Role of IK channels in rat distal colon

描述（由申请人提供）：中间电导Ca2+激活的K+ (IK)通道存在于哺乳动物结肠上皮细胞的顶端（AP）和基底外侧（BL）膜中。虽然先前已经克隆了IK cDNA（我们将其命名为IK1），但我们做了以下初步观察：1)分离到一个剪接变体，其中29个氨基酸的整个第2跨膜结构域被删除，我们将其命名为IK2；2)利用我们针对c端和缺失的29aa基序提出的抗体，将IK1免疫定位到AP膜和IK2免疫定位到BL膜；3)卡巴酚（CCH），一种增加细胞内游离Ca2+的毒蕈碱受体激动剂，刺激K+和Cl-分泌，而K+和Cl-分泌被克曲唑（CLT）（一种IK通道抑制剂）抑制；4)饲粮K+耗竭下调AP膜IK1蛋白和mRNA，而不下调BL膜IK2蛋白和mRNA。我们认为IK2对细胞超极化激活Cl-分泌至关重要，而IK1与K+分泌的激活密切相关，也可能参与Cl-的分泌。我们建议：1)为了确定这些IK亚型在不同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亚型通道与AP和BL膜的天然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，我们建议确定a)整个基序或特定的氨基酸残基是否负责IK1蛋白的AP膜递送，b)新的氨基酸基序或其他辅助蛋白的缺失是否负责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，将在正常大鼠和IK功能选择性“敲除”的三种模型中进行生理研究，包括Isc测定和86Rb通量：饮食中K+消耗，代表功能性IK1敲除；以及IK1敲除小鼠，我们通过先前发表的腺病毒转染方法“敲入”IK1或IK2，分别产生IK2或IK1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.