Serhan Karvar

Medical University of South Carolina, Charleston, South Carolina, United States

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Publications (19)162.44 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Gastric acid secretion is mediated by the K(+) dependent proton pump (H(+),K(+)-ATPase), which requires a continuous supply of K(+) at the luminal side of the apical membrane. Several K(+) channels are implicated in gastric acid secretion. However, the identity of the K(+) channel(s) responsible for apical K(+) supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane upon stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm, but exhibited apical localization after stimulation. Further, knocking down KCNJ15 expression with a shRNA adenoviral construct abolished histamine stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H(+),K(+)-ATPase, was detected in all of the parietal cells by immunofluorescence staining, while only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K(+) supply during stimulated acid secretion. Copyright © 2015, American Journal of Physiology - Cell Physiology.
    AJP Cell Physiology 06/2015; DOI:10.1152/ajpcell.00012.2015 · 3.67 Impact Factor
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    Jo Suda, Don C Rockey, Serhan Karvar
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    ABSTRACT: The most prominent ezrin-radixin-moesin (ERM) protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy and a CMFDA assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 hours, both endogenous and overexpressed wild type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia eliminated the typical secretory response after hypoxia, but the dominant active construct (radixin-T567D) rescued this phenotype. Hypoxia also led to Mrp-2 mislocalization, and caused Mrp-2 to be dissociated from radixin; the radixin phospho mimicking mutant (T564D) abrogated this effect of hypoxia. Taken collectively, these findings strongly suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury. Copyright © 2014, American Journal of Physiology- Gastrointestinal and Liver Physiology.
    AJP Gastrointestinal and Liver Physiology 12/2014; 308(4):ajpgi.00369.2014. DOI:10.1152/ajpgi.00369.2014 · 3.74 Impact Factor
  • Serhan Karvar, Jo Suda, Lixin Zhu, Don C Rockey
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    ABSTRACT: NHERF1 is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains, and with the Ezrin-Radixin-Moesin (ERM) family of proteins in its carboxy-terminus. The role of NHERF-1 in hepatocyte function remains largely unknown. Here we examine the distribution and physiological significance of NHERF1 and Mrp-2 in hepatocytes. A wild-type, radixin binding site mutant (F355R), and NHERF1 PDZ1- and PDZ2-domain adenoviral mutant constructs were tagged with yellow fluorescent protein (YFP) and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A CMFDA assay was used to characterize Mrp-2 function. Similar to Mrp-2, wild type NHERF-1 and the NHERF-1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. The NHERF1 PDZ1 and F355R mutants were devoid of a secretory response using CMFDA, while wild type NHERF1 infected cells exhibited increased GSFM secretion. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1. Further, the findings suggest that NHERF1 is essential for maintaining the localization and function of Mrp-2.
    AJP Cell Physiology 08/2014; 307(8). DOI:10.1152/ajpcell.00011.2014 · 3.67 Impact Factor
  • Gastroenterology 05/2014; 146(5):S-498. DOI:10.1016/S0016-5085(14)61797-2 · 13.93 Impact Factor
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    ABSTRACT: This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued), a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.
    PLoS ONE 02/2012; 7(2):e31789. DOI:10.1371/journal.pone.0031789 · 3.53 Impact Factor
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    ABSTRACT: Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.
    AJP Cell Physiology 04/2011; 301(2):C507-21. DOI:10.1152/ajpcell.00355.2010 · 3.67 Impact Factor
  • Jo Suda, Lixin Zhu, Serhan Karvar
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    ABSTRACT: Radixin, the dominant ezrin-radixin-moesin (ERM) protein in hepatocytes, has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin after a conformational activation by phosphorylation at Thr564. The present studies were undertaken to investigate the cellular changes in expression of radixin in WIF-B cells and to assess radixin distribution and its influence on cell polarity. We used a recombinant adenoviral expression system encoding radixin wild-type and Thr564 mutants fused to cyan fluorescent protein (CFP), as well as conventional immunostaining procedures. Functional analyses were characterized quantitatively. Similar to endogenous radixin, adenovirus-infected radixin-CFP-wild type and nonphosphorylatable radixin-CFP-T564A were found to be expressed heavily in the compartment of canalicular membrane vacuoles, typically colocalizing with multidrug resistance-associated protein 2 (Mrp-2). Expression of radixin-CFP-T564D, which mimics constant phosphorylation, was quite different, being rarely associated with canalicular membranes. The WIF-B cells were devoid of a secretory response, T567D radixin became predominantly redistributed to the basolateral membrane, usually in the form of dense, long spikes and fingerlike projections, and the altered cell polarity involved changes in apical membrane markers. Differences in polar distribution of radixin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections and also suggest that radixin might be an organizer and regulator of Mrp-2 and cell polarity in hepatocytes.
