Huntingtin-associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells.

Key points Huntingtin-associated protein 1 (HAP1) is expressed in neurons and endocrine cells, in which it is thought to regulate vesicle trafficking., HAP1 is a binding partner of the Huntington's disease (HD)-causing protein huntingtin, and binding is stronger in HD., Whether HAP1 regulates a significant end-point of vesicle transport, exocytosis, and what stage of exocytosis HAP1 may regulate, is unknown., We use mouse chromaffin cells to demonstrate that HAP1 regulates exocytosis via two potentially interlinked mechanisms: control of vesicle docking and the readily releasable vesicle pool, and regulation of fusion pore stabilization., These results establish HAP1 as a significant player in exocytosis control with potential relevance for HD and for a number of neuronal and homeostatic pathways., Abstract Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1−/− and HAP1+/+ littermate mice. Numbers of Ca2+-dependent and Ca2+-independent full fusion events in HAP1−/− cells are significantly decreased compared with those in HAP1+/+ cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca2+ entry. Whereas release per full fusion event is unchanged in HAP1−/− cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1−/− cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation. [ABSTRACT FROM AUTHOR]