The voltage-gated sodium channel ß2 subunit associates with lipid rafts by S-palmitoylation.

The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated ß subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that ß2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases ß2 association with detergent-resistant membranes. ß2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of ß2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical ß2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting ß2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical ß2. Although ß2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of ß2.

N-Glycosylation of the voltage-gated sodium channel ß2 subunit is required for efficient trafficking of NaV1.5/ß2 to the plasma membrane.

The voltage-gated sodium channel is critical for cardiomyocyte function and consists of a protein complex comprising a pore-forming α subunit and two associated ß subunits. It has been shown previously that the associated ß2 subunits promote cell surface expression of the α subunit. The major α isoform in the adult human heart is NaV1.5, and germline mutations in the NaV1.5-encoding gene, sodium voltage-gated channel α subunit 5 (SCN5A), often cause inherited arrhythmias. Here, we investigated the mechanisms that regulate ß2 trafficking and how they may determine proper NaV1.5 cell surface localization. Using heterologous expression in polarized Madin-Darby canine kidney cells, we show that ß2 is N-glycosylated in vivo and in vitro at residues 42, 66, and 74, becoming sialylated only at Asn-42. We found that fully nonglycosylated ß2 was mostly retained in the endoplasmic reticulum, indicating that N-linked glycosylation is required for efficient ß2 trafficking to the apical plasma membrane. The nonglycosylated variant reached the cell surface by bypassing the Golgi compartment at a rate of only approximately one-third of that of WT ß2. YFP-tagged, nonglycosylated ß2 displayed mobility kinetics in the plane of the membrane similar to that of WT ß2. However, it was defective in promoting surface localization of NaV1.5. Interestingly, ß2 with a single intact glycosylation site was as effective as the WT in promoting NaV1.5 surface localization. In conclusion, our results indicate that N-linked glycosylation of ß2 is required for surface localization of NaV1.5, a property that is often defective in inherited cardiac arrhythmias.

Trafficking and localisation to the plasma membrane of Nav 1.5 promoted by the ß2 subunit is defective due to a ß2 mutation associated with Brugada syndrome.

BACKGROUND INFORMATION: Cardiac channelopathies arise by mutations in genes encoding ion channel subunits. One example is Brugada Syndrome (BrS), which causes arrhythmias and sudden death. BrS is often associated with mutations in SCN5A, encoding Nav 1.5, the α subunit of the major cardiac voltage-gated sodium channel. This channel forms a protein complex including one or two associated ß subunits as well as other proteins. RESULTS: We analysed regulation of Nav 1.5 localisation and trafficking by ß2, specifically, Nav 1.5 arrival to the cell surface. We used polarised Madin-Darby canine kidney (MDCK) cells and mouse atria-derived HL-1 cells, which retain phenotypic features of adult cardiomyocytes. In both, Nav 1.5 was found essentially intracellular, mainly in the endoplasmic reticulum, whereas ß2 localised to the plasma membrane, and was restricted to the apical surface in MDCK cells. A fraction of ß2 interacted with Nav 1.5, despite their limited overlap. Importantly, ß2 promoted Nav 1.5 localisation to the cell surface. Both ß2 WT and the BrS-associated mutation D211G (substitution of Asp for Gly) effectively reached the plasma membrane. Strikingly, however, ß2 D211G was defective in promoting Nav 1.5 surface localisation. CONCLUSIONS: Our data sustain that ß2 promotes surface localisation of Nav 1.5, which can be affected due to ß2 mutations associated with channelopathies. SIGNIFICANCE: Our findings add to the understanding of ß2 role in Nav 1.5 trafficking and localisation, which must influence cell excitability and electrical coupling in the heart. This study will contribute to knowledge on development of arrhythmias.

Subcellular localisation of retromer in post-endocytic pathways of polarised Madin-Darby canine kidney cells.

