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1 Department Cellular and Molecular Physiology, Yale University Medical School, Mexico City, Mexico
2 Molecular Physiology Unit, Universidad Nacional Autonoma de Mexico, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: steven.hebert{at}yale.edu.
The murine apical bumetanide-sensitive Na+-K+-2Cl- cotransporter gene (mBSC1), exhibits two spliced isoform products that differ at the C-terminal domain. A long C-terminal isoform (L-mBSC1) encodes the Na+-K+-2Cl- cotransporter and a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1 cotransporter activity that is abrogated by cAMP. However, the mechanism of this dominant negative effect was not clear. In this study, we used confocal microscopic analysis of an enhanced green fluorescent protein fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in the Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1 injected oocytes by measuring the bumetanide-sensitive 86Rb+ uptake. Oocytes injected with L-mBSC1-EGFP cRNA developed a distinct plasma membrane-associated fluorescence that colocalized with the fluorescent membrane dye, FM 4-64. The fluorescence intensity in L-mBSC1-EGFP oocytes did not change after cAMP was added to the extracellular medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in a dose-dependent manner with co-expression of S-mBSC1. The inhibitory effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis inhibitor colchicine blocked the effect of cAMP on the L-mBSC1-EGFP/S-mBSC1 co-injected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive 86Rb+ uptake. Our data suggest that the dominant negative effect of S-mBSC1 on L-mBSC1 transport function is due to effects on trafficking of the cotransporter.
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