cAMP-dependent activation of the renal-specific Na+-K+-2Cl- cotransporter is mediated by regulation of cotransporter trafficking.
The murine apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter gene (mBSC1) exhibits two spliced isoform products that differ at the COOH-terminal domain. A long COOH-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 (EGFP) fusion construct (L-mBSC1-EGFP) expressed to characterize the surface expression of the L-BSC1 isoform in Xenopus laevis oocytes. Functional expression was also assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive (86)Rb(+) 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 coexpression 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-coinjected oocytes. All changes in L-mBSC1 surface expression correlated with modification of bumetanide-sensitive (86)Rb(+) uptake. Our data suggest that the dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is due to the effects of the cotransporter on trafficking.
Meade
Patricia
P
Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México.
Hoover
Robert S
RS
Plata
Consuelo
C
Vázquez
Norma
N
Bobadilla
Norma A
NA
Gamba
Gerardo
G
Hebert
Steven C
SC
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D018076:DNA, Complementary / Q000096:biosynthesis; D004789:Enzyme Activation; D005089:Exocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007668:Kidney / Q000201:enzymology* / Q000502:physiology*; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D009865:Oocytes; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000378:metabolism*; D014981:Xenopus
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010726:Phosphodiesterase Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D049452:Green Fluorescent Proteins; D003994:Bucladesine; D000242:Cyclic AMP; D003078:Colchicine; D015056:1-Methyl-3-isobutylxanthine