BACKGROUND INFORMATION: Retromer is required for endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR), allowing delivery of hydrolases into lysosomes. It is constituted by a conserved heterotrimer formed by vacuolar protein sorting (Vps) gene products Vps26, Vps35 and Vps29, which is in charge of cargo selection, and a dimer of phosphoinositide-binding sorting nexins (SNXs), which has a structural role. Retromer has been implicated in sorting of additional cargo. Thus, retromer also promotes polymeric immunoglobulin A (pIgA) transcytosis by the pIgA receptor (pIgR) in polarised cells, and considerable evidence implicates retromer in controlling epithelial cell polarity. However, the precise localisation of retromer along the endocytic pathway of polarised cells has not been studied in detail. RESULTS: Our biochemical analysis using rat liver endosome fractions suggests a distinct distribution pattern. Although subunits of the cargo-selective complex were enriched in early endosomes (EEs), levels of SNX2 were greater in sorting endosomes. We then immunolocalised the retromer subunits in polarised Madin-Darby canine kidney (MDCK) cells by confocal microscopy. An estimated 25% of total Vps26 and SNX2 localised to EEs, with negligible portions in recycling endosomes as well as in late endosomes and lysosomes. Although Vps26 was in structures of more heterogeneous size and shape than SNX2, these markedly overlapped. In consequence, the two retromer subcomplexes mostly colocalised. When we analysed retromer overlap with its cargo, we found that structures retromer and pIgA(+) are independent of those structures retromer and CI-MPR(+) . Remarkably, retromer localised preferentially at the transcytotic pathway. Pharmacological inhibition of phosphoinositide 3-kinase affected the co-distribution of retromer with pIgA and the CI-MPR, delaying pIgA progress to the apical surface. CONCLUSIONS: In polarised MDCK cells, we found retromer associated with certain specialised EE-derived pathways. Our data imply that retromer is largely engaged in pIgA transcytosis in pIgR-expressing MDCK cells, as opposed to endosome-to-Golgi retrieval.

Retromer regulates postendocytic sorting of ß-secretase in polarized Madin-Darby canine kidney cells.

ß-Amyloid (Aß) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the ß-APP cleaving enzyme (BACE or ß-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aß peptides.

A missense mutation in the sodium channel ß2 subunit reveals SCN2B as a new candidate gene for Brugada syndrome.

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium ß2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (ß2WT) or mutant (ß2D211G) ß2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the ß2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that ß2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant ß2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor.

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis.

Tumor suppressor p27(Kip1) undergoes endolysosomal degradation through its interaction with sorting nexin 6.

A large body of evidence supports the hypothesis that proteasomal degradation of the growth suppressor p27(Kip1) (p27) facilitates mammalian cell cycle progression. However, very few studies have addressed the possibility of proteasome-independent mechanisms of p27 proteolysis. Here we provide evidence for a novel pathway of p27 degradation via the lysosome that is mediated by its interaction with the endosomal protein sorting nexin 6 (SNX6), a member of the sorting nexin family of vesicular trafficking regulators. p27 and SNX6 interact in vitro and in vivo in mammalian cells, partially colocalize in endosomes, and are present in purified endosomal fractions. Gain- and loss-of-function studies revealed that SNX6 induces endosomal accumulation of p27. Moreover, p27 is detected in lysosomes and inhibition of lysosome-dependent proteolysis impairs serum-mediated down-regulation of p27 in a SNX6-dependent manner. To validate the localization of p27 in these organelles, we analyzed several cell lines using two different anti-p27 antibodies, several organelle-specific markers [e.g., early endosome antigen 1, lysosomal-associated membrane protein (LAMP) 1, LAMP2, and LysoTracker], and overexpression of fluorescent p27 and SNX6. Remarkably, silencing of SNX6 attenuates p27 down-regulation in the G(1) phase of the mitotic cell cycle and delays cell cycle progression. We therefore conclude that, in addition to the proteasome-dependent pathway, SNX6-mediated endolysosomal degradation of p27 also contributes to cell cycle progression in mammalian cells.

Trafficking and Function of the Voltage-Gated Sodium Channel ß2 Subunit.

The voltage-gated sodium channel is vital for cardiomyocyte function, and consists of a protein complex containing a pore-forming α subunit and two associated ß subunits. A fundamental, yet unsolved, question is to define the precise function of ß subunits. While their location in vivo remains unclear, large evidence shows that they regulate localization of α and the biophysical properties of the channel. The current data support that one of these subunits, ß2, promotes cell surface expression of α. The main α isoform in an adult heart is NaV1.5, and mutations in SCN5A, the gene encoding NaV1.5, often lead to hereditary arrhythmias and sudden death. The association of ß2 with cardiac arrhythmias has also been described, which could be due to alterations in trafficking, anchoring, and localization of NaV1.5 at the cardiomyocyte surface. Here, we will discuss research dealing with mechanisms that regulate ß2 trafficking, and how ß2 could be pivotal for the correct localization of NaV1.5, which influences cellular excitability and electrical coupling of the heart. Moreover, ß2 may have yet to be discovered roles on cell adhesion and signaling, implying that diverse defects leading to human disease may arise due to ß2 mutations.

