Calcium-sensing beyond neurotransmitters: functions of

Biosci. Rep. (2009) / 29 / 245–259 (Printed in Great Britain) / doi 10.1042/BSR20090031

Calcium-sensing beyond neurotransmitters:functions of synaptotagmins in neuroendocrineand endocrine secretionNatalia GUSTAVSSON and Weiping HAN1

Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, A*STAR, Singapore 138667

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SynopsisNeurotransmitters, neuropeptides and hormones are released through the regulated exocytosis of SVs (synapticvesicles) and LDCVs (large dense-core vesicles), a process that is controlled by calcium. Synaptotagmins are a familyof type 1 membrane proteins that share a common domain structure. Most synaptotagmins are located in brainand endocrine cells, and some of these synaptotagmins bind to phospholipids and calcium at levels that triggerregulated exocytosis of SVs and LDCVs. This led to the proposed synaptotagmin–calcium-sensor paradigm, that is,members of the synaptotagmin family function as calcium sensors for the regulated exocytosis of neurotransmitters,neuropeptides and hormones. Here, we provide an overview of the synaptotagmin family, and review the recent mousegenetic studies aimed at understanding the functions of synaptotagmins in neurotransmission and endocrine-hormonesecretion. Also, we discuss potential roles of synaptotagmins in non-traditional endocrine systems.

Key words: calcium sensor, hormone secretion, neuropeptide, neurotransmitter, regulated exocytosis,synaptotagmin

INTRODUCTION

The classical work of Katz and colleagues (see [1–5]) more thanhalf a century ago established the quantum nature of neurotrans-mitter release and the pivotal role of calcium in regulating theprobability of a given quantum being released. These funda-mental findings, particularly the function of calcium in exocytoticsecretion, were later extended and confirmed in neuroendocrineand endocrine systems, including adrenal chromaffin cells andpancreatic β- and α-cells [6–9]. The identification of proteins thatcan transduce calcium signals to exocytotic machineries has beenof tremendous interest among neuroscientists and cell biologists,as this would lead to the elucidation of the fundamental neuronalfunction, namely synaptic transmission, and the general mechan-isms of regulated exocytosis. Synaptotagmin-1, which was des-cribed as a protein on SVs (synaptic vesicles) and LDCVs (largedense-core vesicles) by an antibody-binding study [10], emergedas a leading candidate, and was suggested to function as a calciumsensor for synaptic exocytosis based on molecular and structural

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Abbreviations used: B/K protein, brain/kidney protein; E-Syt, extended synaptotagmin-like protein; GIP, gastric inhibitory polypeptide, also known as glucose-dependent insulinotropicpeptide; GLP-1, glucagon-like peptide 1; LDCV, large dense-core vesicle; MCTP, multiple C2-domain and transmembrane protein; RNAi, RNA interference; SNARE, solubleN-ethylmaleimide-sensitive fusion-protein-attachment protein receptor; Strep14, synaptotagmin XIV-related protein; SV, synaptic vesicle.1To whom correspondence should be addressed (email [email protected]).

analysis [11]. It is now known that synaptotagmin-1 belongs to atype 1 membrane-protein family of at least 15 members (Table 1),and that some synaptotagmins are expressed primarily in neurons,and neuroendocrine and endocrine cells, with calcium-binding af-finities in the range of 1–40 μM, the levels at which regulated exo-cytosis is triggered in these cells [12]. Because of its potential roleas a calcium sensor for SV and LDCV exocytosis, the synaptot-agmin family has attracted considerable interest over the past 20years. A number of bioinformatics studies and in-depth reviewshave been published on various aspects of the synaptotagminfamily, for example, synaptotagmin gene structures and phylo-geny [13–16], protein structure and function [17–19], and mo-lecular mechanisms of its actions in neurotransmission and SVexocytosis [20–26]. In the present review, we provide an over-view of the mammalian synaptotagmin family, and focus on re-cent mouse genetic studies that provide an understanding of thefunctions of synaptotagmins in neurotransmission and endocrine-hormone secretion. In addition, we discuss potential roles of themammalian synaptotagmin family in atypical endocrine systemsand non-classical secretory-granule exocytosis.

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N. Gustavsson and W. Han

Table 1 The synaptotagmin family and related proteins

Protein name Gene name Genbank® accession number Amino acid length Molecular mass (Da)

Synaptotagmin-1 synaptotagmin I NM_00 9306 421 47418

Synaptotagmin-2 synaptotagmin II NM_00 9307 422 47263

Synaptotagmin-3 synaptotagmin III NM_01 6663 587 63254

Synaptotagmin-4 synaptotagmin IV NM_00 9308 425 47659

Synaptotagmin-5 synaptotagmin IX* NM_02 1889 491 56247

Synaptotagmin-6 synaptotagmin VI NM_01 8800 511 57190

Synaptotagmin-7 synaptotagmin VII‡ NM_01 8801 403 45472

Synaptotagmin-8 synaptotagmin VIII NM_01 8802 395 44093

Synaptotagmin-9 synaptotagmin V† NM_01 6908 386 43130

Synaptotagmin-10 synaptotagmin X NM_01 8803 523 59019

Synaptotagmin-11 synaptotagmin XI NM_01 8804 430 48359

Synaptotagmin-12 synaptotagmin XII NM_13 4164 421 46680

Synaptotagmin-13 synaptotagmin XIII NM_03 0725 426 46870

Synaptotagmin-14 synaptotagmin XIV NM_18 1546 555 62044

Synaptotagmin-15 synaptotagmin XV NM_17 6931 389 43523

Strep14 synaptotagmin XVI NM_17 2804 549 61339

B/K synaptotagmin XVII NM_13 8649 470 53293

*The gene name for synaptotagmin-5 is synaptotagmin IX.†The gene name for synaptotagmin-9 is synaptotagmin V.‡Only the short form of synaptotagmin-7 is listed in the Table. Synaptotagmin-7 is expressed in multiple splicing variants, ranging from 122 amino acids for the truncated form to 687amino acids for the long form.

THE SYNAPTOTAGMIN FAMILY

By definition, a synaptotagmin is composed of a short N-terminalsequence followed by an N-terminal transmembrane region, alinker of variable length and two functional C2 domains: C2Aand C2B (see Figure 1 and below for more discussion on C2

domains) [16]. So far, at least 15 synaptotagmins have been iden-tified and cloned, which can be divided into five groups basedon their genomic and sequence similarities (see Figure 1 andFigure 2 for details) [14,16]. Several reports described or re-ferred to Strep14 (synaptotagmin XIV-related protein) and B/Kprotein (brain/kidney protein) as the two latest members of thesynaptotagmin family, namely synaptotagmin-16 and -17 (Fig-ure 2 and Table 1) [14,15,27,28]. Although these two ‘synapto-tagmins’ have some of the features shared by the synaptotagmins,such as two C-terminal C2 domains, they lack an N-terminal trans-membrane region and therefore they should not be classified assynaptotagmins. Two other protein families, rabphilin and Doc2(double C2 protein), also contain two C-terminal C2 domains,and, similar to Strep14 and B/K protein, they lack transmem-brane regions, and thus are related to synaptotagmins, but arenot synaptotagmins in the strict sense of the term [29–32]. Inaddition to synaptotagmins, there are several other families ofmembrane proteins with sequence and structural similarities tosynaptotagmins. E-Syts (extended synaptotagmin-like proteins)contain an N-terminal transmembrane region but more than twoC2 domains [33,34], ferlins contain three to six C2 domains anda C-terminal transmembrane region [35], and MCTPs (multipleC2-domain and transmembrane proteins) contain three C2

domains and two transmembrane regions (Table 2) [36].

Figure 1 Domain structures of mouse synaptotagminsAll synaptotagmins are composed of an N-terminal transmembrane re-gion, a linker of variable length and two functional C2 domains. Synapto-tagmins are divided into five groups based on sequence similarities. The‘Y’ shape in synaptotagmin-1 and -2 denotes N-terminal glycosylation,a feature that is unique to these two synaptotagmins. The linker regionin synaptotagmin-7 is extensively alternatively spliced into at least 12isoforms. A conserved stop codon in one of the alternatively splicedexons results in a truncated form, synaptotagmin-7T. Synaptotagmin-7Lis a long form that results from the usage of all four spliced exons.Synaptotagmin-7S is the standard form of synaptotagmin-7.

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246 C©The Authors Journal compilation C©2009 Biochemical Society

Functions of synaptotagmins in endocrine secretion

Table 2 Other C2-containing membrane proteinsBesides synaptotagmins, three additional families of membrane proteins contain calcium-binding C2 domains: E-Syts (E-Syt1, E-Syt2 and E-Syt3),ferlins (dysferlin, otoferlin and myoferlin), and MCTPs (MCTP1 and MCTP2).

Protein name Gene name Genbank® accession number Amino acid length Molecular mass (Da)

E-Syt1 Fam62a NM_011843 1092 121554

E-Syt2 Fam62b NM_028731 845 94139

E-Syt3 Fam62c NM_177775 891 100168

Dysferlin dysferlin Q9ESD7 2090 237911

Otoferlin otoferlin Q9ESF1 1997 224819

Myoferlin myoferlin Q69ZN7 2048 233324

MCTP1 mctp1 NM_030174 694 80539

MCTP2 mctp2 NM_001024703 878 100178

Figure 2 Phylogenetic tree for mouse synaptotagmins (mSyts),Syt14l and B/K proteinSynaptotagmins are grouped into five subfamilies. Note that Syt14l andB/K protein are grouped together with synaptotagmin-12 to -15. How-ever, these two proteins are not part of the synaptotagmin family due totheir lack of N-terminal transmembrane regions. Syt14l, synaptotagmin14-like protein (also known as synaptotagmin 14-related protein).

The precise tissue distribution, cellular localization and functionsof these other protein families are mostly unknown, althoughcertain members of the ferlins are linked to muscular dystrophy,which may be caused by impaired membrane fusion and con-sequent defects in plasma-membrane repair [33,35,36].

The C2 domain is a widely occurring conserved sequencemotif of 130–140 amino acid residues, which was first definedas the second constant sequence in protein kinase C isoforms[11,17,18]. The C2 domain was first shown to bind to calciumin synaptotagmin-1 [17,18,37,38]. Subsequent atomic-structureanalysis of synaptotagmin-1 at 1.9 A (1 A = 0.1 nm) resolutionindicated that its C2 domains are composed of a stable eight-stranded β-sandwich with flexible loops emerging from the topand bottom (Figure 3) [18,39]. NMR studies of synaptotagmin-1revealed that calcium binds exclusively to the top loops, andthe binding pockets are coordinated by five conserved aspart-ate residues: three calcium ions bind to C2A via Asp172, Asp178,

Asp230, Asp232, Ser235 and Asp238, and two calcium ions bind toC2B via Asp303, Asp309, Asp363, Asp365 and Asp371 (Figure 3)[40–42]. Not all synaptotagmin C2 domains bind to calcium. Infact, based on sequence similarities and subsequent confirma-tion by biochemical analyses, only eight synaptotagmins bindto calcium, namely synaptotagmin-1, -2, -3, -5, -6, -7, -9 and-10. The lack of critical residues involved in calcium binding ac-counts for the majority of the failure of the other synaptotagminsto bind to calcium. This applies to both of the C2 domains ofsynaptotagmin-8, -12, -13, -14 and -15, and the C2A domainof synaptotagmin-4 and -11 (Figure 3) [16,43,44]. Although theC2B domains of synaptotagmin-4 and -11 possess all five acidicresidues in the top loops, they do not bind to calcium due tothe spatial orientation of the calcium ligands, which fail to formproper calcium-binding sites (Figure 3) [45]. For calcium-bindingsynaptotagmins, although amino acid residues in the top loopsother than those mentioned above are not directly involved in co-ordinating calcium binding, they affect calcium-binding affinity,such as Arg233 in synaptotagmin-1 [40,41,46,47]. The diversityof sequences and structures flanking the calcium-coordinatingamino acid residues enables the eight synaptotagmins to bind tocalcium with various affinities, covering the full range of calciumrequirements for regulated exocytosis [11,12,16,48–51].

