Cell biology of Ca2+-triggered exocytosis
Introduction
Ca2+-induced exocytosis initiates many forms of intercellular communication, as exemplified by synaptic transmission, which begins with Ca2+-triggered synaptic vesicle exocytosis that mediates neurotransmitter release (Figure 1) [1]. Similarly, neuroendocrine cells secrete hormones by Ca2+-induced exocytosis [2], mast cells release their granule contents upon stimulation by Ca2+-controlled exocytosis [3], and even in T-lymphocytes, Ca2+-triggered exocytosis is functionally essential [4]. The question of how Ca2+ triggers exocytosis was first raised by Bernhard Katz's seminal discovery that Ca2+ induces synaptic vesicle exocytosis, and thereby initiates synaptic transmission [5]. However, not much progress was made in this question until the discovery of synaptotagmin-1 (Syt1) as a candidate Ca2+-sensor for synaptic exocytosis [6]. Work from many laboratories has provided overwhelming evidence that Syt1 and its homologs function as the primary Ca2+-sensors in most forms of exocytosis, and has elucidated the principal mechanism by which synaptotagmin operates [1]. However, as described below, this work has also raised important new questions about the role of Ca2+ in regulating membrane traffic. The present review focuses on the cell biology of Ca2+-triggered exocytosis in neurons and endocrine cells, and tries to relate the emerging synaptotagmin Ca2+-sensor paradigm to these new unanswered questions.
Section snippets
Synaptic exocytosis
In presynaptic nerve terminals, neurotransmitters are packaged into small synaptic vesicles, and released by Ca2+-triggered exocytosis of synaptic vesicles at the presynaptic active zone (Figure 1). Three different modes of neurotransmitter release exist (Figure 2):
- 1.
Evoked synchronous release initiates within a millisecond after an action potential induces Ca2+-influx into a presynaptic terminal [7, 8]. Fast synchronous release measured as the postsynaptic response can be fitted by a double
Endocrine exocytosis
Hormonal exocytosis of endocrine cells operates on large dense-core vesicles (LDCVs) that are probably similar to neuropeptide LDCVs in neurons. LDCV exocytosis has been studied mostly in adrenal chromaffin cells and in pancreatic β-cells, where Ca2+-triggered exocytosis operates in three phases, referred to as the fast, slow, and sustained phase [28, 29]. The three phases of LDCV exocytosis have been attributed to different vesicle pools, but it is uncertain whether these pools represent
Synaptotagmins as Ca2+-sensors for exocytosis
Synaptotagmins are synaptic and secretory vesicle proteins (although some isoforms may be on the plasma membrane) that contain a single N-terminal transmembrane region, and two C-terminal Ca2+-binding C2-domains [6]. 16 mammalian synaptotagmin isoforms were identified, 8 of which bind Ca2+ with distinct apparent Ca2+-affinities (Syt1-3, Syt5-7, Syt9, and Syt10 [31, 32, 33], Table 1). Synaptotagmins are highly conserved evolutionarily; even all invertebrates express multiple isoforms. However,
Dual-Ca2+-sensor model for synaptic exocytosis
The most precise definition of synaptic transmission was achieved at the giant calyx of Held synapse in the brainstem that allows simultaneous patching of presynaptic and postsynaptic cells [57, 58]. Measurements in the calyx synapse provided estimates of the Ca2+-affinity (10–100 μM) and Ca2+-cooperativity (∼5 Ca2+-ions) of neurotransmitter release [44••, 57, 58]. As shown in mutant mice, this release is mediated by Syt2 as Ca2+-sensor [9, 44••].
Calyx synapses normally exhibit little
Synaptotagmin as a Ca2+-sensor for spontaneous release
At a synapse, lowering the extracellular Ca2+-concentration partially blocks spontaneous mini release; incubating synapses with membrane-permeable Ca2+-buffers, however, or infusing Ca2+-buffers into the calyx presynaptic terminal, blocks almost all spontaneous release [9, 17••]. These results suggested that the majority of spontaneous release is Ca2+-dependent, but raised the question what Ca2+-sensor mediates this effect. Interestingly, knockin mutations in Syt1 that change its apparent Ca2+
Other potential Ca2+-sensors for exocytosis
Which Ca2+-sensor mediates asynchronous release and other Ca2+-dependent types of exocytosis in which Syt1, Syt2, Syt7, and Syt9 do not act as Ca2+-sensors? Naturally, prime candidates are the other four Ca2+-binding synaptotagmins that have no known function (Syt3, Syt5, Syt6, and Syt10). However, at least for asynchronous release, this candidacy is doubtful since these synaptotagmins bind Ca2+ via a mechanism akin to that of Syt1, with a likely Ca2+-binding stoichiometry of ∼5, whereas
Conclusions
A universal mechanism by which Ca2+-binding to synaptotagmins triggers exocytosis has emerged over the past decade. This mechanism mediates most Ca2+-triggered exocytosis using a pas-de-deux of synaptotagmins and complexin acting on SNARE complexes and phospholipid membranes. However, new intriguing questions have emerged. Are forms of exocytosis for which no synaptotagmin Ca2+-sensor has been identified, such as asynchronous release, mediated by an atypical synaptotagmin, or by novel Ca2+
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Note Added in Proof
After this review was accepted for publication, three more papers described single molecule studies demonstrating how synaptotagmin functions as a Ca2+-sensor for Ca2+-dependent vesicle fusion in vitro [77, 78, 79].
Acknowledgements
We wish to thank Drs. Weiping Han and Jianyuan Sun for valuable discussions. Z.P.P. was supported by NARSAD Young Investigator Award and NIH/NINDS Epilepsy Training Grant5T32NS007280.
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