Secretory Vesicle Membrane Research Articles

Abstract P2045: Regulation Of Atrial Natriuretic Peptide Secretion By Palmitoylation And Modulation Of The Rab3a GTPase Cycle In Cardiac Myocytes

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release in cardiomyocytes. We generated transgenic mice overexpressing the Golgi-localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9 by S-acyl proteomics. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9-mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1 R728A ) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling.

Comparing different fluorescent probes to quantify lipid order alterations during Ca2+ mediated synaptotagmin C2 domain binding to membranes.

Changes in membrane order caused by lipids of different acyl-chain saturation or Ca2+ mediated binding of Synaptotagmin 1's C2 domains to the membrane change the equilibrium conformation of the t-SNARE Syntaxin 1a's linker region between SNARE motif and trans membrane domain. A more rigid linker in Syntaxin caused by the disordering of the surrounding lipid bilayer led to more fusion between secretory vesicle membranes and target membranes. This observation suggests a mechanism for Ca2+ stimulated exocytosis in which the Ca2+ sensor Synaptotagmin acts on SNARE proteins through the lipid bilayer. The exact nature of the physical changes in the lipid bilayer that cause the conformational changes in the SNARE protein are not known. Several fluorescent probes have been introduced and applied that are sensitive to one or more environmental properties of the lipid bilayer. We compare the effects of Ca2+ mediated C2 domain binding to model membranes and different acyl-chains on membrane fluidity, order, polarity, and lateral pressure by measuring fluorescence anisotropy, fluorescence lifetime, and general polarity using four different commonly used fluorescent probes, namely NBD, DPH, Flipper TR, and Laurdan. The direct comparisons of these probes in the same systems explore their sensitivities to parameters that are most relevant to changes in SNARE conformation leading to SNARE mediated fusion that have been reported previously.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, Cynops pyrrhogaster.

In adult newts, when a limb is amputated, a mesenchymal cell mass called the blastema is formed on the stump, where blood vessels filled with premature erythrocytes, named polychromatic normoblasts (PcNobs), elongate. We previously demonstrated that PcNobs in the blastema express an orphan gene, Newtic1, and that they secrete growth factors such as BMP2 and TGFβ1 into the surrounding tissues. However, the relationship between Newtic1 expression and growth factor secretion was not clear since Newtic1 was thought to encode a membrane protein. In this study, we addressed this issue using morphological techniques and found that the Newtic1 protein is a component of globular structures that accumulate at the marginal band in the cytoplasm along the equator of PcNobs. Newtic1-positive (Newtic1(+)) globular structures along the equator were found only in PcNobs with a well-developed marginal band in the blastema. Newtic1(+) globular structures were associated with microtubules and potentially incorporated TGFβ1. Based on these observations, we propose a hypothesis that the Newtic1 protein localizes to the membrane of secretory vesicles that primarily carry TGFβ1 and binds to microtubules, thereby tethering secretory vesicles to microtubules and transporting them to the cell periphery as the marginal band develops.

Probing single molecule mechanical interactions of syntaxin 1A with native synaptobrevin 2 residing on a secretory vesicle

Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex formation, which includes an assembly of the intermediate cis binary complex between Sx1A and SNAP25 on the plasma membrane to engage Sb2 on the vesicle. However, whether a trans binary Sx1A-Sb2 complex alone could mediate vesicle docking in a cellular environment remains unclear. To address this issue, we used atomic force microscopy (AFM) in the force spectroscopy mode combined with fluorescence imaging. Using AFM tips functionalized with the full Sx1A cytosolic domain, we probed native Sb2 studding the membrane of secretory vesicles docked at the plasma membrane patches, referred to as “inside-out lawns”, identified based on fluorescence stains and prepared from primary culture of lactotrophs. We recorded single molecule Sx1A-Sb2 mechanical interactions and obtained measurements of force (∼183 pN) and extension (∼21.6 nm) necessary to take apart Sx1A-Sb2 binding interactions formed at tip-vesicle contact. Measured interactive force between a single pair of Sx1A-Sb2 molecules is sufficient to hold a single secretory vesicle docked at the plasma membrane within distances up to that of the measured extension. This finding further advances a notion that native vesicle docking can be mediated by a single trans binary Sx1A-Sb2 complex in the absence of SNAP25.