THE SYNAPTOTAGMIN–CALCIUM-SENSOR PARADIGM

As alluded to earlier, the regulated exocytosis of SVs in neur-ons, and of LDCVs in neuroendocrine and endocrine cells, istriggered by calcium. To function as a calcium sensor in regu-lated exocytosis, a protein must satisfy the following criteria:first, it must have proper calcium affinity to detect a rise inintracellular calcium levels during the release processes;secondly, it must show a calcium cooperativity that is consistentwith the calcium dependence of triggered exocytosis. In the caseof SV exocytosis, neurotransmitter release is composed of twokinetically distinct components: a major synchronous componentand a delayed asynchronous component [20,52,53]. For the syn-chronous component, the required calcium concentrations are in

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Figure 3 For legend see facing page

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the range of ∼10–40 μM and the calcium-cooperativity value is∼5, as determined by the photolysis of caged calcium in the calyxof Held giant synapse, the best and most systematically analysedpreparation to date [24,54–58], whereas the calcium requirementfor the asynchronous component is estimated to be in the rangeof several μM to several tens of μM, with a calcium cooperativityvalue of 2–3 [24,58]. Remarkably, synaptotagmin-1, which waspreviously proposed to function as a calcium transducer for neur-otransmitter release based on biochemical studies [11], meets therequirements for the synchronous calcium sensor, an idea that issupported and confirmed by two important mouse genetic studies.The first genetic study was performed on hippocampal neurons ofsynaptotagmin-1 knockout mice, which showed a complete ab-sence of the major synchronous component of neurotransmitterrelease, demonstrating that synaptotagmin-1 is required for syn-chronous neurotransmitter release [24,59,60]. It is worth notingthat the essential role of synaptotagmin-1 in neurotransmissionwas also shown in synaptotagmin-1 null mutants of Drosophilaand Caenorhabditis elegans [61–64]. The second genetic studywas carried out on hippocampal neurons of a synaptotagmin-1knockin mouse harbouring a point mutation (R233Q) in thesynaptotagmin-1 C2A domain that alters the overall apparent cal-cium affinity of synaptotagmin, which showed an identical shiftin the apparent calcium affinity of SV exocytosis [46]. Besidesthese genetic studies, two other studies that used synaptotagmin-1knockin (D232N and D238N) mice [47,65] and two studies thatused the viral expression of various synaptotagmin-1 mutants insynaptotagmin-1 knockout neurons [66,67] provide further sup-port for the conclusion of the R233Q knockin study: the overallcalcium affinity of synaptotagmin-1 determines the calcium af-finity of SV exocytosis. Together with the calcium-binding prop-erties, these genetic studies of synaptotagmin-1 established itsfunction as a calcium sensor for synchronous neurotransmitterrelease [24].

Subsequent genetic analyses of synaptotagmin-2 and -9 knock-out mouse lines provided unequivocal evidence that these twosynaptotagmins also function as synchronous calcium sensorsfor neurotransmitter release, synaptotagmin-2 for neurons in thecaudal brain regions and synaptotagmin-9 for neurons inthe limbic system, which complement synaptotagmin-1 as thefast calcium sensor for neurons in the rostral brain regions[24,46,48,58,59,68–71]. The differential distribution and dis-tinct properties of the three neuronal calcium sensors supportthe emerging synaptotagmin–calcium-sensor paradigm, namelythat synaptotagmins serve as individually acting Ca2+ sensors inneurotransmitter release [24,46,48,58,59,68–71]. However, thisalso raises several important questions. What are the identities ofthe calcium sensors for asynchronous neurotransmitter release?What is the function of non-calcium-binding synaptotagmins?

Does the synaptotagmin Ca2+-sensor paradigm apply generallyto the exocytosis of LDCVs, such as hormone-containing secret-ory granules? Finally, do synaptotagmins play any role in non-traditional endocrine tissues, such as leptin and adiponectin se-cretion from adipocytes?

CALCIUM SENSORS FORASYNCHRONOUSNEUROTRANSMITTER RELEASE

Synchronous neurotransmitter release is a highly specialized pro-cess and the fastest in cell biology. Thousands of vesicles canbe released with a lag time as short as 60 μs in certain syn-apses [57,72–74]. This process is estimated to require calciumin the 10–40 μM range and a calcium-binding cooperativity of5 [24,54–58]. In comparison, asynchronous neurotransmitter re-lease is considerably slower, and requires slightly lower levelsof calcium and a lower calcium-binding cooperativity [58]. Itis clear that synaptotagmin-1, -2 and -9 function as the prin-cipal calcium sensors for synchronous neurotransmitter releasein their respective synapses [46,58,59,68,69,75,76]. For calcium-sensing during asynchronous neurotransmitter release, additionalproteins must be involved. One possibility is that SVs for fast andslow release are molecularly distinct, i.e. SVs possessing fast cal-cium sensors are destined for fast synchronous release and thosewith slow sensors for delayed asynchronous release. However,there is no evidence that these different pools of SVs with dif-ferent molecular compositions exist. Another possibility, whichalso assumes the existence of SV pools of distinct molecularidentities, is that different ratios and/or combinations of proteinson SVs determine how a particular SV is released. A third pos-sibility, which is supported by experimental data and mathem-atical modelling, is the dual-sensor model: calcium sensors forsynchronous and asynchronous release operate in competitionwith each other, with the slower asynchronous sensor bindingto calcium at lower concentrations and cooperativity, whereasthe faster synchronous sensor binds at higher concentrations andcooperativity [58,77,78]. This model, which predicts that the syn-chronous sensor dominates during pulses of high calcium con-centrations, while the asynchronous sensor dictates release eventsduring sustained phases of lower calcium concentrations, is sup-ported by the demonstration in the calyx of Held synapse thatsynchronous and asynchronous release operate on the same ves-icle pool [58].

