Robson L, Hunter M
Department of Biomedical Science, University of Sheffield, Alfred Denny Building, Sheffield, UK S10 2TN.
J Membr Biol. 2005 Oct;207(3):161-7. doi: 10.1007/s00232-005-0811-2.
K(+) channels in the renal proximal tubule play an important role in salt reabsorption. Cells of the frog proximal tubule demonstrate an inwardly rectifying, ATP-sensitive K(+) conductance that is inhibited by Ba(2+), G(Ba). In this paper we have investigated the importance of phosphorylation state on the activity of G(Ba) in whole-cell patches. In the absence of ATP, G(Ba) decreased over time; this fall in G(Ba) involved phosphorylation, as rundown was inhibited by alkaline phosphatase and was accelerated by the phosphatase inhibitor F(-)(10 mM: ). Activation of PKC using the phorbol ester PMA accelerated rundown via a mechanism that was dependent on phosphorylation. In contrast, the inactive phorbol ester PDC slowed rundown. Inclusion of the PKC inhibitor PKC-ps in the pipette inhibited rundown. These data indicate that PKC-mediated phosphorylation promotes channel rundown. Rundown was prevented by the inclusion of PIP-2 in the pipette. PIP-2 also abrogated the PMA-mediated increase in rundown, suggesting that regulation of G(Ba) by PIP-2 occurred downstream of PKC-mediated phosphorylation. G-protein activation inhibited G(Ba), with initial currents markedly reduced in the presence of GTPgammas. These properties are consistent with G(Ba) being a member of the ATP-sensitive K(+) channel family.
肾近端小管中的钾离子通道在盐重吸收中起重要作用。青蛙近端小管细胞表现出一种内向整流、对ATP敏感的钾离子电导，该电导受钡离子（Ba(2+)）抑制，即G(Ba)。在本文中，我们研究了磷酸化状态对全细胞膜片中G(Ba)活性的重要性。在没有ATP的情况下，G(Ba)随时间下降；G(Ba)的这种下降涉及磷酸化，因为碱性磷酸酶可抑制其衰减，而磷酸酶抑制剂氟化物（10 mM：）可加速其衰减。使用佛波酯PMA激活蛋白激酶C（PKC）通过一种依赖于磷酸化的机制加速了衰减。相反，无活性的佛波酯PDC减缓了衰减。将PKC抑制剂PKC-ps加入微管中可抑制衰减。这些数据表明PKC介导的磷酸化促进通道衰减。通过在微管中加入磷脂酰肌醇-4,5-二磷酸（PIP-2）可防止衰减。PIP-2还消除了PMA介导的衰减增加，这表明PIP-2对G(Ba)的调节发生在PKC介导的磷酸化下游。G蛋白激活抑制G(Ba)，在存在GTPγS的情况下初始电流明显降低。这些特性与G(Ba)属于ATP敏感钾离子通道家族一致。
