Plata C, Meade P, Hall A, Welch R C, Vázquez N, Hebert S C, Gamba G
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.
Am J Physiol Renal Physiol. 2001 Apr;280(4):F574-82. doi: 10.1152/ajprenal.2001.280.4.F574.
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.
在缺乏血管加压素的情况下，髓质厚壁升支细胞表达一种对呋塞米敏感的、与钾离子无关的钠离子 - 氯离子共转运体，该共转运体受高渗抑制。小鼠肾脏特异性的钠离子 - 钾离子 - 2氯离子共转运体基因（SLC12A1）产生六种可变剪接异构体。其中三种具有长的COOH末端结构域，编码对布美他尼敏感的钠离子 - 钾离子 - 2氯离子共转运体（BSC1 - 9/NKCC2），另外三种具有短的COOH末端结构域，称为mBSC1 - A4、B4或F4（19）。在此，我们确定了非洲爪蟾卵母细胞中表达的mBSC1 - A4的功能特性。当在正常卵母细胞渗透压（约200 mosmol/kgH₂O）下孵育时，注射了mBSC1 - 4的卵母细胞相对于注射水的对照未表现出显著的钠离子摄取增加，免疫组织化学分析表明，大多数mBSC1 - 4蛋白位于卵母细胞细胞质中而非质膜上。相反，当mBSC1 - 4卵母细胞暴露于低渗环境（约100 mosmol/kgH₂O）时，观察到钠离子摄取显著增加，但铷离子（⁸⁶Rb⁺）摄取未增加。增加的钠离子摄取依赖于氯离子、对呋塞米敏感且对环磷酸腺苷（cAMP）敏感，但与钾离子无关。在120至70 mosmol/kgH₂O之间，随着渗透压降低，钠离子摄取增加（r = 0.95，P < 0.01）。免疫组织化学分析表明，在低渗条件下，mBSC1 - A4蛋白表达于质膜上。这些研究表明，SLC12A1基因的mBSC1 - A4异构体编码一种低渗激活、对cAMP和呋塞米敏感的钠离子 - 氯离子共转运体。因此，BSC1基因的可变剪接有可能提供了一种分子机制，使髓质厚壁升支细胞能够实现从钠离子 - 氯离子共转运体到钠离子 - 钾离子 - 2氯离子共转运体的转换。
