Abstract
Regulated secretion is an intracellular pathway that is highly conserved from protists to humans. Granin family proteins were proposed to participate in the biogenesis, maturation and release of secretory granules in this pathway. However, the exact molecular mechanisms underlying the intracellular functions of the granin family proteins remain unclear. Here, we show that chromogranin B (CHGB), a secretory granule protein, inserts itself into membrane and forms a chloride-conducting channel. CHGB interacts strongly with phospholipid membranes through two amphipathic α helices. At a high local concentration, CHGB insertion in membrane causes significant bilayer remodeling, producing protein-coated nanoparticles and nanotubules. Fast kinetics and high cooperativity for anion efflux from CHGB vesicles suggest that CHGB tetramerizes to form a functional channel with a single-channel conductance of ∼125 pS (150/150 mM Cl-). The CHGB channel is sensitive to an anion channel blocker and exhibits higher anion selectivity than the other six known families of Cl- channels. Our data suggest that the CHGB subfamily of granin proteins forms a new family of organelle chloride channels.
Highlights
Cells rely on secretory pathways to send out specific bioactive molecules to their surroundings (Grimes & Kelly, 1992)
chromogranin B (CHGB) inserts into membranes and induces nanoparticles and nanotubules from bilayers To study CHGB function, recombinant murine CHGB was purified from sf9 cells (Fig S1A)
In sizeexclusion chromatography (SEC), purified CHGB was eluted as a single, symmetric peak (Fig 1A) with an apparent size equivalent to an ~300 kD globular protein
Summary
Cells rely on secretory pathways to send out specific bioactive molecules to their surroundings (Grimes & Kelly, 1992). Granin family proteins are by default granule proteins that chaperone other secretory molecules through regulated secretion (Hosaka & Watanabe, 2010; Bartolomucci et al, 2011). They have both intracellular and extracellular functions. It was proposed that CHGs interact with cargos and serve as a low-affinity, high-capacity Ca2+ reserve Their extracellular functions are executed by CHG-derived peptides that are associated with various human diseases (Bartolomucci et al, 2011). Native chromogranin B (CHGB) forms high-order aggregates at low pH and with milliMolar Ca2+ (Yoo, 1995a, 1995b). A companion article will study the CHGB channel function in neuroendocrine cells
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