CRACM3 regulates the stability of non-excitable exocytotic vesicle fusion pores in a Ca(2+)-independent manner via molecular interaction with syntaxin4.
Ca(2+) release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca(2+)-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 Ca(2+) influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca(2+), 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 Ca(2+) channel.
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan.
Shuang
S
Liu
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan.
Muhammad Novrizal Abdi
MN
Sahid
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan.
Erika
E
Takemasa
Department of Bioscience, Integrated Center for Sciences, Ehime University, Shitsukawa, Toon-shi, Ehime, 791-0295, Japan.
Takeshi
T
Kiyoi
Department of Molecular and Cellular Physiology, Osaka City University Graduate School of Medicine, 1-5-7 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.
Miyuki
M
Kuno
Department of Molecular Medicine for Pathogenesis, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan.
Yusuke
Y
Oshima
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsugawa, Toon-shi, Ehime 791-0295, Japan.
Kazutaka
K
Maeyama
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D000071739:Calcium Release Activated Calcium Channels / Q000378:metabolism; D045744:Cell Line, Tumor; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism; D059010:Single-Cell Analysis; D019284:Thapsigargin / Q000494:pharmacology
D015220:Calcium Channels; D000071739:Calcium Release Activated Calcium Channels; C573659:Orai3 protein, rat; D050765:Qa-SNARE Proteins; D019284:Thapsigargin; D002118:Calcium