A comprehensive review of the influence of Epigallocatechin gallate on Sjögren's syndrome associated molecular regulators of exocytosis (Review)

Sjögren's syndrome (SS) is an autoimmune disorder that affects the salivary glands, leading to reduced secretory functions and oral and ocular dryness. The salivary glands are composed of acinar cells that are responsible for the secretion and production of secretory granules, which contain salivary components, such as amylase, mucins and immunoglobulins. This secretion process involves secretory vesicle trafficking, docking, priming and membrane fusion. A failure during any of the steps in exocytosis in the salivary glands results in the altered secretion of saliva. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors, actin, tight junctions and aquaporin 5 all serve an important role in the trafficking regulation of secretory vesicles in the secretion of saliva via exocytosis. Alterations in the expression and distribution of these selected proteins leads to salivary gland dysfunction, including SS. Several studies have demonstrated that green tea polyphenols, most notably Epigallocatechin gallate (EGCG), possess both anti-inflammatory and anti-apoptotic properties in normal human cells. Molecular, cellular and animal studies have indicated that EGCG can provide protective effects against autoimmune and inflammatory reactions in salivary glands in diseases such as SS. The aim of the present article is to provide a comprehensive and up-to-date review on the possible therapeutic interactions between EGCG and the selected molecular mechanisms associated with SS.

VNUT Is a Therapeutic Target for Type 2 Diabetes and NASH

ATP, used in cells as an energy currency, also acts as an extracellular signaling molecule. Studies of purinergic receptor subtypes have revealed that purinergic chemical transmission plays important roles in various cell types. The vesicular nucleotide transporter (VNUT), the ninth transporter in the SLC17 organic anion transporter family, is essential for vesicular ATP storage and its subsequent release. The VNUT is localized on the membrane of secretory vesicles and actively transports ATP into vesicles using an electrochemical gradient of protons supplied by vacuolar proton ATPase (V-ATPase) as a driving force. ATP acts as a damage-associated molecular pattern (DAMPs), contributing to the persistence of chronic inflammation. Chronic inflammation induces systemic insulin resistance, which is the underlying pathology of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). We previously demonstrated that ATP transported in insulin granules via the VNUT negatively regulates insulin secretion. We also found that hepatocytes release ATP in a VNUT-dependent manner, which elicits hepatic insulin resistance and inflammation. VNUT-knockout mice exhibited improved glucose tolerance and were resistant to the development of high fat diet-induced NAFLD. In this article, we summarize recent advances in our understanding of the mechanism of the VNUT, the development of inhibitors, and its pathological involvement in type 2 diabetes and NAFLD. The pharmacological inhibition of the VNUT may represent a potential therapeutic approach for the treatment of metabolic diseases.

Impaired insulin exocytosis in chronic hepatitis C infection: contributory role of p38δ MAPK-protein kinase D-golgi complex axis.

Hepatitis C virus (HCV) infection and chronic hepatitis C (CHC) are associated with a measurable risk of insulin resistance (IR)/impaired glucose tolerance (IGT)/diabetes mellitus (DM). While loss of hepatic endocrine function contributes to liver cirrhosis in diabetic patients, onset and progression of IR/IGT to diabetes and exacerbation of incident hyperglycemia are ostensibly linked with chronic HCV infection. In this regard, the study by Chen J et al. appearing in Clinical Science (2020) (134(5) https://doi.org/10.1042/CS20190900) attempts to understand the mechanisms underlying the savaging effects of chronic HCV infection on insulin-producing pancreatic β-cells and hence diabetic onset. The study investigated the role of mitogen-activated protein kinase (MAPK) p38δ-protein kinase D (PKD)-golgi complex axis in impacting insulin exocytosis. It was inferred that an insulin secretory defect of pancreatic β-cells, owing to disrupted insulin exocytosis, to an extent explains β-cell dysfunction in HCV-infected or CHC milieu. HCV infection negatively regulates first-phase and second-phase insulin secretion by impinging on PKD-dependent insulin secretory granule fission at trans-golgi network and insulin secretory vesicle membrane fusion events. This commentary highlights the study in question, that deciphered the contribution of p38δ MAPK-PKD-golgi complex axis to β-cell dysfunction in CHC milieu. This pivotal axis proffers a formidable therapeutic opportunity for alleviation of double burden of glucose abnormalities/DM and CHC.