Retromer and sorting nexins in development.

Trafficking and signaling processes involve common molecular components. The machinery that controls intracellular trafficking is vital in ensuring that signaling mechanisms take place correctly. An illustrative example of this relationship is the sustained signaling of endocytosed membrane receptors, such as receptor Tyr kinases and G-protein coupled receptors, after ligand-induced activation. An intriguing role in controlling the fate of these and other receptors at the endosome has been attributed to members of the sorting nexin protein family. The best characterized sorting nexins are subunits of a multimeric complex, termed retromer. It was first found in yeast that retromer mediates endosome-to-Golgi retrieval of receptors after they have delivered soluble hydrolase precursors into the vacuole, the organelle equivalent to the mammalian lysosome. Work in cultured mammalian cells later demonstrated that retromer performs an analogous function in higher eukaryotes. Data from genetically modified mice, and from a simpler organism such as the nematode Caenorhabtidis elegans, has revealed that retromer performs an essential role during embryogenesis. This review will discuss implications of recent work on this subject.

Retromer in Polarized Protein Transport.

Retromer is an evolutionary conserved protein complex required for endosome-to-Golgi retrieval of receptors for lysosomal hydrolases. It is constituted by a heterotrimer encoded by the vacuolar protein sorting (VPS) gene products Vps26, Vps35, and Vps29, which selects cargo, and a dimer of phosphoinositide-binding sorting nexins, which deforms the membrane. Recent progress in the mechanism of retromer assembly and functioning has strengthened the link between sorting at the endosome and cytoskeleton dynamics. Retromer is implicated in endosomal sorting of many cargos and plays an essential role in plant and animal development. Although it is best known for endosome sorting to the trans-Golgi network, it also intervenes in recycling to the plasma membrane. In polarized cells, such as epithelial cells and neurons, retromer may also be utilized for transcytosis and long-range transport. Considerable evidence implicates retromer in establishment and maintenance of cell polarity. That includes sorting of the apical polarity module Crumbs; regulation of retromer function by the basolateral polarity module Scribble; and retromer-dependent recycling of various cargoes to a certain surface domain, thus controlling polarized location and cell homeostasis. Importantly, altered retromer function has been linked to neurodegeneration, such as in Alzheimer's or Parkinson's disease. This review will underline how alterations in retromer localization and function may affect polarized protein transport and polarity establishment, thereby causing developmental defects and disease.

Formation of multicellular epithelial structures.

The kidney is primarily comprised of highly polarized epithelial cells. Much has been learned recently about the mechanisms of epithelial polarization. However, in most experimental systems the orientation of this polarity is determined by external cues, such as growth of epithelial cells on a filter support. When Madin-Darby canine kidney (MDCK) cells are grown instead in a three-dimensional (3D) collagen gel, the cells form hollow cysts lined by a monolayer of epithelial cells, with their apical surfaces all facing the central lumen. We have found that expression of a dominant-negative (DN) form of the small GTPase Rac1 causes an inversion of epithelial polarity, such that the apical surface of the cells instead faces the periphery of the cyst. This indicates that the establishment of polarity and the orientation of polarity can be experimentally separated by growing cells in a 3D collagen gel, where there is no filter support to provide an external cue for orientation. DN Rac1 causes a defect in the assembly of laminin into its normal basement membrane network, and addition of a high concentration of exogenous laminin rescues the inversion of polarity caused by DN Rac1.

A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor.

Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.

Retromer: multipurpose sorting and specialization in polarized transport.

Retromer is an evolutionary conserved protein complex required for endosome-to-Golgi retrieval of lysosomal hydrolases' receptors. A dimer of two sorting nexins-typically, SNX1 and/or SNX2-deforms the membrane and thus cooperates with retromer to ensure cargo sorting. Research in various model organisms indicates that retromer participates in sorting of additional molecules whose proper transport has important repercussions in development and disease. The role of retromer as well as SNXs in endosomal protein (re)cycling and protein targeting to specialized plasma membrane domains in polarized cells adds further complexity and has implications in growth control, the establishment of developmental patterns, cell adhesion, and migration. This chapter will discuss the functions of retromer described in various model systems and will focus on relevant aspects in polarized transport.

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor.

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein.

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.