Evidence for Involvement of the Putative First Extracellular Loop in Differential Volume Sensitivity of the Na⁺/H⁺ Exchangers NHE1 and NHE2

Abstract

We studied hyperosmolarity-induced changes in cell volume and cytoplasmic pH in PS120 cells expressing Na(+)/H(+) exchanger (NHE) isoforms and their mutants. Change in cell volume was estimated by measuring change in cell height by means of confocal microscopy. Regulatory volume increase (RVI) and cytoplasmic alkalinization were observed in cells expressing NHE1 but not in cells expressing NHE2 or NHE3. Studies using chimeric exchangers revealed that the membrane domain of the exchanger is responsible for the difference in volume sensitivity between NHE1 and NHE2. Although deletion or point mutation within the first extracellular loop of NHE1 did not affect RVI and alkalinization, point mutations within the corresponding region of NHE2, particularly a region containing aa 41-53, as well as replacement of the N-terminus of NHE2 with the corresponding region of NHE1, rendered NHE2 responsive to the activating effect of cell shrinkage. Thus, the membrane domain plays an important role in the response of the exchanger to cell shrinkage. The data suggest that the putative first extracellular loop of NHE2, but not that of NHE1, may exert an inhibitory influence on hyperosmolarity-induced activation of the exchanger and thereby block RVI.

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

“…Next, we examined the volume sensitivity of NHE1 (29)(30)(31)(32)(33)(34) in this whole-cell mode with extensive dialysis of the cytoplasm. Cell volume was changed both by application of pressure to the pipette and application of osmotic gradients to the cells.…”

Dysfunction of regulatory volume increase is a key component of apoptosis

“…Hypertonic stimulation is known to stimulate NHE, thereby eliciting significant intracellular alkalinization in HCO À 3 -free conditions [16,[26][27][28]. Thus, there exists a possibility that impaired intracellular alkalinization upon hypertonic stimulation is involved in apoptosis induction in HeLa cells treated with an NHE1 blocker (amiloride) together with an AE blocker (DIDS) and in NHE1-deficient PS120 cells.…”

Physiology of Cell Volume Regulation in Vertebrates

“…Again pointing to the involvement of regulatory cofactors, the volume sensitivity of NHE2 seems to be cell-type specific (Table 4). In PS120 cells, Su et al (964) found that replacing the first extracellular loop with that of NHE1 rendered NHE2 volume sensitive, suggesting that in NHE2, yet not in NHE1, this region plays an inhibitory role in shrinkage-activation. NHE4 is robustly activated by cell shrinkage in a manner dependent on the actin cytoskeleton and has been suggested to play a special role in RVI in tissues exposed to very high osmolarity, such as the kidney medulla (69, 70).…”

“…However, divergent views have also been reported (432,964), likely indicating that the volume sensitivity of NHE3 may be dependent on the cell type in which it is expressed. This may reflect the requirement for a cell type-specific regulatory intermediate, in accordance with the substantial number of cell type-specific NHE3 regulators (see Ref.…”