As only synaptotagmin-1, -2 and -9 are principal calciumsensors for neurotransmitter release in their respective brain

Figure 3 Sequence alignment for mouse synaptotagmin (mSyt)-family proteinsC2A (A) and C2B (B) of synaptotagmins were aligned using the AlignX® program of Vector NTI® (Invitrogen). Red barslabelled β1–β8 mark the eight β-strands in the C2 domains. Blue bars mark the two α-helices in the C2B domain. Invertedfilled triangles denote the acidic residues that form the calcium-binding pockets. Colour highlighting of the sequences:yellow and blue denote identical amino acids in all (yellow) or most (blue) synaptotagmins, and green indicates conservedamino acid substitutions.

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Table 3 Comparison of neurotransmitter release with peptide secretion

Characteristic Neurotransmitter release Peptide secretion

Cargo Neurotransmitters (e.g. glutamate, GABA) Peptides/hormones (e.g. insulin, catecholamines)

Storage Synaptic vesicles LDCVs or secretory granules

Vesicle size 40–50 nm 200–350 nm

Regeneration Multiple pathways* Recycled via Golgi complex

Speed Fast (∼60 μs to 6 ms) Slow (up to tens of s)

Release site Synaptic active zone Plasma membrane†

Targets Postsynaptic receptors May be far from release site

Duration of action Short Long

Calcium requirement Tens of μM Lower (several to tens of μM)

*SVs may be recycled locally or regenerated through the endosomal pathway.†Hot spots, analogous to the synaptic active zone, were reported to be present on the plasma membrane of neuroendocrine cells [189].

regions, other calcium-binding synaptotagmins (synaptotagmin-3, -5, -6, -7 and -10) may function as slow sensors in cooperationwith one of the principal sensors. Support for this hypothesiscomes from the observations that these five other synaptotag-mins bind to calcium at higher affinities [12,24,79]. Of these fivesynaptotagmins, only synaptotagmin-7 was investigated for itsrole as a calcium sensor in GABAergic neurons from a mouseknockout model: deletion of synaptotagmin-7 did not changefast synchronous release, slow asynchronous release or short-term synaptic plasticity of release of neurotransmitters [80]. Thisstudy indicated that synaptotagmin-7 is unlikely to be a slowsensor for inhibitory neurons in the cortex, but may function inother forms of calcium-dependent synaptic exocytosis or peptide-hormone secretion [80].

FUNCTIONS OF NON-CALCIUM-BINDING SYNAPTOTAGMINS

Seven of the 15-member synaptotagmin family (synaptotagmin-4, -8, -11, -12, -13, -14 and -15) do not bind to calcium, andsome of these non-calcium-binding synaptotagmins are presentat high levels in neurons and endocrine cells (see below for adiscussion on particular non-calcium-binding synaptotagmins inendocrine secretion) [12,16,23,24,81,82]. As they do not bind tocalcium, these synaptotagmins cannot function as individuallyacting calcium sensors for various release processes. They may,however, regulate calcium-independent vesicle exocytosis, suchas calcium-independent voltage-dependent secretion in dorsalroot ganglia neurons [83], or partner with a calcium-bindingsynaptotagmin to regulate calcium-dependent neurotransmitterrelease.

Among the seven non-calcium-binding synaptotagmins, verylittle information is available for synaptotagmin-13, -14 and -15beyond descriptions of molecular cloning and basic character-izations [28,43,84]. Synaptotagmin-12 forms a stable complexwith synaptotagmin-1 on SVs, but regulates spontaneous vesicle

release independent of synaptotagmin-1 [85]. Synaptotagmin-11has been linked to neurological and neurodegenerative diseases,but its molecular functions are unclear [86,87]. Synaptotagmin-8is present in kidney epithelia and sperm acrosomes, but isabsent or present at very low levels in the brain [82,88,89].Synaptotagmin-4 is the best-studied non-calcium-binding syn-aptotagmin, but it is expressed at rather low levels (lower than,for example, its close relative, synaptotagmin-11 [82]), and its tis-sue distribution, cellular localization and function remain incon-clusive or even controversial. For example, synaptotagmin-4 wasreported to be located on LDCVs to regulate secretory-granulematuration and fusion-pore kinetics during regulated exocytosis[90–93], on SVs in hippocampal neurons but without a role in reg-ulating fusion-pore kinetics or calcium-dependent SV exocytosis[94], on Golgi and non-LDCV vesicular structures in PC12 cells(a tumour cell line derived from rat adrenal medulla) [95], in thepostsynaptic compartment [96,97], and on astrocytes to regulatecalcium-dependent vesicle exocytosis [98,99]. More studies areneeded to understand whether and how the non-calcium-bindingsynaptotagmins function in regulated exocytosis.

CALCIUM SENSORS INNEUROENDOCRINE AND ENDOCRINESECRETION

Although neurotransmitter release and peptide-hormone secre-tion have distinct characteristics (Table 3), the fundamental mech-anisms of regulated exocytosis in neuronal, neuroendocrine andendocrine cells are quite similar, in that they all depend on aconserved core machinery for vesicle fusion at the plasma mem-brane. The proteins involved in regulated exocytosis in synapsesand neuroendocrine cells, and the distinct characteristics of thesetwo types of regulated exocytosis, have been extensively studiedand reviewed (for reviews, see [22–25,100–107]). This sectionof the review focuses on recent studies, particularly mouse ge-netic studies on calcium-sensing in primary neuroendocrine and

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endocrine cells, with the occasional mention of relevant studiesusing derived tumour cell lines.

