Figure 4. Cytoplasmic perfusion of acyl CoA or CoA circumvents four mitochondrial MEND blocks.
(A) Typical experiment in the presence of cyclosporine A (CsA, 3 μM) to block MEND. Ca influx cause a 40% increase of Cm via exocytosis, and Cm remains stable thereafter for minutes. Cytoplasmic perfusion of myristoyl CoA (mCoA, 15 μM) via the micro-capillary within the patch pipette causes a MEND response that begins within 10 s and internalizes 70% of the plasmalemma within 2.5 min. (B) Composite results quantifying MEND that occurs when mCoA or CoA is perfused into the cytoplasm of cells in which MEND has been blocked by interventions acting on mitochondria. From left to right, bar graphs present results for pipette perfusion of mCoA (15 μM) in cells in which MEND was blocked by cyclosporine A (CsA, 3 μM; black and white), by opening cells at 22°C with CCCP (20 μM) and oligomycin (5 μM; purple), by opening cells at 22°C with KSP (blue), and by pretreatment of cells with OAG (15 μM) for 30 min (green). The penultimate results quantify MEND caused by pipette perfusion of CoA (20 μM) into cyclosporine A-blocked cells (yellow). The final data set shows results for cells in which MEND was blocked by a high cytoplasmic CoA concentration (3 mM). Ca influx caused on average 38% exocytic responses, and Cm was then stable. When CoA was perfused out to the cytoplasm (‘wash-out’), endocytosis started within 15 s and amounted to 56% of the plasmalemma on average. For all results, n > 6.