Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells

Abstract

Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and aBin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-␤-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.

18F-FDOPA PET/CT Uptake Parameters Correlate with Catecholamine Secretion in Human Pheochromocytomas

“…Two days after culture, chromaffin cells originating from PHEO were washed with Locke's solution (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 0.01 mM EDTA, 15 mM HEPES, and 11 mM glucose; pH 7.5) and processed for catecholamine release measurements by amperometry as previously described [14].…”

Phosphatidic acid metabolism regulates neuroendocrine secretion but is not under the direct control of lipins

“…Catecholamine's secretion was induced by 10 s pressure ejection of 100 mM K + solution from a micropipette positioned at 10 μm from the cell and recorded during 60 s. Amperometric recordings were performed with an AMU130 (Radiometer Analytical) amplifier, sampled at 5 kHz, and digitally low‐passed filtered at 1 kHz. Analysis of amperometric recordings was done as previously described with a macro (obtained from Dr. R. Borges laboratory; http://webpages.ull.es/users/rborges/) written for Igor software (Wavemetrics), allowing automatic spike detection and extraction of spike parameters. The number of amperometric spikes was counted as the total number of spikes with an amplitude >5 pA within 60 s. The spike parameters analysis was restricted to spikes with amplitudes >5 pA. Quantal size (Q) of individual spike and foot is measured by calculating the spike area above the baseline.…”

αII‐spectrin controls calcium‐regulated exocytosis in neuroendocrine chromaffin cells through neuronal Wiskott–Aldrich Syndrome protein interaction

“…Catecholamine's secretion was induced by 10 s pressure ejection of 100 mM K + solution from a micropipette positioned at 10 μm from the cell and recorded during 100 s. Amperometric recordings were performed with an AMU130 (Radiometer Analytical) amplifier, sampled at 5 kHz, and digitally low‐pass filtered at 1 kHz. Analysis of amperometric recordings was performed as previously described with a custom‐written macro (obtained from Dr. R. Borges laboratory) running under the Igor software (Wavemetrics), allowing automatic spike detection and extraction of spike parameters. The number of amperometric spikes was counted as the total number of spikes with an amplitude >5 pA within 100 s. The spike parameter analysis was restricted to spikes with amplitudes of 5 pA. Data of amperometric spikes were averaged by individual cell.…”