Effects of NBI-98782, a selective vesicular monoamine transporter 2 (VMAT2) inhibitor, on neurotransmitter efflux and phencyclidine-induced locomotor activity: Relevance to tardive dyskinesia and antipsychotic action

Valbenazine, a vesicular monoamine transporter 2 (VMAT2, SLC18A2) inhibitor, is a newly approved treatment for tardive dyskinesia. VMAT2 is present in the membrane of secretory vesicles and transports dopamine (DA), norepinephrine (NE), serotonin (5-HT), histamine, glutamate (Glu), and GABA into vesicles for presynaptic release. We utilized microdialysis in awake, freely moving mice to determine the effect of NBI-98782, the active metabolite of valbenazine, alone, or in combination with several antipsychotic drugs (APDs), to influence neurotransmitter efflux in the medial prefrontal cortex (mPFC), dorsal striatum (dSTR), hippocampus and nucleus accumbens (NAC); we also compared it with tetrabenazine, the prototypical VMAT2 inhibitor. Acute NBI-98782 and tetrabenazine decreased mPFC, dSTR, hippocampus, and NAC DA, 5-HT, and NE efflux, while increasing that of DOPAC, HVA, and 5-HIAA. Sub-chronic NBI-98782 (7 days) decreased baseline DA and 5-HT efflux in both mPFC and dSTR. NBI-98782 elicited similar effects on neurotransmitter efflux in sub-chronic NBI-98782-treated mice but also enhanced ACh and GABA; the decrease in DA efflux in mPFC and dSTR was not significant in the sc-treated animals. NBI-98782 suppressed clozapine-, olanzapine- and risperidone-induced DA efflux in both mPFC and dSTR, and ACh efflux in mPFC. NBI-98782 suppressed the haloperidol-induced DA efflux in dSTR, with minimal effect on GABA efflux. NBI-98782 attenuated PCP-induced DA, 5-HT, NE and Glu efflux, and AMPH-induced DA and NE efflux, in both mPFC and dSTR, as well as PCP- and AMPH-induced hyperlocomotion, suggesting possible beneficial antipsychotic effects.

VH+-ATPase-induced intracellular acidification is critical to glucose-stimulated insulin secretion in beta cells.

Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells.

Aquaporin regulation: Lessons from secretory vesicles.

Secretory vesicle swelling has been demonstrated to be a requirement in cell secretion. In the past 25 years, the quest to elucidate the molecular mechanism of secretory vesicle swelling, serendipitously revealed the presence of water channels or aquaporins at the secretory vesicle membrane and their involvement in rapid gaiting of water into secretory vesicles and their swelling during secretion. These studies further provided an understanding of aquaporin regulation at the secretory vesicle membrane. Secretory vesicles within live cells, isolated secretory vesicles, single vesicle electrophysiological patch clamp studies and the swelling complex reconstituted into liposomes, have all been utilized to elucidate the mechanism and regulation of secretory vesicle swelling. Results from these studies collectively demonstrate the involvement of β-adrenergic receptor, heterotrimeric GTP-binding G-proteins such as GαI; PLA2 and potassium and chloride channels, in the regulation of aquaporins at the secretory vesicle membrane.

