Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis.

Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

A conformational switch in the Piccolo C2A domain regulated by alternative splicing.

C2 domains are widespread Ca2+-binding modules. The active zone protein Piccolo (also known as Aczonin) contains an unusual C2A domain that exhibits a low affinity for Ca2+, a Ca2+-induced conformational change and Ca2+-dependent dimerization. We show here that removal of a nine-residue sequence by alternative splicing increases the Ca2+ affinity, abolishes the conformational change and abrogates dimerization of the Piccolo C2A domain. The NMR structure of the Ca2+-free long variant provides a structural basis for these different properties of the two splice forms, showing that the nine-residue sequence forms a beta-strand otherwise occupied by a nonspliced sequence. Consequently, Ca2+-binding to the long Piccolo C2A domain requires a marked rearrangement of secondary structure that cannot occur for the short variant. These results reveal a novel mechanism of action of C2 domains and uncover a structural principle that may underlie the alteration of protein function by short alternatively spliced sequences.

The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells.

Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Calpha C2 (PKCalphaC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCalphaC2 domain backbones. In response to an intracellular calcium increase, PKCalphaC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCalphaC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCalphaC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1.

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.

Molecular determinants of regulated exocytosis.

Regulated exocytosis forms the basis for many intercellular signaling processes, for example, in hormone secretion or neurotransmitter release. During regulated exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane in a tightly controlled reaction that is most often triggered by calcium. Recent advances have allowed major insights into the molecular mechanisms that mediate regulated exocytosis. In the present review, we will briefly discuss two key features of regulated exocytosis that have been particularly well studied recently. First, we will examine the current understanding of the membrane fusion reaction that underlies regulated exocytosis and that is effected by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and munc18-like proteins similar to other membrane fusion reactions. Second, we will describe the role of the major candidates for the calcium sensors that trigger exocytosis, a protein family called synaptotagmins. Although our understanding of regulated exocytosis is as yet incomplete, the results from the studies of SNAREs, munc18s, and synaptotagmins have provided a molecular anchor for a more complete future description.

Role of electrostatic and hydrophobic interactions in Ca(2+)-dependent phospholipid binding by the C(2)A-domain from synaptotagmin I.

Most C(2)-domains bind to phospholipid bilayers as a function of Ca(2+). Although phospholipid binding is central for the normal functions of C(2)-domain proteins, the precise mechanism of phospholipid binding is unclear. One of the key questions is whether phospholipid binding by C(2)-domains is primarily governed by electrostatic or hydrophobic interactions. We have now examined this question for the C(2)A-domain of synaptotagmin I, a membrane protein of secretory vesicles with an essential function in Ca(2+)-triggered exocytosis. Our results confirm previous data showing that Ca(2+)-dependent phospholipid binding by the synaptotagmin C(2)A-domain is exquisitely sensitive to ionic strength, suggesting an essential role for electrostatic interactions. However, we find that hydrophobic interactions mediated by exposed residues in the Ca(2+)-binding loops of the C(2)A-domain, in particular methionine 173, are also essential for tight phospholipid binding. Furthermore, we demonstrate that the apparent Ca(2+) affinity of the C(2)A-domain is determined not only by electrostatic interactions as shown previously, but also by hydrophobic interactions. Together these data indicate that phospholipid binding by the C(2)A-domain, although triggered by an electrostatic Ca(2+)-dependent switch, is stabilized by a hydrophobic mechanism. As a result, Ca(2+)-dependent phospholipid binding proceeds by a multimodal mechanism that mirrors the amphipathic nature of the phospholipid bilayer. The complex phospholipid binding mode of synaptotagmins may be important for its role in regulated exocytosis of secretory granules and synaptic vesicles.

Conformational switch of syntaxin-1 controls synaptic vesicle fusion.

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

Injection of nerve growth factor into stellate ganglia improves norepinephrine reuptake into failing hearts.

An impairment of cardiac norepinephrine reuptake through the neuronal norepinephrine transporter promotes depletion of cardiac norepinephrine stores and local cardiac sympathetic activation in heart failure. Nerve growth factor regulates differentiation and survival of adult sympathetic cells and is decreased in failing hearts. We hypothesized that injection of nerve growth factor into stellate ganglia normalizes cardiac norepinephrine homeostasis in experimental heart failure. Rats with transverse aortic constriction characterized by heart failure, depleted cardiac norepinephrine stores, and impaired cardiac norepinephrine reuptake were used as an experimental model. Nerve growth factor (20 microg) or saline was directly injected into left stellate ganglia 4 weeks after transverse aortic constriction. Thirty-two hours after injection, determinants of cardiac norepinephrine homeostasis were measured. As compared with saline, nerve growth factor refilled depleted cardiac norepinephrine stores and improved cardiac [3H]-norepinephrine uptake into isolated perfused hearts of transverse aortic constricted rats. In addition, pharmacological blockade of the norepinephrine transporter led to a higher increase in the overflow of endogenous norepinephrine from hearts of nerve growth factor-injected than saline-injected transverse aortic constricted rats. Norepinephrine transporter mRNA levels and the density of cardiac sympathetic nerves were not changed. Thirty-two hours after nerve growth factor injection, echocardiography revealed an increase in fractional shortening as compared with 2 days before injection. In conclusion, nerve growth factor attenuates local cardiac sympathetic overdrive of hypertrophic hearts by improving cardiac norepinephrine reuptake and might represent a novel therapeutic principle in the treatment of heart failure.

Analysis of the catalytic domain of phosphatidylinositol 4-kinase type II.

Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.