Munc-18-2 regulates exocytosis of H⁺-ATPase in rat inner medullary collecting duct cells

Abstract

-Exocytic insertion of Hϩ -ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H ϩ -ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H ϩ -ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 Ϯ 7% and increased the interaction between GFP-syntaxin 1A and H ϩ -ATPase by 170 Ϯ 23%. Apical membrane Munc-18-2 decreased by 27.5 Ϯ 4.6% and H ϩ -ATPase increased by 246 Ϯ 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H ϩ -ATPase. In a pull-down assay of H ϩ -ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H ϩ -ATPase pulled down by 64 Ϯ 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H ϩ -ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H ϩ -ATPase, synaptosomeassociated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H ϩ -ATPase vesicles.soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion THE SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE factor (NSF) attachment protein target receptor (SNARE) hypothesis of vesicle transport postulates that proteins in vesicular and target membranes form a core complex that leads to the docking and fusion of these membranes. Synaptobrevin or vesicle-associated membrane proteins (VAMPs) are primarily located on vesicles, and syntaxins are located on the target membranes. VAMP and syntaxin along with synaptosome-associated protein 23 or 25 (SNAP-23, the isoform expressed in somatic tissue, or SNAP-25, the isoform expressed in neuroexcitable tissue) form a parallel four-helix bundle. VAMP and syntaxin each contribute one helix and SNAP-23 or -25 contributes two helices (16) to form the four-helix fusion complex. The formation of this complex is thought to provide the energy required to overcome the energetic barrier of lipid bilayer fusion (16). Syntaxin has been found to have three different conformational arrangements: one in isolation, another when bound to Munc, and a third as part of the core SNARE complex (16). It has been proposed that when syntaxin is bound to Munc, it is in a "closed conformation," which does not allow...

AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b

“…This role of Munc18a, in contrast to the one we propose here, could either be synapsespecific or might result from the simplification of the cellular milieu -which could exalt a particular function of Munc18. By contrast, a negative regulatory role for non-neuronal Munc18 isoforms has been proposed for GLUT4 exocytosis in adipocytes (Macaulay et al, 2002;Tamori et al, 1998;Thurmond et al, 1998), insulin exocytosis in β-cells (Zhang et al, 2000), exocytosis of human platelets (Houng et al, 2003), the apical delivery of influenza virus hemagglutinin (HA) in CaCo-2 cells (Riento et al, 2000) and H + -ATPase exocytosis in rat IMCD (Nicoletta et al, 2004). More recently, it has clearly been shown that adipocytes derived from Munc18c -/-mice display an increased sensitivity to insulinstimulated fusion of GLUT4 with the plasma membrane (Kanda et al, 2005).…”

“…Although it has been reported that, in yeast, fusion cannot proceed to the formation of the SNARE complex in the absence of Sec proteins (Bryant and James, 2001;Sato et al, 2000), it has been suggested that, in mammals, Munc18c acts as a negative regulator of GLUT4-containing vesicles in 3T3-L1 adipocytes (Kanda et al, 2005;Tamori et al, 1998;Thurmond et al, 1998). A possible role of the Munc18b isoform has been proposed in the regulation of apical membrane transport in CaCo-2 epithelial cells (Riento et al, 1998;Riento et al, 2000), exocytosis of H + -ATPase in rat inner medullary collecting-duct cells (Nicoletta et al, 2004) and in gastricacid secretion (Liu et al, 2007).…”

Angiotensin II Stimulates Vacuolar H+-ATPase Activity in Renal Acid-Secretory Intercalated Cells from the Outer Medullary Collecting Duct

“…Immunohistochemistry demonstrated the expression of PKC-␣, PKC-␤1, PKC-, and PKC-␦ in IC of the collecting duct. 25,27 Evidence from freshly isolated OMCD IC as well as from cell culture models 19,31,54 suggests that PKC has a stimulatory effect on H ϩ -ATPase activity. In addition, our experiments indicate that intact ERK1/2 mitogen-activated protein kinases and PI3-K are required for the stimulation to occur.…”

A Cytosolic Splice Variant of Cab45 Interacts with Munc18b and Impacts on Amylase Secretion by Pancreatic Acini

“…Munc18c was reported to localize on the basal membrane where it interacts with syn4, and release of this SM protein from the basal surface by supramaximal cholecystokinin stimulation was suggested to redirect apical exocytosis to the basal membrane (Gaisano et al, 2001). Munc18b has been shown to control apical exocytosis in several epithelial cell types (Riento et al, 2000;Kauppi et al, 2002), H ϩ -ATPase insertion into the apical membrane of kidney inner medullary collecting duct cells (Nicoletta et al, 2004), and mast cell granule secretion (Martin-Verdeaux et al, 2003). Furthermore, Munc18b was recently shown to impact on amylase release from rat parotid acinar cells by controlling the interaction of the synaptotagmin-like protein Slp4-a/granuphilin with syn2 and -3 (Fukuda et al, 2005).…”