Role of membrane cholesterol in exocytosis at the neuromuscular junction: interplay of Ca2+ and ROS

Petrov et al, Role of membrane cholesterol in spontaneous exocytosis at frog neuromuscular synapses: reactive oxygen species-calcium interplay, J Physiol, 2014, 592.22:4995-5009


Cholesterol is a ubiquitous component of the cell membrane, adjusting membrane fluidity and stiffness and directly interacting with membrane proteins to affect their localization and function.  Petrov et al. sought to explain how cholesterol depletion leads to an increase in exocytosis at the frog neuromuscular junction.  Methyl-beta-cyclodextrin (MBCD) was used to mobilize cholesterol.  Exposure to MBCD caused a rapid increase in reactive oxygen species (ROS) production, which was measured by a variety of fluorescence reporter molecules.  This increase in ROS could be inhibited by pre-treatment with antioxidants and oxidase inhibitors.  At the same time, MBCD increased intracellular calcium.  Several blockers were used to show that this primarily arose from TRPV channel activity, though the blockers used would also inhibit voltage-gated calcium channels.  Block of Ca2+ increase did not affect ROS production, so ROS was not produced via the increased intracellular Ca2+.  It is less clear to me whether the increased calcium can be attributed to ROS vs a direct action on TRPV.  And it is less clear whether TRPV is the sole source of increased calcium flux since the blockers used are also inhibitors of voltage-gated calcium channels.

Why It’s Important

Though there are some open questions about the specificity of blockers used in the study, the primary results are concrete, namely that cholesterol depletion has impact on excitability by activating several processes that are of special interest to our ototoxicity study.  We hypothesize that HPBCD-induced ototoxicity involves activation of stress pathways and modulation of membrane ion channels, culminating in the induction of cell death pathways.  It is unlikely that one specific mechanism is the link between HPBCD injection and OHC death.  This paper highlights the possibility that oxidative stress and calcium influx may play a role.

Link to PDF of paperPetrov_JPhys_2014-MBCD-ROS-antioxidants-synapse

PPT fileLabMeeting_111315


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