Sarker Rafiquel, Boronia Tatiana B, Cole Robert N, Singh Varsha, Lin Ruxian, McNamara George, Donowitz Mark
Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
Am J Physiol Cell Physiol. 2025 Aug 1;329(2):C560-C573. doi: 10.1152/ajpcell.00046.2025. Epub 2025 Jun 26.
The epithelial brush border (BB) Cl/[Formula: see text] exchanger SLC26A3 [down-regulated in adenoma (DRA)] is part of two separate intestinal transport processes, neutral NaCl absorption (linked to NHE3) and anion secretion (interacting with CFTR). There is a gap in understanding the regulation of DRA in digestive physiology and in the secretory diarrheal diseases, in which there is elevation of cAMP and/or Ca. The acute stimulatory regulation of DRA in Caco-2 cells by elevated cAMP (forskolin) and Ca (ATP) was studied. As previously reported, DRA was maximally stimulated similarly by forskolin, ATP, and their combination at concentrations that alone maximally stimulated DRA (not additive) and also by their combination at concentrations that alone had no effect (called synergistic stimulation). DRA was phosphorylated at baseline on a single amino acid (S) and this markedly decreased with acute cAMP/ATP stimulation. Immunoprecipitation (IP) of DRA identified two associating proteins, inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) and LiM domain and actin-binding protein 1 (LIMA1), which were shown involved in the cAMP/Ca stimulation. KD of IRBIT1 or LIMA1 reduced the cAMP plus ATP stimulation of DRA but did not alter basal DRA activity. Maximum ATP, but not maximum forskolin stimulation of DRA, was IRBIT dependent. Also, the elevation in intracellular Ca by cAMP/ATP was IRBIT dependent. IRBIT and LIMA1 bound DRA under basal conditions, and the amount bound increased with cAMP/ATP stimulation. cAMP/ATP stimulation increased the coprecipitation of LIMA1 with both IRBIT and DRA. With acute stimulation, there was also more BB DRA, IRBIT, but not LIMA1. These results identify a DRA-IRBIT-LIMA1 complex that is involved in acute stimulation of DRA activity. Insights into acute DRA regulation relevant to secretory diarrhea studied synergistic cAMP and Ca-induced DRA stimulation. DRA was phosphorylated under baseline conditions, which decreased with acute stimulation. Acute stimulation, but not basal activity, required the presence of both IRBIT and LIMA1 (an actin-binding scaffold), involved more plasma membrane DRA, and also increased DRA association with IRBIT and LIMA1, indicating a role for a DRA-IRBIT-LIMA1 plasma membrane complex in acute DRA stimulation.
上皮刷状缘（BB）氯离子/[公式：见原文]交换体SLC26A3[腺瘤下调（DRA）]是两个独立的肠道转运过程的一部分，即中性氯化钠吸收（与NHE3相关）和阴离子分泌（与CFTR相互作用）。在消化生理学和分泌性腹泻疾病中，对DRA调节的理解存在空白，在这些疾病中cAMP和/或钙离子会升高。研究了在Caco-2细胞中，cAMP（福斯高林）和钙离子（ATP）升高对DRA的急性刺激调节作用。如先前报道，福斯高林、ATP及其组合在单独能最大程度刺激DRA的浓度下（无相加作用）对DRA的刺激作用最大，并且在单独无作用的浓度下其组合也能最大程度刺激DRA（称为协同刺激）。DRA在基线时在单个氨基酸（S）上发生磷酸化，急性cAMP/ATP刺激后这种磷酸化明显减少。对DRA进行免疫沉淀（IP）鉴定出两种相关蛋白，即与肌醇1,4,5-三磷酸一起释放的肌醇1,4,5-三磷酸受体结合蛋白（IRBIT）和LiM结构域及肌动蛋白结合蛋白1（LIMA1），它们参与了cAMP/钙离子刺激过程。IRBIT1或LIMA1的敲低降低了cAMP加ATP对DRA的刺激作用，但未改变DRA的基础活性。ATP对DRA的最大刺激作用（而非福斯高林的最大刺激作用）依赖于IRBIT。此外，cAMP/ATP引起的细胞内钙离子升高也依赖于IRBIT。在基础条件下IRBIT和LIMA1与DRA结合，结合量随cAMP/ATP刺激而增加。cAMP/ATP刺激增加了LIMA1与IRBIT和DRA两者的共沉淀。急性刺激时，刷状缘DRA和IRBIT增多，但LIMA1未增多。这些结果确定了一个参与DRA活性急性刺激的DRA-IRBIT-LIMA1复合物。对与分泌性腹泻相关的DRA急性调节的深入了解研究了协同的cAMP和钙离子诱导的DRA刺激。DRA在基线条件下发生磷酸化，急性刺激后磷酸化减少。急性刺激（而非基础活性）需要同时存在IRBIT和LIMA1（一种肌动蛋白结合支架），涉及更多的质膜DRA，并且还增加了DRA与IRBIT和LIMA1的结合，表明质膜DRA-IRBIT-LIMA1复合物在DRA急性刺激中起作用。