    AJP Cell Physiology 03/2011; 300(3):C416-24. DOI:10.1152/ajpcell.00467.2010 · 3.67 Impact Factor
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    Jo Suda, Lixin Zhu, Serhan Karvar
    AJP Cell Physiology 12/2010; 300(3):416-24. · 3.67 Impact Factor
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    ABSTRACT: BACKGROUND & AIMS: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation. METHODS: We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. RESULTS: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. CONCLUSIONS: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
    Gastroenterology 10/2010; 140(3):868-78. DOI:10.1053/j.gastro.2010.09.044 · 13.93 Impact Factor
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    ABSTRACT: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation. We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
    Gastroenterology 09/2010; 140(3):868-78. · 13.93 Impact Factor
  • Jo Suda, Lixin Zhu, Serhan Karvar
    Gastroenterology 05/2010; 138(5). DOI:10.1016/S0016-5085(10)63603-7 · 13.93 Impact Factor
  • Jo Suda, Lixin Zhu, Serhan Karvar
    Gastroenterology 05/2009; 136(5). DOI:10.1016/S0016-5085(09)61072-6 · 13.93 Impact Factor
  • Serhan Karvar, Jo Suda, Lixin Zhu
    Gastroenterology 04/2008; 134(4). DOI:10.1016/S0016-5085(08)62673-6 · 13.93 Impact Factor
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    ABSTRACT: Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.
    Traffic 09/2005; 6(8):654-66. DOI:10.1111/j.1600-0854.2005.00306.x · 4.71 Impact Factor
  • Gastroenterology 04/2003; 124(4). DOI:10.1016/S0016-5085(03)82236-9 · 13.93 Impact Factor
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    ABSTRACT: A newly designated procedure for high-pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel-coated aluminium plates for conventional subsequential cryoimmobilization by high-pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron-dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine-treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H+/K+-ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H+/K+-ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron-dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high-pressure freezing of primary culture cells.
    Journal of Microscopy 01/2003; 208(Pt 3):158-66. DOI:10.1046/j.1365-2818.2002.01085.x · 2.15 Impact Factor
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    ABSTRACT: The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.
    Journal of Biological Chemistry 01/2003; 277(51):50030-5. DOI:10.1074/jbc.M207694200 · 4.60 Impact Factor
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    ABSTRACT: Acid secretion by parietal cells involves secretagogue-dependent recycling of the H+-K+-ATPase. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated as participants in membrane trafficking, docking, and fusing processes. Here we studied the intracellular distribution and functional importance of one SNARE protein, vesicle associated membrane protein-2 (VAMP-2), in gastric parietal cells. Using an adenoviral recombinant expression system encoding VAMP-2 (synaptobrevin-2) fused to the green fluorescent protein (GFP), we expressed the GFP-VAMP-2 protein in primary cultures of rabbit parietal cells, which enables us to visualize the dynamics of GFP-VAMP-2 in a variety of functional states by fluorescence microscopy. To ascertain the function of VAMP-2 in parietal cell activation, streptolysin-O permeabilized gastric glands were treated with tetanus toxin, a potent and preferential protease for VAMP-2, and acid secretion was measured. In resting parietal cells GFP was detected throughout the cytoplasm in a pattern of distribution that was very similar to that of H+-K+-ATPase. After stimulation, we observed that the GFP-VAMP-2 translocated to the apical plasma membrane along with the H+-K+-ATPase. A relatively high degree of co-localization was detected between GFP-VAMP-2 and H+-K+-ATPase. Tetanus toxin inhibited cAMP/ATP-stimulated acid secretion by about 45% in permeabilized gastric glands with a concomitant reduction in the level of immunoreactive VAMP-2. Adenovirus-based GFP reporter fusion proteins can be used to efficiently study the functional dynamics of SNAREs. VAMP-2 is associated with tubulovesicle membranes in the parietal cell and plays a role in stimulation-associated membrane recruitment and acid secretion.
    Gastroenterology 07/2002; 123(1):281-90. DOI:10.1053/gast.2002.34217 · 13.93 Impact Factor
  • Gastroenterology 04/2001; 120(5). DOI:10.1016/S0016-5085(01)83242-X · 13.93 Impact Factor

Publication Stats

126 Citations
162.44 Total Impact Points

Institutions

  • 2014
    • Medical University of South Carolina
      • Division of Gastroenterology and Hepatology
      Charleston, South Carolina, United States
  • 2011–2012
    • University of Southern California
      • Keck School of Medicine
      Los Angeles, California, United States
  • 2010–2011
    • University of California, Los Angeles
      Los Ángeles, California, United States
  • 2001–2005
    • University of California, Berkeley
      • Department of Molecular and Cell Biology
      Berkeley, California, United States