Adrenal chromaffin cellsAdrenal chromaffin cells and the PC12 tumour cell line derivedfrom them are one of the most widely used cell systems instudying regulated exocytosis [2,6,100–103,105,108–112]. Pan-creatic β-cells are another popular cell model, which will be dis-cussed below [113–115]. Many fundamental studies regardingregulated exocytosis and the identities of calcium sensors wereperformed in PC12 cells (e.g. [12,79,116–122]), some of whichhave been confirmed by subsequent genetic studies [123–129].However, there are discrepancies between studies performed onPC12 and adrenal chromaffin cells in certain cases, due to dif-ferent molecular compositions of protein isoforms in these twocell types [130–133]. Regarding calcium-sensing in adrenal chro-maffin cells, the first functional genetic analysis was carried out insynaptotagmin-1 knockout mice [134]. Similar to neurotransmit-ter release, catecholamine release from adrenal chromaffin cellsalso follows rapid and delayed phases [135–137]. Using a com-bination of calcium-uncaging and membrane-capacitance meas-urements, Voets et al. [134] showed that synaptotagmin-1 deletionspecifically abolished the fast burst of exocytosis that is supportedby the readily releasable pool of vesicles in chromaffin cells. Con-sistent with SV exocytosis studies of synaptotagmin-1 knockout,this report demonstrated that synaptotagmin-1 was required forthe rapid calcium-dependent fusion of LDCVs, and suggestedthat synaptotagmin-1 functions as a calcium sensor during therapid phase of LDCV exocytosis in chromaffin cells [134].

Similar to neurotransmitter-release studies, the specific effectsof synaptotagmin-1 deletion on the rapid phase of LDCV exocyt-osis prompted strong interest in the search for an additional cal-cium sensor that complements synaptotagmin-1 to regulate thedelayed phase of LDCV exocytosis. Besides synaptotagmin-1,synaptotagmin-7, but not synaptotagmin-2 or -9, is also expressedin adrenal chromaffin cells [132]. Synaptotagmin-7 is ubiquit-ously expressed during early stages of development, but be-comes restricted to secretory cells with regulated exocytosis afterbirth [138]. Among the eight calcium-binding synaptotagmins,synaptotagmin-7 shows maximal calcium-dependent phosphol-ipid binding in the low-micromolar calcium range [12], whichfits well with the calcium requirement during delayed exocyt-osis in chromaffin cells [134]. Schonn et al. [132] studied therole of synaptotagmin-7 in chromaffin-granule exocytosis onthree genetically modified mice and found that: (i) deletion ofsynaptotagmin-7 alone impaired calcium-triggered exocytosis by∼50%; (ii) calcium-binding to synaptotagmin-7 C2B was essen-tial for the normal function of synapthotagmin-7, as the impairedexocytosis observed in synaptotagmin-7 knockout mice was re-produced in synaptotagmin-7 knockin mice that expressed nor-mal levels of synaptotagmin-7 with a mutated C2B domain thatresulted in the complete loss of calcium binding; (iii) both thefast and the delayed LDCV exocytosis were nearly abolishedin synaptotagmin-1 and -7 double-knockout mice. These res-ults established that synaptotagmin-7 is a major calcium sensor

for chromaffin-granule exocytosis, and functions in complementwith synaptotagmin-1 in the regulation of calcium-dependentLDCV exocytosis in adrenal chromaffin cells [132].

Pancreatic islet β-cellsAmong endocrine cells, pancreatic β-cells are probably the mostextensively studied cell model because of the great importanceof insulin secretion in normal physiology and in diabetes [113–115]. Insulin is stored and secreted from β-cells via a complex,highly regulated process. Under physiological conditions, an el-evation of blood glucose triggers rapid uptake of glucose intopancreatic β-cells. Glucose metabolism inside β-cells resultsin an increased ATP/ADP ratio, which leads to KATP-channelclosure, membrane depolarization and the subsequent opening ofvoltage-gated calcium channels and a rise in cytoplasmic calciumconcentration [114]. Glucose-stimulated insulin secretion has abiphasic pattern, consisting of a 10–15-min rapid first phase anda less prominent but sustained second phase [139]. The first phaseof insulin secretion requires a rapid and marked elevation of in-tracellular calcium concentration, whereas the second phase re-quires amplifying signals from glucose metabolism in additionto oscillatory intracellular calcium [140]. Insulin secretion bycalcium-dependent LDCV exocytosis is probably executedby SNARE (soluble N-ethylmaleimide-sensitive fusion-protein-attachment protein receptor) proteins, similar to neurotransmitterrelease in central neurons and neuropeptide secretion from chro-maffin cells [7,22]. In addition to SNARE proteins, numerousexocytotic proteins that may be involved in insulin-secretion reg-ulation have been identified [104,113], although functional rolesof these proteins in insulin secretion remain to be established.

Regarding calcium-sensing in insulin secretion, most of thestudies were performed using insulin-secreting cell lines, for ex-ample, INS-1, RINm5F and MIN6 cells. These studies on thepotential roles of individual synaptotagmins and other proteins,such as calpain and calcium channels, in insulin secretion ininsulin-secreting cell lines were recently reviewed or reported[113,141,142]. The rest of this section will focus on two calcium-binding synaptotagmin isoforms that show the highest transcriptlevels in pancreatic islets: synaptotagmin-7 and -9 [81].

