L-type calcium currents conducted by Ca V 1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of Ca V 1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for ␤-adrenergic regulation in the fight-or-flight response. Mice expressing Ca V 1.2 channels with the distal C terminus deleted (DCT ) current in cardiomyocytes, where Ca 2ϩ enters through the channel and initiates excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release (1). Normal expression of Ca V 1.2 channels is required for cardiac contractile function and for survival beyond embryonic day 14 (2). Lack of the Ca V 1.2 channel also abolishes the development of myogenic tone and disrupts hormonal regulation of blood pressure (3). In contrast, deletion of Ca V 1.3, which also conducts L-type Ca 2ϩ currents, causes sinoatrial nodal dysfunction and cardiac arrhythmias but does not impair contractility or cause premature death (4). Overall, these gene deletion studies illustrate that L-type Ca 2ϩ currents are essential for normal cardiovascular function and for normal development.Ca V 1 channels are multisubunit complexes composed of a pore-forming ␣1 subunit and auxiliary ␤, ␣2␦, and in some cases ␥ subunits (5-7). They are a primary target for regulation by numerous hormones, protein kinases, and phosphoprotein phosphatases (5-7). In the "fight-or-flight" response, increased force of contraction is achieved largely through regulation of Ca V 1.2 channels in the heart by the sympathetic nervous system through activation of ␤-adrenergic receptors, adenylyl cyclase, and cyclic AMP-dependent protein kinase (PKA) and resulting phosphorylation of Ca V 1.2 channels (1,5,6,8). ␤-Adrenergic regulation of Ca V 1.2 channels requires A-kinase anchoring protein 15 (AKAP15), 2 which anchors the kinase to the distal C terminus of Ca V 1.2 via a modified leucine zipper (LZ) motif (9 -11).The C terminus of Ca V 1 channels undergoes proteolytic processing in vivo in skeletal and cardiac muscle (12-15). In cardiac muscle, the ␣1 subunit of Ca V 1.2 channels is present in two size forms of ϳ240 and 210 kDa, which differ by truncation of the distal C terminus (DCT) (15). This truncation leads to enhanced activity of Ca V 1.2 channels expressed in Xenopus oocytes and mammalian cell lines (16,17). Single channel conductance, modulation by ␤ and ␣2␦ subunits, and sensitivity to Ca 2ϩ channel agonists such as Bay K8644 remain unchanged (16, 17). The proteolytically cleaved DCT binds to the truncated channel and acts as potent autoinhibitor (18). Mutations of key charged residues at the interface between distal and proximal C-terminal domains of Ca V 1.2 relieves autoinhibition (18). Moreover, recent studies indicate that regulation of Ca V 1.2 channels can be reconstituted in transfect...

Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8+ T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient lytic granule secretion at the IS.

VAMP8 is associated with the recycling endosome compartment rather than with cytotoxic granules and is required for a fusion step between recycling endosomes and the plasma membrane that brings syntaxin-11 to the immune synapse for cytotoxic granule exocytosis.

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTLmediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.

Regulation of Ca V 1.2 channels in cardiac myocytes by the ␤-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ␤-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for Ca V 1.2 and Ca V 1.3 channels in neurons. AKAP15 co-immunoprecipitates with Ca V 1.2 and Ca V 1.3 channels. AKAP15 has overlapping localization with Ca V 1.2 and Ca V 1.3 channels in cell bodies and proximal dendrites and is closely co-localized with Ca V 1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with Ca V 1.2 and Ca V 1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of Ca V 1.2 channels (Ca V 1.2⌬DCT) reveal that AKAP15 interacts with neuronal Ca V 1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of Ca V 1.2 channels via that interaction. Ca V 1.2⌬DCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from Ca V 1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of Ca V 1.2 and Ca V 1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal Ca V 1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.Voltage-gated calcium channels of the Ca V 1 subfamily conduct L-type calcium currents that transduce cell-surface depolarization into calcium transients and initiate excitation-contraction coupling, excitation-secretion coupling, protein phosphorylation, and gene regulation (1-5). Calcium influx via postsynaptic Ca V 1 channels supports sustained phosphorylation of cAMP response element-binding protein (CREB) 3 and CREB-dependent gene expression in hippocampal neurons (6 -13).Functional Ca V 1 channels are multimeric complexes composed of pore-forming ␣ 1 and associated ␣ 2 ␦, ␤, and in some cases, ␥ subunits (14 -19). These channels have an extended C terminus containing many protein interaction sites for regulation (5). In brain, Ca V 1 channels are composed of 70% Ca V 1.2 and 22% Ca V 1.3 with minor contributions from other Ca V 1 channels, as indicated by immunoprecipitation with specific antibodies (20). Ca V 1.2 and Ca V 1.3 channels are primarily localized in the soma and proximal dendrites (20, 21).The ␤-adrenergic pathway activates cAMP-dependent protein kinase (PKA) and increases the activity of Ca V 1 channels in skeletal and cardiac myocytes and neurons (1-5, 22, 23). PKAmediated regulatio...

