Calcium-sensing Receptor Decreases Cell Surface Expression of the Inwardly Rectifying K+ Channel Kir4.1
Calcium-sensing Receptor Decreases Cell Surface Expression of the Inwardly Rectifying K+ Channel Kir4.1
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DOI：
10.1074/jbc.m110.160390
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发表时间：
2011-01-21
影响因子：
4.8
通讯作者：
Miller, R. Tyler
中科院分区：
文献类型：
--
作者：
Cha, Seung-Kuy;Huang, Chunfa;Miller, R. Tyler
Ca 2+敏感受体（CaR）调节肾脏中的盐和水转运，如通过功能CaR突变获得与Bartter综合征样盐耗表型的关联所证明的，但这种作用的精确机制尚未完全确定。我们以前发现，CaR与内向整流K+通道Kir4.1相互作用并使其失活，Kir4.1在远端肾单位中表达，有助于基底外侧K+传导，并且其中功能缺失突变与包括肾盐耗的复杂表型相关。我们现在发现CaR通过减少Kir4.1的细胞表面表达来使其失活。突变型汽车降低了HEK-293细胞中Kir4.1细胞表面表达和电流密度，与其信号传导活性成比例。突变的、激活的G alpha（q）降低了Kir4.1的细胞表面表达和电流密度，这些作用被RGS 4（一种阻断通过G alpha（i）和G alpha（q）的信号传导的蛋白质）阻断。其他α亚基的影响不显著。敲低小窝蛋白-1阻断了G α（q）对Kir4.1的作用，而敲低网格蛋白重链则没有作用。CaR对肾外髓K+通道没有类似的影响，这是一种顶端膜远端肾单位K+通道，由网格蛋白包被的囊泡内化。免疫共沉淀研究表明，在HEK细胞和肾脏提取物中，CaR和Kir4.1与小窝蛋白-1物理相关。因此，CaR通过涉及G α（q）和小窝蛋白的机制降低Kir4.1通道的细胞表面表达。这些结果为CaR抑制肾脏NaCl转运提供了新的分子基础。
The Ca2+-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K+ channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K+ conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated G alpha(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via G alpha(i) and G alpha(q). Other alpha subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of G alpha(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K+ channel, an apical membrane distal nephron K+ channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves G alpha(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.