Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods
Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods
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DOI：
10.1073/pnas.0403930101
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发表时间：
2004-07-13
影响因子：
11.1
通讯作者：
Hilgemann, DW
中科院分区：
文献类型：
--
作者：
Fuster, D;Moe, OW;Hilgemann, DW
钠/氢交换器 (NHE) 是普遍存在的离子转运蛋白，具有多种细胞功能。我们通过在全细胞膜片钳和细胞内部灌注过程中测量细胞外质子 (H+) 梯度，研究了两种哺乳动物亚型 NHE1（普遍存在）和 NHE3（上皮特异性）。最大 Na+ 依赖性 H+ 通量 (J(H+)) 相当于豚鼠心室肌细胞中 NHE1 的 200 pA 电流，以及负鼠肾细胞中 NHE3 的 5-10 pA 电流。该通量被 NHE 抑制剂乙基异丙米洛利阻断，并且在 NHE 缺陷的 AP-1 细胞中不存在。 NHE1 活性在灌注不可水解的 ATP [腺苷 5'-(β,γ-亚氨基)三磷酸]时保持稳定，通过 ATP 耗竭（用寡霉素或灌注腺苷三磷酸酶进行 2 脱氧-D-葡萄糖）消除，可以用磷酸肌醇 4,5-二磷酸恢复，并且不受肌动蛋白细胞骨架破坏（latrunculin 或移液管灌注凝溶胶蛋白）。 NHE3（但不是 NHE1）被磷酸肌醇 3,4,5-三磷酸可逆激活。在千兆海豹形成过程中，NHE1 和 NHE3 的活动都会受到巨大斑块的破坏。即使在没有 ATP 的中性细胞质 pH 值下，NHE1（但不是 NHE3）也会被细胞收缩可逆地激活，并被细胞肿胀抑制。中国仓鼠卵巢成纤维细胞中的 NHE1（但负鼠肾细胞中的 NHE3 除外）会被使细胞膜变薄的物质（L-a-溶血磷脂酰胆碱和辛基-13D-吡喃葡萄糖苷）抑制，并被胆固醇富集激活，从而使细胞膜增厚。然而，在 AP-1 细胞中表达的 NHE1 对这些试剂不敏感，但对体积变化仍然敏感。因此，疏水性错配的变化可以调节 NHE1，但并不是其体积敏感性的基础。
Sodium/hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H+) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na+-dependent H+ fluxes (J(H+)) are equivalent to currents 200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pAforNHE3 in opossum kidney cells. The fluxes are blocked by an NHE inhibitor, ethylisopropylamiloride, and are absent in NHE-cleficient AP-1 cells. NHE1 activity is stable with perfusion of nonhydrolyzable ATP [adenosine 5'-(beta,gamma-imido)triphosphate], is abolished by ATP depletion (2 deOXY-D-glucose with oligomycin or perfusion of apyrase), can be restored with phosphaticlylinositol 4,5-bisphosphate, and is unaffected by actin cytoskeleton disruption (latrunculin or pipette perfusion of gelsolin). NHE3 (but not NHE1) is reversibly activated by phosphaticlylinositol 3,4,5-trisphosphate. Both NHE1 and NHE3 activities are disrupted in giant patches during gigaohnn seal formation. NHE1 (but not NHE3) is reversibly activated by cell shrinkage, even at neutral cytoplasmic pH without ATP, and inhibited by cell swelling. NHE1 in Chinese hamster ovary fibroblasts (but not NHE3 in opossum kidney cells) is inhibited by agents that thin the membrane (L-a-lysophosphatidylcholine and octyl-13D-glucopyranoside) and activated by cholesterol enrichment, which thickens membranes. Expressed in AP-1 cells, however, NHE1 is insensitive to these agents but remains sensitive to volume changes. Thus, changes of hydrophobic mismatch can modulate NHE1 but do not underlie its volume sensitivity.