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS).Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level.Keywords: Cytotoxic T lymphocytes r FHL r Lytic granule fusion r Syntaxin11 clusters r TIRFM Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCTLs kill their target cells through the polarized fusion of lytic granules (LGs) at the immunological synapse (IS). The IS is initiated when the TCRs recognize specific MHC peptide complexes on the target cell. The recognition results in the clustering of molecules that leads to the formation of supramolecular activation clusters (SMACs) [1]. TCRs and associated signaling proteins accumulate to form a central SMAC while adhesion proteins Correspondence: Prof. Jens Rettig e-mail: jrettig@uks.eu needed for stable target cell contact form the peripheral SMAC. After IS formation, LGs polarize, dock, prime, and finally fuse to release their contents into the cleft between CTL and target cell [2].Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are known executors of almost all intracellular fusion events [3]. The SNARE proteins that execute synaptic vesicle fusion in neurons and neuroendocrine cells have been well characterized [4,5]. Vesicle fusion is initiated by the formation of a four helical bundle that consists of one R-SNARE (v-SNARE) that resides on one membrane (mostly vesicle membranes) and three Q-SNAREs (Qa, Qb, Qc; t-SNARE) that reside on another membrane (mostly the plasma membrane) of the cell [6]. paired Student t-test). (B) Cell lysates from naïve CTLs (N), CTLs stimulated for 3 days by anti-CD3/anti-CD28 antibody-coated beads (S) andCTLs overexpressing TFP-Stx11 (OE) were assessed for Stx11 expression by Western blot using a polyclonal anti-Stx11 antibody. (C) Lysates from CTLs transfected with either control or three different Stx11 siRNAs (#1, #2, and #6, respectively) were blotted for Stx11 (top) and GAPDH (bottom) as loading control. (D) Qua...

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8 1 T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8 1 T cells, laying the groundwork for further understanding their potential role in T-cell function.Key words: Exocytosis . Immunological synapse . SNARE proteins Supporting Information available online IntroductionCytotoxic T lymphocytes (CTLs) kill pathogen-infected and/or tumorigenic cells. To do so, they first establish contact with the target cells. The interface between CTLs and target cells is termed the immunological synapse (IS). Formation of the IS is initiated when CD3/T-cell receptor (TCR) complexes on the surface of the CTL recognize antigens that are presented by the MHC class I molecules on the surface of the target cell. This initiates a cascade of signaling events including the clustering of TCR complexes at the IS. Finally, lytic granules (LGs) reorient towards the IS and the cytotoxic components contained therein are being released into the cleft at the contact zone. This constitutes the granulemediated pathway of target cell killing, which is one of the most important pathways used by CTLs for killing target cells [1][2][3]. Dysfunction in any step of LG release, including reorientation to the IS, priming, docking or fusion at the IS, may lead to lifethreatening immune disorders, such as familial hemophagocytic lymphohistiocytosis (FHL) [4]. Several molecules, which are essential for LG trafficking or fusion, have been identified: syntaxin11 [5][6][7], Munc13-4 [8], Munc18-2 [9, 10] and Rab27a [11,12]. Interestingly, most of those proteins either belong to the SNARE (soluble NSF attachment receptors) family or execute their function via interaction with SNARE proteins.SNAREs belong to a family of membrane proteins that have been implicated as the conserved core protein machinery required for all intracellular membrane fusion events that mediate intracellular trafficking [13,14]. There are at present 38 known members of the mammalian SNARE family [15]. SNAREs were initially classified functionally as v-SNAREs and t-SNAREs SHORT COMMUNICATIONÃ ...

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusioncompetent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.