Dynamin-2 Mutations Linked to Neonatal-onset Centronuclear Myopathy impair exocytosis and endocytosis in adrenal chromaffin cells

Abstract

ABSTRACTDynamins are large GTPases whose primary function is to catalyze membrane scission during endocytosis, but also modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T and p.S619L, impair Ca2+-induced exocytosis of GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of NPY-labelled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with DMPP. They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the CNM- linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.