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      CLCA : chloride channel or modulator?

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      LoewenMEThesis.pdf (2.043Mb)
      Date
      2004-04-12
      Author
      Loewen, Matthew Eric
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
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      Abstract
      A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Veterinary Biomedical Sciences
      Program
      Veterinary Biomedical Sciences
      Supervisor
      Forsyth, George W.
      Committee
      Janz, David M.; Hamilton, Donald L.; Cotton, Calvin U.; Appleyard, Greg D.; Muir, Gillian D.
      Copyright Date
      April 2004
      URI
      http://hdl.handle.net/10388/etd-04142004-142723
      Subject
      secretory diarrhea
      bestrophin
      anion transport
      endothelial adhesion molecule
      mCLCA3
      hCLCA1
      CLCA
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      • Graduate Theses and Dissertations
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