Bevensee M O, Schmitt B M, Choi I, Romero M F, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA.
Am J Physiol Cell Physiol. 2000 Jun;278(6):C1200-11. doi: 10.1152/ajpcell.2000.278.6.C1200.
We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) 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 (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-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 microM 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.
我们筛选了大鼠脑cDNA文库，并使用5' cDNA末端快速扩增技术从大鼠脑中克隆了两种电生性钠-碳酸氢根协同转运体（NBC）亚型（rb1NBC和rb2NBC）。在氨基酸水平上，一个克隆（rb1NBC）与人胰腺NBC的同源性为96%。另一个克隆（rb2NBC）与rb1NBC相同，只是其COOH末端有61个独特的氨基酸，这是由于开放阅读框3'端附近97 bp的缺失所致。通过逆转录-聚合酶链反应（RT-PCR），我们证实大鼠脑mRNA含有这种97 bp的缺失。此外，我们制备了兔多克隆抗体，可区分rb1NBC（alpharb1NBC）和rb2NBC（alpharb2NBC）独特的COOH末端。alpharb1NBC标记的一种约130 kDa的蛋白主要来自肾脏，而alpharb2NBC标记的一种约130 kDa的蛋白主要来自脑。alpharb2NBC标记的蛋白在大鼠脑皮质神经元中的表达高于培养的星形胶质细胞；alpharb1NBC则呈现相反模式。在表达研究中，将1.5% CO₂/10 mM HCO₃⁻应用于注射了rb2NBC cRNA的非洲爪蟾卵母细胞，会导致：1）细胞内pH（pH(i)）从最初由CO₂诱导的酸化中恢复；2）细胞超极化。随后，去除细胞外钠离子会逆转pH(i)的升高并引发快速去极化。在存在450 μM二异丙基氟磷酸（DIDS）的情况下，去除细胞外钠离子对pH(i)无影响，并引发轻微超极化。去除钠离子引起的pH(i)下降速率对去除细胞外氯离子不敏感。因此，rb2NBC是一种对DIDS敏感的电生性NBC，至少在大鼠脑中主要表达。
