Abstract
Ca2+ release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca2+-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca2+ influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca2+, led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca2+ channel.
Highlights
Through the binding of the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM) to the CRAC channel proteins CRACM1, CRACM2 and CRACM3, of which CRACM1 is the major pore-forming subunit of the CRAC channel[5,6]
Electrophysiological and single-vesicle monitoring results suggest that CRACM3 plays a role in the membrane-vesicle fusion process that is distinct from its function as a compensatory CRAC channel subunit
While studying sensitive factor attachment receptor proteins (SNAREs) proteins expressed in a rat basophilic leukaemia (RBL-2H3) cell line, a commonly used non-excitable exocytosis model that allows exocytosis to be monitored via secretory histamine release, we noticed that the CRACM3 signal appeared to be enriched in syntaxin4-immunoprecipitated blots
Summary
Through the binding of the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM) to the CRAC channel proteins CRACM1, CRACM2 and CRACM3, of which CRACM1 is the major pore-forming subunit of the CRAC channel[5,6]. CRACM3 is generally considered to be compensatory for the loss of CRACM1. We dissected the role of CRACM3 in the molecular interaction network of non-excitable exocytotic vesicle fusion, which is generally considered a distinct signal-transduction pathway from that of store-operated Ca2+ entry, with intracellular Ca2+ playing an intermediary role between them. Electrophysiological and single-vesicle monitoring results suggest that CRACM3 plays a role in the membrane-vesicle fusion process that is distinct from its function as a compensatory CRAC channel subunit
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