Diacylglycerols regulate distribution of the muscle-type nicotinic acetylcholine receptor

KAMERBEEK, C. K.; MATEOS, M. V.; PEDICONI, M. F.; BARRANTES, F. J.

Buenos Aires

Congreso; SAIB-Molecular mechanisms in cell signaling and gene expression; 2013

The effects of exogenous and endogenously-generated diacylglycerols (DAG) on the density and distribution of the muscle-type nicotinic acetylcholine receptor (AChR) in CHO-K1/A5 cells and their involvement in possible downstream pathways were evaluated by [125I]-α-bungarotoxin binding, fluorescence microscopy, cell transfection and Western blots. Treatment with dioctanoylglycerol (DOG) for 4h augmented cell-surface AChR levels without altering total AChR levels. Co-incubation of DOG with drugs that increase intracellular calcium (A23187 or Thapsigargin) and thus activate classical PKC, did not produce the same effect. Incubation with Gö 6976, a classical PKC inhibitor, mimicked the DOG effect, increasing AChR levels. Longer exposure to DOG (18 h) led to intracellular accumulation of AChR without affecting the total amount of AChR. DOG changes on AChR distribution were blocked when cells were co-incubated with Gö 6976 and Rottlerin (inhibitor of PKC δ). Decreasing DAG levels at the Golgi, by silencing Nir2 protein, led to accumulation of AChR intracellularly. In conclusion, exogenous and endogenous DAG appear to modulate the distribution of muscle-type AChR. Short-term exposure to DOG may lead to classical PKC inhibition, whereas long-term effects of DOG may involve activation of both classical and novel PKC enzymatic mechanisms.