We investigated the inhibitory role of the nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by ∼70%) by a NO donor SNAP (100 μm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 μm) or after G-protein activation with intracellular perfusion of GTPγS (100 μm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 μm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of 32 P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg 8 -vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO-cGMP-PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

The vesicle docking protein p115 was found to be phosphorylated in a cell cycle-specific manner; it was found phosphorylated in interphase but not in mitotic cells. During interphase, however, two forms of p115 were detected in the cells; the phosphorylated form was found exclusively in cytosol, whereas the unphosphorylated form was associated with membranes, mostly of the Golgi complex. The latter form was released from the membranes upon phosphorylation. Mutational analysis revealed that the phosphorylation site of p115 was the Ser 942 residue in the C-terminal acidic domain. A mutant with a single substitution of Ser 942 3 Ala markedly increased its association with the Golgi membrane. Another mutant with Ser 942 3 Asp was able to associate with the membrane, although at a decreased level, indicating that the dissociation of p115 from the membrane is not simply due to the negative charge of phosphorylated Ser 942 . Taken together, these results suggest that the phosphorylation of Ser 942 at the C-terminal acidic domain regulates the interaction of p115 with the Golgi membrane, possibly taking part in the regulatory mechanism of vesicular transport.Vesicular transport of proteins is carried out by the formation of coated vesicles from a donor compartment, followed by their uncoating and subsequent docking and fusion of the vesicles with a target compartment membrane, in which a number of soluble and membrane proteins are involved (1). The docking of vesicles to target membranes is accomplished through the specific interaction between membrane proteins named v-and t-SNAREs (vesicle and target SNAP receptors) (1, 2). This is followed by binding of SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins) and N-ethylmaleimide-sensitive factor, which lead to membrane fusion. Vesicle docking is also controlled by interactions of SNAREs with other proteins including Rab proteins (3).p115, a peripheral membrane protein localized to the Golgi apparatus and also present in the cytoplasm, was first identified as a component required for intra-Golgi transport (4) and found to be identical to the transcytosis-associated protein TAP (5). Structural analysis also indicates that it is a homolog to Uso1p, a yeast protein required for transport from the endoplasmic reticulum to the Golgi (6). p115/TAP exits as a parallel homodimer with two globular heads followed by a rod-like domain containing a C-terminal acidic tail (5, 7). p115/TAP and Uso1p have been shown to function in a docking step prior to membrane fusion (8). Recently, Warren and his colleagues (9) demonstrated that p115 binds to Golgi membranes with high affinity during interphase but not during mitosis. In addition, GM130, a peripheral protein tightly associated with the cisGolgi membrane (10), was identified as the binding site for p115 on the Golgi membrane (11). GM130 was modified by phosphorylation only during mitosis, resulting in no binding of GM130 to p115.The mechanism of membrane binding inhibition by p115 during mitosis would provide a molecu...

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a precursor (pro-C3), which was processed to the mature form with a-and /?-subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro-C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro-C3 to the Golgi complex where prcK3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.Brefeldin A; Secretion-blocking agent; Complement C3; Complement precursor accumulation; (Rat hepatocyte)

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex proteinlipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic ␣-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.Cellular organelles like the Golgi apparatus and the endoplasmic reticulum adopt characteristic structures depending on their function (1, 2). For example, extensive membrane nanotubules, typically 50 -70 nm in diameter and up to several m in length, have been observed to form the Golgi complex, the trans Golgi network, and the connections between the Golgi stacks. The morphological engineering of these membranes involve complex interactions between proteins and lipids that are not yet understood (3, 4). However, there must be some fundamental machinery required to form such structures.We recently described the properties of a de novo-designed 18-mer peptide, Hel 13-5 (5, 6). This peptide can adopt an ideal ␣-helix having a 240°hydrophobic sector region (Fig. 1A). It forms a self-association state in buffer solution by adopting this amphipathic structure (70% ␣-helical structure by CD), and it binds to model-and biomembranes with high affinity. In the present study, we show that Hel 13-5 induces nanotubular structures, not only for PC liposomes, but also for various naturally occurring phospholipids. Most importantly, we demonstrate that Hel 13-5 transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of, and resembling the shape of, the nanotubules of the Golgi apparatus. MATERIALS AND METHODSReagents-Peptide was synthesized by the Fmoc 1 strategy based on the solid phase technique starting from Fmoc-PAL-PEG resin using a PerSeptive 9050 automatic peptide synthesizer described previously (5). The stock solutions of Hel peptides were prepared as follows: the powders were damped with a small amount of 30% acidic acid and then diluted in buffer (5 mM Tes/100 mM NaCl, pH 7.4). The peptide concentrations in the buffer solution were determined from the UV absorbance of Trp at 280 nm (⑀ ϭ 5500).Turbidity Measurement-A lipid solution in chloroform was filmed in a round bottom flask by drying in a stream of N 2 gas. The lipid film was hydrated with the Tes buffer by vortexing. The turbid liposome solution obtained was then dilu...

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments in vitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 microM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 microM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction in vitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.