The role of pH in the intracellular acetylcholine receptor ligand processing

Previous studies have shown that the internalized AChRs are transported through many vesicular compartments: Golgi associated vesicles, coated vesicles, smooth vesicles, endosome-like structures and lysosomes. These compartments have an acidic pH ranging from 4.5 to 6.5. The pH differences between organelles suggests that these differences may influence the sorting and final expression of AChRs. To test this hypothesis, we measured the number of counts of 125I-alpha BTX or 125I-Mab35 dissociated from myotube membranes containing AChRs as a function of pH. Neither the 125I-alpha BTX nor 125I-Mab35 showed an enhanced dissociation in the pH range 4.0-7.0, whereas lowering the pH to 6.0 or below enhanced the dissociation of 125I-alpha 2-macroglobulin from myotubes. In other experiments using Torpedo membrane we showed that neither 125I-alpha BTX nor 125I-Mab35 appreciably dissociated from the AChR unless the pH was less than 4 or above 11. Double-label studies using a novel membrane permeable acidotropic molecule DAMP (3-(2,4 nitroanilino) 3'amino-N-methyl-dipropylamine), facilitated mapping the pH of the intracellular compartments containing internalized AChRs. This molecule accumulates inside acidic compartments in the cell and has a dinitrophenol (DNP) group recognized by DNP specific antibodies. Cells were treated with 30 micrograms DAMP for 30 min and allowed to internalize Mab35-gold (15 nm) for various periods (0-15 h). At each time point we fixed and washed the cells, and incubated with anti-DNP monoclonal antibodies followed by incubation with anti-mouse IgG and protein A colloidal gold (5 nm). Different sized gold particles allowed us to simultaneously identify the AChR compartments and estimate their pH. Sister cultures were exposed to acidotropic drugs to destroy pH gradients. Under those conditions, AChR delivery to lysosomes was blocked. Our studies show that AChRs are transported through acidic compartments ranging from pH 4.5 to 6.5 and in contrast to other ligands they do not dissociate from the intracellular membranes at low pH.