Park C S, Fanestil D D
Am J Physiol. 1983 Dec;245(6):F716-25. doi: 10.1152/ajprenal.1983.245.6.F716.
Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.
在蟾蜍膀胱顶膜中发现了两个可滴定基团，它们对Na⁺转运有影响，其表观酸解离常数（pKa）分别为4.2和6.7，初步确定为一个羧基和一个咪唑基。N - 乙氧羰基 - 2 - 乙氧基 - 1,2 - 二氢喹啉（EEDQ）是一种对羧基残基有选择性的试剂，它能抑制蟾蜍膀胱中的Na⁺转运。研究了EEDQ通过潜在修饰羧基残基产生抑制作用的潜在化学反应。EEDQ对Na⁺转运的抑制作用取决于反应介质的pH值和亲核试剂的可用性，这表明共价酰基 - 亲核试剂键的形成可能参与了对Na⁺转运的不可逆抑制。抑制动力学表明，每关闭一个Na⁺转运位点（推测为Na⁺通道）形成一个酰基 - 亲核试剂键的化学计量关系。初步确定参与酰基 - 亲核试剂键形成的亲核试剂的表观pKa为6.7。添加到黏膜林格溶液中的氨氯吡咪及其两种类似物（但不是浆膜侧的氨氯吡咪）可防止EEDQ对Na⁺转运的抑制——这一发现与以下假设一致：EEDQ激活的羧基与氨氯吡咪在顶膜上特异性结合位点或其附近的亲核试剂发生反应，最有可能是在Na⁺通道处。我们的研究结果使我们推测，氨氯吡咪必须与Na⁺通道上至少两个位点相互作用才能阻断通道。这两个位点之一似乎是Na⁺通道上的阴离子羧基与氨氯吡咪的阳离子胍基之间的离子相互作用。
