Alternatively spliced isoform of apical Na(+)-K(+)-Cl(-) cotransporter gene encodes a furosemide-sensitive Na(+)-Cl(-)cotransporter.
In the absence of vasopressin, medullary thick ascending limb cells express a K(+)-independent, furosemide-sensitive Na(+)-Cl(-) cotransporter that is inhibited by hypertonicity. The murine renal specific Na(+)-K(+)-2 Cl(-) cotransporter gene (SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na(+)-K(+)-2 Cl(-) cotransporter (BSC1-9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (approximately 200 mosmol/kgH(2)O), mBSC1-4-injected oocytes do not express significant Na(+) uptake over H(2)O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1-4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1-4 oocytes are exposed to hypotonicity (approximately 100 mosmol/kgH(2)O), a significant increase in Na(+) uptake but not in (86)Rb(+) uptake is observed. The increased Na(+) uptake is Cl(-) dependent, furosemide sensitive, and cAMP sensitive but K(+) independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH(2)O (r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na(+)-Cl(-) cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na(+)-Cl(-)-to-Na(+)-K(+)-2Cl(-) switching in thick ascending limb cells.
Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City CP 14000, Mexico.
C
C
Plata
P
P
Meade
A
A
Hall
R C
RC
Welch
N
N
Vázquez
S C
SC
Hebert
G
G
Gamba
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005665:Furosemide / Q000494:pharmacology*; D015870:Gene Expression / Q000502:physiology; D007038:Hypotonic Solutions / Q000494:pharmacology; D007536:Isomerism; D007546:Isoquinolines / Q000494:pharmacology; D008138:Loop of Henle / Q000201:enzymology; D008322:Mammals; D009865:Oocytes / Q000502:physiology; D009994:Osmolar Concentration; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012979:Sodium Radioisotopes / Q000493:pharmacokinetics; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; D014316:Tritium; D014982:Xenopus laevis
D002352:Carrier Proteins; D004232:Diuretics; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D007546:Isoquinolines; D010726:Phosphodiesterase Inhibitors; D012979:Sodium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; D002034:Bumetanide; D014316:Tritium; D005665:Furosemide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine