NHE1 inhibition by amiloride- and benzoylguanidine-type compounds - Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity
NHE1 inhibition by amiloride- and benzoylguanidine-type compounds - Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity
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DOI：
10.1074/jbc.m701637200
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发表时间：
2007-07-06
影响因子：
4.8
通讯作者：
Cala, Peter M.
中科院分区：
文献类型：
--
作者：
Pedersen, Stine F.;King, Scott A.;Cala, Peter M.
普遍存在的Na+/H+交换剂NHE 1与其常用的抑制剂阿米洛利和苯甲酰胍（Hoechst型抑制剂（HOE））型化合物的相互作用尚未完全了解。我们以前克隆NHE 1从Amphiuma tridactylum（AtNHE 1）和美洲鲽（PaNHE 1）。虽然与阿米洛利和HOE敏感的人NHE 1（hNHE 1）高度同源，但AtNHE 1对HOE型不敏感，PaNHE 1对阿米洛利和HOE型化合物均不敏感。在这里，我们产生了嵌合体来“敲入”阿米洛利和HOE对PaNHE 1的敏感性，从而确定了几个参与抑制剂相互作用的NHE 1区域。hNHE 1、AtNHE 1和PaNHE 1的显著不同的抑制剂敏感性不能通过跨膜（TM）区域9的差异来解释。通过PaNHE 1的C-末端尾部用AtNHE 1的相应区域替换TM 10，部分恢复了对阿米洛利和相关化合物5 '-（N-乙基-N-异丙基）阿米洛利（EIPA）的敏感性，但对HOE 694的敏感性没有恢复。这种效应不是由于尾部区域，而是依赖于TM 10 -11，因为仅用AtNHE 1替换该区域也部分恢复了阿米洛利和EIPA，但不恢复HOE敏感性。匡威的突变体（AtNHE 1的TM 10 -11被PaNHE 1的TM 10 - 11取代）表现出更高的阿米洛利和EIPA敏感性，并且也是HOE敏感性的。用hNHE 1（VFFLF）或AtNHE 1（TFFLF）的相应残基替换PaNHE 1的TM区4中的LFFFY基序大大增加了对阿米洛利和HOE型化合物的敏感性，尽管AtNHE 1对HOE 694不敏感。阿米洛利敏感性的增加似乎与Na+/H+交换率的增加相关。可以得出结论，TM 4和TM 10 -11内的区域有助于阿米洛利和HOE敏感性，这两个区域赋予部分抑制剂敏感性NHE 1。
The interaction of the ubiquitous Na+/H+ exchanger, NHE1, with its commonly used inhibitors, amiloride- and benzoylguanidine ( Hoechst type inhibitor ( HOE))-type compounds, is incompletely understood. We previously cloned NHE1 from Amphiuma tridactylum (AtNHE1) and Pleuronectes americanus (PaNHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1 regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl) amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10-11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity. The converse mutant (TM10-11 of AtNHE1 replaced with those of PaNHE1) exhibited even higher amiloride and EIPA sensitivity and was also HOE-sensitive. Replacing an LFFFY motif in TM region 4 of PaNHE1 with the corresponding residues of hNHE1 (VFFLF) or AtNHE1 (TFFLF) greatly increased sensitivity to both amiloride- and HOE-type compounds, despite the fact that AtNHE1 is HOE694- insensitive. Gain of amiloride sensitivity appeared to correlate with increased Na+/H+ exchange rates. It is concluded that regions within TM4 and TM10-11 contribute to amiloride and HOE sensitivity, with both regions imparting partial inhibitor sensitivity to NHE1.