Active K plus secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon
Active K plus secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon
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DOI：
10.1152/ajpgi.00337.2002
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发表时间：
2003-07-01
影响因子：
4.5
通讯作者：
Rajendran, VM
中科院分区：
文献类型：
--
作者：
Joiner, WJ;Basavappa, S;Rajendran, VM
胆碱能药物刺激结肠K+分泌需要激活毒蕈碱受体和释放细胞内Ca2+。然而，Ca2+的上升导致K+通过顶膜外排的确切机制尚不清楚。在本研究中，对大鼠近端结肠的Northern blot分析显示，存在编码rSK2[小电导（SK）]、rSK4[中电导（IK）]和rSlo[大电导（BK）] Ca2+激活的K+通道的转录本。在饮食中缺乏K+的动物中，只有rSK4 mRNA在结肠中减少。在此基础上，从大鼠结肠cDNA文库中克隆了一个编码K+通道rSK4的cDNA。将该cDNA转染到中国仓鼠卵巢（CHO）细胞中，导致Ca2+激活的K+通道的表达，该通道被IK通道抑制剂氯霉唑（CLT）阻断。共聚焦免疫荧光证实了近端结肠隐窝中IK通道的存在，Western blotting显示IK蛋白分选到结肠上皮的顶端和底外侧表面。此外，利用单向Rb-86（+）通量研究了大鼠近端结肠上皮条的跨细胞活性K+分泌。在浆膜表面添加thapsignarin或carbachol可增强净Rb-86（+）分泌。粘膜添加CLT完全抑制了碳甾醇诱导的净Rb-86（+）分泌。相比之下，BK和SK通道抑制剂、iberiotoxin和apamin分别只有部分抑制作用。最后，在缺乏K+的动物中观察到SK4信息的减少，碳水化合物诱导的Rb-86（+）分泌在缺乏K+的动物中被消除。这些结果表明，在大鼠近端结肠中，rSK4通道介导毒菌碱激动剂诱导的K+分泌，并且rSK4通道的转录下调以防止饮食中K+消耗时K+的损失。
Colonic K+ secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca2+. However, the precise mechanisms by which this rise in Ca2+ leads to K+ efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [ small conductance (SK)], rSK4 [ intermediate conductance (IK)], and rSlo [ large conductance (BK)] Ca2+-activated K+ channels. In dietary K+-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K+ channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca2+-activated K+ channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K+ secretion was studied on epithelial strips of rat proximal colon using unidirectional Rb-86(+) fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net Rb-86(+) secretion. The mucosal addition of CLT completely inhibited carbachol-induced net Rb-86(+) secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K+, carbachol-induced Rb-86(+) secretion was abolished in dietary K+-depleted animals. These results suggest that the rSK4 channel mediates K+ secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent K+ loss during dietary K+ depletion.