Synaptotagmin-7 is expressed in LDCVs of pancreatic β-cells[81,143]. Although no consensus has been reached in terms of theprecise calcium levels that support insulin-granule exocytosis, itis estimated to be in the low-micromolar range, similar to thatin neuroendocrine cells, such as adrenal chromaffin cells [144–147]. Studies have shown that synaptotagmin-7 is present andappears to regulate insulin secretion in insulin-secreting cell lines[143,148]. Physiological analysis of synaptotagmin-7 knockoutmice demonstrates that deletion of synaptotagmin-7 leads to gluc-ose intolerance and impaired insulin secretion in vivo, as wellas markedly reduced insulin secretion from isolated islets [81].Synaptotagmin-7 knockout mice exhibit normal insulin sensitiv-ity, insulin production, islet architecture and β-cell ultrastructuralorganization [81,149]. Furthermore, the knockout mice displaynormal metabolic and calcium responses after glucose stimula-tion [81]. These results show that reduced insulin secretion in

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synaptotagmin-7 knockout mice is not caused by cellular defectsor impaired signalling upstream of calcium triggering of insulin-granule exocytosis, and reveal that synaptotagmin-7 functions asa positive regulator of insulin secretion, consistent with its pro-posed role as a high-affinity calcium sensor regulating insulinsecretion.

As synaptotagmin-7 deletion only reduces insulin secretionby ∼40%, other protein(s) must be involved in regulating theremaining LDCV exocytosis in pancreatic β-cells. One probablecandidate is synaptotagmin-9, which is expressed at similar levelsto synaptotagmin-7 in mouse islets, and at least 6–8-fold higherthan the two next-highest-expressed calcium-binding synaptot-agmins, synaptotagmin-2 and -3 [81]. Synaptotagmin-9 bindsto calcium and phospholipids with an affinity of 10–30 μM [51].Moreover, synaptotagmin-9 is present in β-cells, and knockdownof synaptotagmin-9 by adenoviral-mediated RNAi (RNA inter-ference) results in impaired insulin secretion in rat islet cells[113,150]. The in vitro results appear to support synaptotagmin-9as another calcium sensor in regulating insulin-granule exocyt-osis. However, RNAi often has off-target effects [151,152], anda more specific method, such as the analysis of synaptotagmin-9knockout mice, is needed to test the functions of synaptotag-min-9 in regulating insulin secretion at the final calcium-dependent exocytotic steps.

Since synaptotagmin-7 knockout and synaptotagmin-9 knock-down each reduces insulin secretion by ∼40–50%, the questionremains as to whether synaptotagmin-7 and -9 act independ-ently from each other, each being responsible for a subset ofinsulin secretion, or whether synaptotagmin-7 and -9 work asa team and together are responsible for the ∼40–50% secre-tion of insulin. To address this issue, it will be necessary togenerate synaptotagmin-7 and -9 double-knockout mice to test:(i) whether deletion of both synaptotagmin-7 and -9 causes fur-ther inhibition of insulin secretion beyond the 40–50% level, asobserved in synaptotagmin single-knockout mice. If the inhibi-tion effect is additive, i.e. the deletion of both synaptotagmin-7and -9 abolishes insulin-granule exocytosis, then synaptotagmin-7 and -9 function independently. If the inhibition remains at40–50%, then synaptotagmin-7 and -9 function as partners toregulate the same pool of LDCVs. The situation is complicatedgreatly if the inhibition falls in between the two scenarios, as par-tial compensation by other synaptotagmins or proteins may beinvolved; (ii) whether there is any residual calcium-dependentinsulin secretion in the absence of synaptotagmin-7 and -9.The presence of a residual calcium-dependent component wouldindicate the existence of a third calcium sensor.

Pancreatic islet α-cellsGlucagon is stored in LDCVs of pancreatic α-cells, and issecreted in response to low blood-glucose levels to stimulate gluc-ose production and glucose release in the liver to restore normalglucose levels [153]. Although both insulin and glucagon regu-late glucose homoeostasis, far fewer studies have been devoted tothe regulation of glucagon secretion than that of insulin release:currently, there is only one glucagon-secretion study for every

five insulin-secretion reports. It is no wonder that after almostfifty years since the description of the glucagon-quantificationmethod [154–156], many aspects of glucagon-secretion regu-lation remain unclear or are just beginning to be unravelled[9,157]. The control of glucagon secretion is very complic-ated, and involves direct calcium-mediated stimulus-secretioncoupling [158], paracrine regulation by insulin, γ-aminobutyricacid, zinc and other factors released from neighbouring β- andδ-cells, and circulating hormones and the autonomic nervous sys-tem [9]. An in-depth and comprehensive review on many aspectsof glucagon secretion and α-cell biology was published recently[9].

Although the precise cellular mechanism of glucagon-granuleexocytosis remains incompletely defined, it is clear that KATP

channels and N-type calcium channels are intimately involvedin the process [158–162], and calcium provides the trigger forLDCV exocytosis in α-cells [8,9]. Very recently, it was repor-ted that synaptotagmin-7 functions as an individually acting cal-cium sensor that mediates nearly all calcium-triggered glucagon-granule exocytosis [163]. To date, this remains the only studyon calcium sensors for glucagon secretion. This study showedthat deletion of synaptotagmin-7 nearly abolished (a reduction of∼80%) glucagon secretion induced by hypoglycaemia in vivo,by low glucose in isolated islets or by membrane depolarizationsin single α-cells, whereas all other physiological and morpho-logical parameters, for example, glucagon content, glucagon-granule number and distribution, and α-cell electrical proper-ties, are unchanged, and thus identified synaptotagmin-7 as theprincipal synaptotagmin isoform required for glucagon secretion[163].

