Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent.
Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent.
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DOI：
10.1152/ajprenal.90227.2008
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发表时间：
2008-09
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通讯作者：
P. Welker;Alexandra Böhlick;K. Mutig;M. Salanova;T. Kahl;H. Schlüter;D. Blottner;José Ponce‐Coria;G. Gamba;S. Bachmann
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作者：
P. Welker;Alexandra Böhlick;K. Mutig;M. Salanova;T. Kahl;H. Schlüter;D. Blottner;José Ponce‐Coria;G. Gamba;S. Bachmann
顶端布美他尼敏感性Na（+）-K（+）-2Cl（-）协同转运蛋白（NKCC 2）是阳离子-氯离子协同转运蛋白超家族的肾脏特异性成员，是负责NaCl跨上皮重吸收的完整膜蛋白。NKCC 2的作用对于肾脏容量调节至关重要。加压素（AVP）控制亨利氏袢（TAL）粗升支细胞中NKCC 2表面表达。我们发现40-70%的Triton X-100不溶性NKCC 2存在于大鼠肾脏和培养的TAL细胞中富含胆固醇的脂筏（LR）中。大鼠肾脏Na +-Cl-协同转运蛋白（NCC）也分布于LR中。在细胞内和质膜中都检测到含有NKCC 2的LR。甲基-β-环糊精（MbetaCD）诱导的胆固醇耗竭显著降低了非洲爪蟾卵母细胞中（86）Rb（+）内流分析的布美他尼敏感的NKCC 2转运。在TAL中，短期AVP应用诱导顶囊泡运输沿着与NKCC 2从非筏LR组分的转变。同时，通过共聚焦分析评估了NKCC 2与LR神经节苷脂GM 1的共定位增加及其极性易位。顶端生物素化显示NKCC 2表面表达增加两倍。这些作用被甲羟戊酸-洛伐他汀/MbetaCD诱导的胆固醇剥夺减弱。总的来说，这些发现表明，NKCC 2池分布在筏。结果与LR介导极性插入、活性和AVP诱导的NKCC 2转运控制跨上皮NaCl转运的模型一致。
Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)-Cl(-) cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by (86)Rb(+) influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.