An electrogenic Na+-HCO3- cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain
An electrogenic Na+-HCO3- cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain
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DOI：
10.1152/ajpcell.2000.278.6.c1200
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发表时间：
2000-06-01
影响因子：
5.5
通讯作者：
Boron, WF
中科院分区：
文献类型：
--
作者：
Bevensee, MO;Schmitt, BM;Boron, WF
我们筛选了大鼠脑cDNA文库，并利用5'端快速扩增技术从大鼠脑中克隆了两个产电Na ~+-HCO_3-协同转运蛋白（NBC）亚型（rb_1NBC和rb_2NBC）。在氨基酸水平上，一个克隆（rb 1 NBC）与人胰腺NBC具有96%的相同性。另一个克隆（rb 2NBC）与rb 1 NBC相同，除了61个独特的COOH末端氨基酸，这是开放阅读框3'端附近97-bp缺失的结果。使用RT-PCR，我们证实，从大鼠脑的mRNA含有这个97 bp的缺失。此外，我们产生了兔多克隆抗体，区分rb 1 NBC（α rb 1 NBC）和rb 2NBC（α rb 2NBC）的独特COOH-末端。α rb 1 NBC标记与主要来自肾脏的130-kDa蛋白相似的蛋白，α rb 2NBC标记与主要来自大脑的130-kDa蛋白相似的蛋白。α rb 2NBC标记的蛋白质在皮层神经元中比从大鼠脑培养的星形胶质细胞中表达更高; α rb 1 NBC表现出相反的模式。在表达研究中，将1.5%CO2/10 mM HCO 3-应用于注射rb 2NBC cRNA的非洲爪蟾卵母细胞，导致1）pH（i）从最初的CO2诱导的酸化恢复，2）细胞过度增殖。随后，去除外部Na+逆转pH（i）的增加并引起快速去极化。在450 μ M DIDS的存在下，除去外部Na+对pH（i）没有影响，并引起小的超极化。去除Na+引起的pH（i）降低速率对去除外部Cl-不敏感。因此，rb 2NBC是主要在至少大鼠的脑中表达的DIDS敏感的产电NBC。
We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na+-HCO3- cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpha rb1NBC) and rb2NBC (alpha rb2NBC). alpha rb1NBC labels an similar to 130-kDa protein predominantly from kidney, and alpha rb2NBC labels an similar to 130-kDa protein predominantly from brain. alpha rb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpha rb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO2/10 mM HCO3- to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO2-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na+ reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 mu M DIDS, removing external Na+ has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na+ is insensitive to removing external Cl-. Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.