Inhibition of rat Na+(-)HCO3(-) cotransporter (NBCn1) function and expression by the alternative splice domain.
Inhibition of rat Na+(-)HCO3(-) cotransporter (NBCn1) function and expression by the alternative splice domain.
复制标题
选择性剪接结构域对大鼠 Na (-)HCO3(-) 协同转运蛋白 (NBCn1) 功能和表达的抑制。
DOI：
10.1113/expphysiol.2009.048603
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发表时间：
2009
影响因子：
2.7
通讯作者：
Choi,Inyeong
中科院分区：
文献类型：
--
作者：
Yang,HanSoo;Cooper,DeborahS;Rajbhandari,Ira;Park,HaeJeong;Lee,Soojung;Choi,Inyeong
Na+-HCO 3-协同转运蛋白NBCn 1（SLC 4A 7）具有多种变体，具体取决于蛋白质胞质氨基和羧基末端的剪接结构域。在这项研究中，我们研究了含有123个氨基酸的氨基末端剪接结构域（盒II）在NBCn 1功能和表达调控中的作用。聚合酶链反应检测到NBCn 1 mRNA在各种组织中含有盒II。在异种卵母细胞中表达两种变体，即含有盒II的NBCn 1-B和缺乏盒II的NBCn 1-E，并通过双电极电压钳进行评估，以测量转运蛋白介导的离子电流。当在RNA注射后3-4天测量时，两种变体显示出相似的电流-电压（I-V）关系。用葡萄糖酸盐代替Cl-不影响I-V关系。当暴露于含有20-50 mmNa+的溶液时，NBCn 1-B产生的电流比NBCn 1-E产生的电流稍微更正。在100 mmNa+下，两种电流相似。两种变体的斜率电导在较高的Na+水平下逐渐增加，并且增加是平行和叠加的。在RNA注射后的不同时间点测量，NBCn 1-B在24-48 h产生的电导低于NBCn 1-E。通过卵母细胞膜制备物的免疫印迹测定，在这些时间点NBCn 1-B的蛋白表达也较低。在负鼠肾（OK）细胞中表达，与对照制剂相比，NBCn 1-E导致哇巴因敏感的对硝基苯酚从磷酸对苯酯的产生增加1.5倍，而NBCn 1-B的影响可以忽略不计。我们的结论是，盒II的主要功能是减少NBCn 1蛋白的表达。
The Na+–HCO3–cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino‐ and carboxy‐termini of the protein. In this study, we examined the role of the amino‐terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1‐B containing cassette II and NBCn1‐E lacking cassette II, were expressed inXenopusoocytes and assessed by two‐electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current–voltage (I–V) relations when measured 3–4 days after RNA injection. Replacment of Cl−with gluconate did not affect theI–Vrelations. When exposed to solutions containing 20–50 mmNa+, the current produced by NBCn1‐B was slightly more positive than that produced by NBCn1‐E. The two currents were similar at 100 mmNa+. The slope conductances for the two variants were progressively increased at higher Na+levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1‐B produced lower conductance than NBCn1‐E at 24–48 h. Protein expression of NBCn1‐B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1‐E caused a 1.5‐fold increase in ouabain‐sensitive production ofp‐nitrophenol fromp‐phenyl phosphate compared with control preparations, whereas NBCn1‐B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression.