypsin-treated cells approach steady-state distributions indicates a receptor diffusion constant of ≈ 1.2 × 10-9 cm2/sec, consistent with a diffusion trap mechanism of receptor aggregation. These results are the first conclusive demonstration that the local concentration of receptors is neither necessary nor sufficient to induce receptor clustering. Our observations suggest that receptor clustering is triggered by the accumulation of some other molecules, or by a voltage-sensitive mechanism.