Role of IK channels in rat distal colon

描述（由申请人提供）：中等电导Ca 2+激活的K+（IK）通道存在于哺乳动物结肠上皮细胞的顶膜（AP）和基底膜（BL）中。虽然IK cDNA（我们命名为IK 1）之前已经被克隆，我们已经进行了以下初步观察：1）分离剪接变体，其中29个氨基酸的整个第二跨膜结构域被缺失，我们命名为IK 2; 2）用针对C-末端和缺失的29 aa基序的抗体将IK 1免疫定位于AP膜和IK 2免疫定位于BL膜; 3）卡巴胆碱（CCH），一种增加细胞内游离Ca 2+的毒蕈碱受体激动剂，刺激K+分泌和Cl-分泌，而K+分泌和Cl-分泌均被一种IK通道抑制剂ctotrimazol（CLT）抑制; 4）膳食K+耗竭下调AP膜IK 1蛋白、mRNA，但不下调BL膜IK 2蛋白和mRNA。我们建议，IK 2是关键的激活Cl-分泌细胞超极化，而IK 1是密切相关的激活K+分泌，也可能有助于Cl-分泌。我们建议：1）为了确定这些IK同种型在不同的膜中表达时是否表现出相同和/或不同的特征，我们提出与AP和BL膜的天然IK通道相比，表征体外表达的IK 1和IK 2同种型通道。2)为了鉴定29个氨基酸基序（在IK 2中缺失）的特定作用，我们建议确定a）整个基序或特定氨基酸残基是否负责IK 1蛋白的AP膜递送和B）新氨基酸基序或其它辅助蛋白的缺失是否负责IK 2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，将在正常大鼠和三种IK功能选择性“敲低”模型中进行生理学研究，包括Isc测定和86 Rb通量：和IK敲除小鼠，其中我们已经通过先前公开的腺病毒转染方法“敲入"IK 1或IK 2，这将分别导致IK 2或IK 1敲除小鼠。因此，CCH诱导的K+通道的明显不同的作用将进行调查，并与功能研究。

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.