Role of IK channels in rat distal colon

描述(申请人提供)：哺乳动物结肠上皮细胞的顶膜(AP)和基侧膜(BL)中均存在中等电导的钙激活K+(IK)通道。虽然我们以前已经克隆了IK基因(我们将其命名为IK1)，但我们做了以下的初步观察：1)分离了一个剪接变异体，其中29个氨基酸的第二跨膜结构域被删除，我们将其命名为IK2；2)我们针对C末端和缺失的29个氨基酸基序的抗体免疫定位了IK1到AP膜和IK2到BL膜；3)卡巴胆碱(CCH)，一种增加细胞内游离钙离子的M受体激动剂，刺激K+分泌和Cl-分泌，而这两种分泌都被IK通道抑制剂克霉唑(CLT)抑制；(4)K+缺乏可下调AP膜IK1蛋白和mRNA的表达，但不能下调BL膜IK2蛋白和mRNA的表达。我们认为，Ik2在细胞超极化激活Cl-分泌中起关键作用，而Ik1与K+分泌的激活密切相关，也可能参与了Cl-的分泌。我们建议：1)为了确定这些IK异构体在不同的膜中表达时是否表现出相同和/或不同的特征，我们建议将体外表达的IK1和IK2异构体通道与AP和BL膜上的天然IK通道进行比较。2)为了确定29个氨基酸基序(在IK2中缺失)的具体作用，我们提出了a)整个基序或特定氨基酸残基(S)是否负责IK1蛋白的AP膜递送，b)新氨基酸基序的缺失或其他辅助蛋白是否负责IK2蛋白的BL膜递送。3)为了确定这两种IK通道蛋白在K+吸收、K+分泌和Cl-分泌中的作用，我们将在正常大鼠和三种选择性“敲除”IK功能的模型中进行包括ISC测定和86Rb通量在内的生理学研究：饮食中的K+消耗代表功能性的IK1基因敲除；以及IK基因敲除小鼠，在该模型中，我们通过先前发表的腺病毒转染法将IK1或IK2基因“敲入”，该方法将分别导致IK2或IK1基因敲除小鼠。因此，CCH诱导的K+通道的明显不同作用将被研究，并与功能研究相关联。

DESCRIPTION (provided by applicant): Intermediate conductance Ca2+-activated K+ (IK) channels are present in both apical (AP) and basolateral (BL) membranes of mammalian colonic epithelial cells. Although IK cDNA (that we have designated as IK1) has previously been cloned, we have made the following preliminary observations: 1) Isolation of a splice variant in which the entire 2nd transmembrane domain of 29 amino acids is deleted that we have designated as IK2; 2) Immunolocalization of IK1 to AP membrane and IK2 to BL membrane with antibodies that we have raised against the C-terminal end and the missing 29 aa motif; 3) Carbachol (CCH), a muscarinic-receptor agonist that increases intracellular free Ca2+, stimulates both K+ secretion and Cl- secretion that are inhibited by ctotrimazol (CLT), an IK channel inhibitor; and 4) Dietary K+ depletion down-regulates AP membrane IK1 protein, mRNA but not BL membrane IK2 protein and mRNA. We propose that IK2 is critical for activation of Cl- secretion by cell hyperpolarization while IK1 is closely associated with activation of K+ secretion and may also contribute to Cl- secretion. We propose: 1) To establish whether these IK isoforms when expressed in distinct membranes manifest the same and/or different characteristics we propose to characterize in vitro expressed IK1 and IK2 isoform channels compared to those of native IK channels of AP and BL membranes. 2) To identify the specific role of the 29 amino acid motif (deleted in IK2), we propose to establish whether a) entire motif or specific amino acid residue(s) is responsible for AP membrane delivery of IK1 proteins and b) whether deletion of the novel amino acid motif or other accessory proteins are responsible for BL membrane delivery of IK2 proteins. 3) To determine the role of these two IK channel proteins in K+ absorption, K+ secretion and Cl- secretion, physiological studies that include Isc determination and 86Rb fluxes will be performed in normal rats and in three models of selective 'knock-down' of IK function: dietary K+ depletion which represents a functional IK1 knockout; and IK knock-out mice in which we have 'knocked-in' either IK1 or IK2 by a previously published adenoviral transfection method that will result in IK2 or IK1 knockout mice, respectively. Therefore, the apparent distinct roles of CCH-induced K+ channels will be investigated and correlated with functional studies.