Membrane Binding of Synaptotagmin-Like Protein 4: Insight from Molecular Dynamics Simulations

Synaptotagmin-like proteins (Slps) are a family of Rab GTPase effectors involved in secretory vesicle trafficking and membrane docking. Slps possess an N-terminal domain that binds Rab27 on large dense-core secretory vesicles and one or two C-terminal C2 domains that dock to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). In particular, Slp-4, also known as granuphilin, docks secretory vesicles to the plasma membrane primarily via its C2A domain, which has a high affinity for anionic lipid bilayers. Although the structure of the Slp-4 C2A domain is known and some of its key PIP2-binding residues have been identified, the full structural mechanism of its strong membrane binding remains unclear. Here, we employ molecular dynamics simulations of the Slp-4 C2A domain to determine contributions of particular residues in binding to a lipid bilayer containing physiologically relevant levels of the anionic target lipids PS and PIP2. Simulations include mutations of two important residues: Lys398, which is known to participate in PIP2 coordination, and Phe452, which is suspected to insert into target membranes hydrophobically. Preliminary results support the experimental finding that both the K398A and F452A mutants are capable of binding membranes but suggest a different balance of lipid interactions in the two mutants. Overall, experimental and computational results indicate that high-affinity membrane binding of the Slp-4 C2A domain is sustained via many lipid-interacting residues distributed over a large electropositive protein surface.

Sphingolipids modulate docking, Ca2+ sensitivity and membrane fusion of native cortical vesicles.

Docking, priming, and membrane fusion of secretory vesicles (i.e. regulated exocytosis) requires lipids and proteins. Sphingolipids, in particular, sphingosine and sphingosine-1-phosphate, have been implicated in the modulation of exocytosis. However, the specific exocytotic steps that sphingolipids modulate and the enzymes that regulate sphingolipid concentrations on native secretory vesicle membranes remain unknown. Here we use tightly coupled functional and molecular analyses of fusion-ready cell surface complexes and cortical vesicles isolated from oocytes to assess the role of sphingolipids in the late, Ca2+-triggered steps of exocytosis. The molecular changes resulting from treatments with sphingolipid modifying compounds coupled with immunoblotting analysis revealed the presence of sphingosine kinase on native vesicles; the presence of a sphingosine-1-phosphate phosphatase is also indicated. Changes in sphingolipid concentrations on vesicles altered their docking/priming, Ca2+-sensitivity, and ability to fuse, indicating that sphingolipid concentrations are tightly regulated and maintained at optimal levels and ratios to ensure efficient exocytosis.

The Rab3A-22A Chimera Prevents Sperm Exocytosis by Stabilizing Open Fusion Pores

At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.

Cytochemical localization of H2O2 in pigment glands of cotton (Gossypium hirsutum L.)

Programmed cell death (PCD) plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species (ROS) produced by the aerobic metabolism of cells, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands of Gossypium hirsutum L. with 3,3-diaminobenzidine (DAB) staining, 2',7'-dichlorodihydrofluorescein diacetate (DCFH2)-DA fluorescent labeling and CeCl3 cytochemical localization techniques. The results showed that the pigment glands of G. hirsutum could generate H2O2, and the amount and localization of H2O2 varied at different developmental stages. At the early developmental stage, a small amount of H2O2 accumulated in the vacuole membrane of pigment gland cells. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cell walls started to accumulate a small amount of H2O2. When pigment gland cell degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cells. With the degradation of the sheath cells, H2O2 was detected in cell wall and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2 are consistent with the process of PCD of individual gland cells, which started from the degradation of intracellular membrane and ended with the degradation of cell walls. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.

Oligophrenin-1: the link between calcium-regulated exocytosis and compensatory endocytosis in neuroendocrine cells

In neuroendocrine cells, hormones and neuropeptides are released from large-dense core vesicles (secretory granules) by calcium-regulated exocytosis. Following exocytosis, compensatory uptake of membrane is required to maintain membrane homeostasis and allow recycling of secretory vesicle membranes. How these cells initiate and regulate this compensatory endocytosis remains poorly understood. Our recent data suggests that oligophrenin-1 (OPHN1) is a link coupling calcium-regulated exocytosis to compensatory endocytosis of secretory granules in the adrenal chromaffin cells (Houy et al., 2015, J Neurosci. 2015, 35:11045-55). Here, we highlight the major evidence and discuss how OPHN1 could couple these two processes.

Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

The role of myosin 1c and myosin 1b in surfactant exocytosis.

ABSTRACTActin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression.

Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo.

The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo.

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.