Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes
Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes
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DOI：
10.1007/s004240050916
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发表时间：
1999-08-01
影响因子：
4.5
通讯作者：
Frömter, E
中科院分区：
文献类型：
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作者：
Heyer, M;Müller-Berger, S;Frömter, E
在非洲爪蟾卵母细胞中表达大鼠肾脏Na+-HCO 3-协同转运蛋白（rkNBC），并采用膜片钳技术在巨大的内/外（i/o）和外/外（o/o）膜上研究了rkNBC的转运。在仅含有Na+和HCO 3-作为可渗透离子的溶液中测定单个贴片的电流/电压（I/V）关系。通过rkNBC（I-NBC）携带的电流通过其对改变浴Na+浓度的响应来鉴定，并定量为被4，4 '-二异硫氰酸根合芪二磺酸盐（DIDS）阻断的电流。从零电流（反转）电位确定HCO 3-向Na+转运的化学计量比（q）。结果和结论如下。首先，DIDS（250 μ mol/l）从细胞外和细胞内表面不可逆地阻断I-NBC。其次，在Na+和HCO 3-浓度梯度的存在下，与rkNBC通常在肾小管细胞中遇到的浓度梯度相似，q接近2。当HCO 3-浓度始终为25 mmol/l时，也观察到相同的值，但在贴片的细胞外或细胞内表面上，Na+浓度高（100 mmol/l）或低（10 mmol/l）。这些数据表明，在卵母细胞膜中，rkNBC在q=2的情况下起作用，如先前在分离的微灌注小管的研究中观察到的（Seki等人，Pflugers Arch 425：409，1993），然而，他们不排除在不同的膜和细胞质环境中rkNBC可以以不同的化学计量起作用的可能性。第三，在大多数实验中，高达2 mmol/l ATP的浴应用增加了I/O贴片的DIDS可恢复电导高达两倍，半饱和常数接近0.5 mmol/l。这种增加与q的变化无关，也与I/V关系的变化无关，这表明诱导主动转运（泵电流）。由于去除ATP后该效应持续存在，并且在不可水解的ATP类似物AMP-PNP中未观察到，因此可能通过蛋白激酶磷酸化激活rkNBC，蛋白激酶可能粘附在膜补丁的细胞质表面。
The rat kidney Na+-HCO3- cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3- as permeable ions. The current carried by rkNBC (I-NBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4,4'-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3- to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 mu mol/l) blocks I-NBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3- concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3- concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflugers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.