Complex structure and regulation of expression of the rat gene for inward rectifier potassium channel Kir7.1

Genomic organization of the rat inward rectifier K(+) channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long ( approximately 3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.     

Cleft Palate,Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel

Kir7.1 is an inwardly rectifying K⁺ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na⁺/K⁺-pump probably serving to recycle K⁺ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree.     

Genetic inactivation of Kcnj16 identifies Kir5.1 as an important determinant of neuronal PCO2/pH sensitivity

The molecular identity of ion channels which confer PCO(2)/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO(2) chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16((+/+)) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16((-/-)) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO(2)/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。