Moore M L, George J N, Turner R J
Clinical Investigation and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):637-42. doi: 10.1042/bj3090637.
The anion dependence of [3H]bumetanide binding and 22Na+ transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter was studied in acinar basolateral membrane vesicles (BLMVs). Cl-, Br- and NO3- have a biphasic effect on binding consistent with the presence of two anion sites associated with the bumetanide binding event, a high-affinity stimulatory site and a lower-affinity inhibitory site. We show that formate shares only the stimulatory site and SO4(2-) only the inhibitory site. The initial rate of [3H]bumetanide binding was stimulated by formate or low [Cl-] and inhibited by SO4(2-) or high [Cl-], but the rate of [3H]bumetanide dissociation was not affected by the presence of these anions in the dissociation medium. However, when [3H]bumetanide was bound to BLMVs in the presence of formate its rate of dissociation was more than four times faster than when binding took place in the presence of Cl-. These observations indicate that the binding of bumetanide and the stimulatory anion are ordered such that the anion must necessarily bind first and subsequently cannot dissociate until after bumetanide dissociates. In zero-trans-flux experiments, extravesicular SO4(2-) and formate had no effect on 22Na+ transport via the co-transporter [Turner and George (1988) J. Membr. Biol. 102, 71-77]. Thus neither of the anion sites associated with bumetanide binding is a Cl- transport site. However, we show here that SO4(2-) inhibits transport when present in the intravesicular space. Since the BLMV preparation is predominantly oriented cytosolic-side-in, this observation indicates the existence of an inhibitory cytosolic anion modifier site. Our data suggest that this site is identical to the inhibitory anion site associated with bumetanide binding.
在兔腮腺腺泡基底外侧膜囊泡（BLMVs）中研究了[³H]布美他尼结合以及兔腮腺钠钾氯协同转运体对²²Na⁺转运的阴离子依赖性。氯离子、溴离子和硝酸根离子对结合有双相效应，这与布美他尼结合事件中存在两个阴离子位点一致，即一个高亲和力的刺激位点和一个低亲和力的抑制位点。我们发现甲酸根离子只共享刺激位点，硫酸根离子只共享抑制位点。[³H]布美他尼结合的初始速率受甲酸根离子或低浓度氯离子刺激，受硫酸根离子或高浓度氯离子抑制，但[³H]布美他尼解离速率不受解离介质中这些阴离子存在的影响。然而，当[³H]布美他尼在甲酸根离子存在下与BLMVs结合时，其解离速率比在氯离子存在下结合时快四倍多。这些观察结果表明布美他尼与刺激阴离子的结合是有序的，即阴离子必须先结合，随后在布美他尼解离后才会解离。在零转运通量实验中，囊泡外的硫酸根离子和甲酸根离子对通过协同转运体的²²Na⁺转运没有影响[特纳和乔治（1988年）《膜生物学杂志》102卷，71 - 77页]。因此，与布美他尼结合相关的两个阴离子位点都不是氯离子转运位点。然而，我们在此表明，当硫酸根离子存在于囊泡内空间时会抑制转运。由于BLMV制剂主要是胞质侧向内取向，这一观察结果表明存在一个抑制性的胞质阴离子修饰位点。我们的数据表明该位点与与布美他尼结合相关的抑制性阴离子位点相同。