The deletion of synaptotagmin-7 results in ∼40% reductionin insulin secretion, an effect that is reminiscent of neuropeptidesand catecholamine release in synaptotagmin-7 knockoutchromaffin cells, which rely on two synaptotagmin isoforms:synaptotagmin-1 and -7 [81,132]. This raised the questionof the relative importance of synaptotagmin-7 in regulatedexocytosis. On the one hand, synaptotagmin-7 is essential forcalcium-dependent LDCV exocytosis in chromaffin and pan-creatic β-cells, as synaptotagmin-7 deletion causes significantimpairment to regulated exocytosis [81,132]. On the other hand,it appears that synaptotagmin-7 always relies on another calciumsensor, for example, synaptotagmin-1 in chromaffin cells, to exertits actions in calcium sensing, thus making synaptotagmin-7an accessory calcium sensor to the principal calcium sensor[132]. These observations raised further questions: whether thesynaptotagmin–calcium-sensor theory applies only to neuronalcalcium sensors, i.e. synaptotagmin-1, -2 and -9, but not toneuroendocrine or endocrine secretion; and whether regulatedexocytosis in neuroendocrine and endocrine cells alwaysinvolves the collaboration of two synaptotagmin isoforms. Theglucagon-secretion study in synaptotagmin-7 knockout miceidentified synaptotagmin-7 as an individually acting calciumsensor and the principal synaptotagmin isoform regulating gluca-gon secretion, and thus validates the synaptotagmin–Ca2+-sensorparadigm, and more importantly, extends the theory beyondthe established neuronal Ca2+-sensors for synaptic vesicle

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Figure 4 Comparison of adipocyte models(A) Differentiated 3T3-L1 cells. Lipid droplets are shown in red. (B) Differentiated mouse stromal vascular cells. (C) Primaryadipocytes from mouse epididymal fat pad. Arrows denote adipocyte nuclei. Scale bars for (A), (B) and (C) are 40 μm,20 μm and 40 μm respectively. Images of adipocytes were kindly provided by Dr Parasuraman Padmanabhan, Dr WenjieQiu and Mr Chun-Yan Lim, Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, A*STAR.

exocytosis, i.e. synaptotagmin-1, -2 and -9, to includesynaptotagmin-7 as an individually acting high-affinityCa2+-sensor for LDCV exocytosis [163].

Clearly, synaptotagmin-7 does not always function as an in-dividually acting calcium sensor, as in the case of adrenal chro-maffin and pancreatic β-cells, where multiple synaptotagminsfunction together to regulate neuropeptide and hormone secretion[81,132]. Besides a calcium-sensing role in endocrine secre-tion, synaptotagmin-7 was also proposed to regulate Ca2+-dependent lysosome fusion during wound repair [164–166], gluc-ose transporter 4 trafficking [149], and osteoclast and osteoblastsecretion [167], although alternative interpretations were offeredregarding some of the proposed functions [168,169]. Nonethe-less, these studies suggest that synaptotagmin-7 may functionas a non-selective regulator for various membrane-fusion eventsoutside neuroendocrine and endocrine systems.

AdipocytesNeuroendocrine and endocrine systems perform their regulatoryfunctions in maintaining physiological homoeostasis by releas-ing signalling molecules, such as adrenaline, growth hormoneand insulin, to act on target sites, often far away from the releasesites. These include the well characterized adrenal chromaffincells and insulin-secreting β-cells. Neuroendocrine and endo-crine systems are distinguished from exocrine and paracrine sys-tems by at least three major characteristics: (i) the signallingmolecules are released directly into the blood or interstitialfluids; (ii) the signalling molecules act on remote targets; and(iii) the signalling molecules are released via regulated exocyt-osis of LDCVs.

After the discovery of leptin in 1994, adipocytes, from whichleptin is secreted, have also been classified as part of the endocrinefamily [170–174]. Besides leptin, several other hormones andcytokines are also secreted from adipocytes, including adipon-

ectin, tumour necrosis factor α and interleukin-6 [173,175,176].These hormones and cytokines that are released from adipo-cytes are collectively referred to as adipokines [177]. Similarlyto neuroendocrine and endocrine systems, adipokines secretedfrom adipocytes are also transported via the blood and act on re-mote targets, for example, leptin regulates hypothalamic neuronsafter passing through the blood–brain barrier [178–180]. How-ever, it is not clear whether adipokines are secreted through theconstitutive pathway or regulated exocytosis, another commonfeature of neuroendocrine and endocrine systems. Moreover, ithas not yet been determined how adipokines are stored insideadipocytes prior to their release, and whether different adipokinesshare common storage systems or secretory pathways. Finally,there has not yet been a systematic analysis of the molecular reg-ulation of adipokine secretion in primary adipocytes or a mousemodel. Previous studies relied on differentiated 3T3-L1 and otherderived cells as model systems for adipocytes; however, thesecell models have dramatically different morphology and molecu-lar composition from primary adipocytes (Figure 4). Therefore,it is necessary to examine the following in primary adipocytes:(i) how adipokines are stored (are they stored in LDCVs or sim-ilar subcellular structures, or simply produced when needed?);(ii) whether various adipokines are secreted through distinctpathways (leptin and adiponectin secretion follows very differ-ent patterns, particularly in obese animals), and whether thereis regulation beyond the levels of transcription and translation;(iii) whether adipokine secretion follows regulated exocytosisand/or the constitutive pathway, which cellular signals triggerthe release and whether calcium is involved; (iv) the nature ofthe precise molecular machinery that is used in the transport andfusion of adipokine-containing structures.

At present, none of these questions have been addressed. Inter-estingly, similar questions on cellular and molecular mechanismsand the regulation of cytokine secretion in macrophages were alsoproposed, and recent progress in cytokine secretion was reviewed

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by Stow et al. [181]. Although adipocytes and macrophages arequite different, the secretion mechanisms of adipokines fromadipocytes may be more similar to cytokine secretion in mac-rophages than to insulin/glucagon secretion from pancreaticβ- and α-cells. Thus, the cytokine studies in macro-phages may offer clues on understanding adipokine-secretionmechanisms.

Regarding a potential role of calcium in leptin secretion, onestudy found that blocking calcium entry inhibits acute leptin se-cretion in cultured adipocytes [182], whereas another study usingrat adipocytes showed that calcium elevations inhibited insulin-induced leptin secretion [183]. This discrepancy may reflect dif-ferences between the cultured and freshly isolated primary adipo-cytes. Nonetheless, calcium appears to have an active role inleptin secretion. Interestingly, several synaptotagmins, includingnon-calcium-binding synaptotagmin-13, are expressed in adipo-cytes, and the expression of some synaptotagmins is develop-mentally regulated during adipocyte differentiation, for example,the long form of synaptotagmin-7 is significantly up-regulated at3–7 days after adipocyte differentiation (Y. Wang and W. Han, un-published work). Further analysis is needed to establish whethercalcium is a triggering signal for adipokine secretion, and whethersynaptotagmins play a role in adipokine exocytosis.

Enteroendocrine cellsEnteroendocrine, or gut endocrine, cells refer mainly to two celllineages, K cells, found in mucosa of the duodenum and the je-junum of the gastrointestinal tract, and L cells, located at the distalgut, predominantly the ileum and colon. K cells secrete GIP (gast-ric inhibitory polypeptide, also known as glucose-dependent in-sulinotropic peptide) and L cells secrete GLP-1 (glucagon-likepeptide 1). GIP and GLP-1 belong to a family of molecules calledincretins, which potentiate glucose-stimulated insulin secretion,suppress glucagon secretion, inhibit gastric emptying, and reduceappetite and food intake [184]. Owing to the potential significanthealth benefits and implications in diabetes/obesity treatment,the incretins have received attention from pharmaceutical indus-tries and academic researchers, with pharmaceutical companiesfocusing on developing long-lasting analogues of incretins andinhibitors of the enzyme that degrades incretins, and academicresearchers studying the cellular and molecular mechanisms ofthe effects of incretins on the brain, liver, muscle, adipose tissueand pancreas.

The fact that K and L cells are scattered in the gastrointestinaltract without distinct morphology from neighbouring cells makesit very difficult to study cell biology and molecular mechanisms ofsecretion regulation in primary cells. Thus, most of the previousstudies to understand the cellular regulation of incretin secretionwere performed on cell lines, such as GLUTag cells, a stableimmortalized murine enteroendocrine cell line that expresses theglucagon gene and secretes GLPs in a regulated manner [185].Recently, Reimann et al. [186] used a transgenic approach to spe-cifically label L cells by expressing a fluorescent protein underthe control of the proglucagon promoter, and found that GLP-1secretion is regulated by the activity of sodium–glucose cotrans-

porter 1 and ATP-sensitive K+-channels. When considering thisfinding and a previous report from the same group that GLP-1secretion requires membrane depolarization and calcium entrythrough L-type calcium channels [187], it is tempting to spec-ulate that GLP-1 secretion may operate in a similar fashion toinsulin secretion from pancreatic β-cells: both insulin and GLP-1secretion are stimulated by glucose, and require a glucose trans-porter and glucose metabolism, ATP-sensitive K+-channels, andmembrane depolarizations and Ca2+ entry through L-type Ca2+-channels.

There is no report on the calcium-sensing mechanisms gov-erning GIP or GLP-1 secretion. We have identified the expressionof several synaptotagmins in GLP-1-secreting cells. In particular,we found that synaptotagmin-7, the high-affinity calcium sensorfor insulin secretion, is expressed in mouse L cells (N. Gustav-sson and W. Han, unpublished work). Further studies are neces-sary to understand whether synaptotagmins, particularly synapto-tagmin-7, regulate GLP-1 secretion.

CONCLUDING REMARKS AND FUTUREPERSPECTIVES

Since the initial cloning and biochemical characterizations[11,71,188] that led to the proposed synaptotagmin–calcium-sensor paradigm, intensive research studies, particular those ongenetically modified knockout and knockin animals, have es-tablished the roles of synaptotagmin-1, -2 and -9 as principalcalcium sensors for neurotransmitter release in their respect-ive synapses [46,59,68–70]. Here, we have reviewed mouse ge-netic studies that provide clear evidence on the functions ofseveral synaptotagmins in SV and LDCV exocytosis, particu-larly for synaptotagmin-7 and its function in insulin and gluca-gon secretion [46,59,81,163]. These studies in pancreatic α- andβ-cells of synaptotagmin-7 knockout mice, along with a num-ber of in vitro and cell-line reports (reviewed in [113]), valid-ate and further extend the synaptotagmin–calcium-sensor theorybeyond the three established fast calcium sensors in neurotrans-mitter release, namely synaptotagmin-1, -2 and -9, to includesynaptotagmin-7 as an individually acting calcium sensor in hor-mone secretion [46,59,68–70,81,113,163].

It is an exciting time to witness the rapid progress of themolecular dissection of regulated exocytosis, in particularthe molecular understanding of calcium sensing in neurotrans-mitter, neuropeptide and hormone secretion. There remain manyimportant unanswered questions, and future studies are neededto address: (i) the significance of co-existing calcium-sensingmechanisms (individually acting versus collaborating/competingcalcium sensors); (ii) the identities of all of the calcium-sensingproteins involved in regulated exocytosis, for example, slowsensors in neurotransmitter release and minor sensors for gluca-gon secretion; (iii) whether non-calcium-binding synaptotagminsparticipate in calcium sensing in collaboration with a calcium-binding synaptotagmin, perhaps for a regulated exocytosis that

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requires low calcium concentration and/or cooperativity; (iv) theinvolvement of synaptotagmins in less-studied or atypical endo-crine systems, such as GLP-1-secreting L cells, and leptin andadiponectin secretion from adipocytes.

ACKNOWLEDGEMENTS

We thank Sir George Radda for advising on and supporting ourstudies, Dr Tom Sudhof for advice and encouragement for manyof our studies and the present review, Dr Jianyuan Sun andDr Zhiping Pang for enjoyable discussions and constructive sugges-tions, and members of the Laboratory of Metabolic Medicine, Singa-pore Bioimaging Consortium, A*STAR, Singapore for discussions.

FUNDING

This work was supported by an intramural program of SingaporeA*STAR Biomedical Research Council.

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Received 6 March 2009/16 April 2009; accepted 17 April 2009Published on the Internet 8 June 2009, doi 10.1042/BSR20090031

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