Bianchini L, Kapus A, Lukacs G, Wasan S, Wakabayashi S, Pouysségur J, Yu F H, Orlowski J, Grinstein S
Centre de Biochimie-Centre National de la Recherche Scientifique, Université de Nice, France.
Am J Physiol. 1995 Oct;269(4 Pt 1):C998-1007. doi: 10.1152/ajpcell.1995.269.4.C998.
Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be activated by shrinking after an acid load. Jointly, these results suggest that the H+ affinity and high cooperativity of the antiporter, earlier attributed to a single "modifier site," can be varied independently and are probably controlled by different regions of the molecule. The data indicate that volume or osmolarity-sensitive site(s) exist between the NH2-terminus and residue 566. This putative volume-sensitive site is therefore different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors.
NHE1（普遍存在的Na⁺/H⁺交换体）的高渗激活在细胞体积调节中起核心作用，但对其潜在机制知之甚少。我们探究了转染到缺乏内源性反向转运活性细胞中的全长和截短形式的NHE1的渗透反应性。在全长NHE1或在第698位或703位截短的构建体进行异源转染后，NHE1的高渗刺激得以保留。相比之下，在第635位截短的突变体（δ635）对渗透挑战无反应。转染体（δ635）表现得好像组成性激活，在等渗、未刺激条件下具有永久升高的胞质pH（pHi）。δ635突变体表现出高亲和力和低协同性的H⁺结合。构建体δ582或δ566的H⁺敏感性降低，因此在静息pHi时无活性。此类细胞在生理pHi附近对渗透应激无反应，但在酸负荷后细胞收缩时可被激活。综合这些结果表明，先前归因于单个“调节位点”的反向转运体的H⁺亲和力和高协同性可以独立变化，并且可能由分子的不同区域控制。数据表明，在NH2末端和第566位残基之间存在体积或渗透压敏感位点。因此，这个假定的体积敏感位点不同于推测介导钙和生长因子刺激作用的位点。
