Kinetic and Pharmacological Properties of Human Brain Na+/H+ Exchanger Isoform 5 Stably Expressed in Chinese Hamster Ovary Cells

Abstract

The recently cloned Na ؉ /H ؉ exchanger isoform 5 (NHE5) is expressed predominantly in brain, yet little is known about its functional properties. To facilitate its characterization, a full-length cDNA encoding human NHE5 was stably transfected into NHE-deficient Chinese hamster ovary AP-1 cells. Pharmacological analyses revealed that H ؉ i -activated 22 Na ؉ influx mediated by NHE5 was inhibited by several classes of drugs (amiloride compounds, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate, cimetidine, and harmaline) at half-maximal concentrations that were intermediate to those determined for the high affinity NHE1 and the low affinity NHE3 isoforms, but closer to the latter. Kinetic analyses showed that the extracellular Na ؉ dependence of NHE5 activity followed a simple hyperbolic relationship with an apparent affinity constant (K Na ) of 18.6 ؎ 1.6 mM. By contrast to other NHE isoforms, NHE5 also exhibited a first-order dependence on the intracellular H ؉ concentration, achieving half-maximal activation at pH 6.43 ؎ 0.08. Extracellular monovalent cations, such as H ؉ and Li ؉ , but not K ؉ , acted as effective competitive inhibitors of 22 Na ؉ influx by NHE5. In addition, the transport activity of NHE5 was highly dependent on cellular ATP levels. Overall, these functional features distinguish NHE5 from other family members and closely resemble those of an amiloride-resistant NHE isoform identified in hippocampal neurons.Transient oscillations in the extra-and intracellular pH (pH o and pH i , respectively) environments of neurons and other cell types of the nervous system can profoundly modulate neuronal membrane excitability by inhibiting various ligand receptormediated currents (1, 2), voltage-gated cation channels (3-6), and gap junction coupling (7), as well as by activating a depolarizing inward Na ϩ current (ASIC3) (8). Thus, precise control of the neuronal pH milieu is an important biological process and may fulfill a regulatory role in brain function (for review, see Refs. 9 and 10).The ion transporters responsible for pH i regulation in the nervous system are not as well characterized as in peripheral cell types, and this is particularly true for neurons, which are often difficult to isolate and/or maintain in culture in a differentiated state. Despite this limitation, accumulating evidence indicates that the acid-base transport systems in brain are heterogeneous but comparable to other organ systems. In cultured fetal or freshly isolated neonatal pyramidal CA1 neurons from rat hippocampus, restoration of steady-state pH i following intracellular acidification involves two principal ion carriers: a Na ϩ -dependent HCO 3 Ϫ /Cl Ϫ exchanger and a novel amilorideresistant Na ϩ /H ϩ exchanger (NHE) 1 (11-13). Later in development, acutely dissociated hippocampal CA1 neurons from adults rats exhibit an acidifying mechanism mediated by a Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchanger, most likely the AE3 isoform (14). Na ϩ /H ϩ and Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchangers also make distinct...

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…Expression is greatest in the brain but was also observed in testis, spleen and skeletal muscle [78,7,5]. NHE5 shows intermediate sensitivity to amiloride inhibition (less than NHE1 but more than NHE3) [161]. Like its closest homologue NHE3, NHE5 localizes to both the plasma membrane and endomebrane compartments(s) and recycles between them [162].…”

Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

“…Briefly, cells were grown to confluence in 24-well polystyrene tissue culture dishes, washed with isotonic choline chloride solution (130 mM choline chloride, 1 mM MgCl 2 , 2 mM CaCl 2 , 20 mM HEPES-Tris, pH 7.4) and incubated with 2 Ci/ml of 22 Na ϩ in the same solution containing 1 mM ouabain (to inhibit Na ϩ efflux by the Na ϩ /K ϩ -ATPase) in the absence or presence of the specific NHE antagonist EIPA (200 M) at room temperature for 5 min. This concentration of EIPA is sufficient to completely block NHE5 activity (IC 50 ϳ 0.42 M) (22). EIPA and its related analogues inhibit NHE activity by competitively blocking Na ϩ influx.…”

“…To measure basal plasmalemmal NHE5 activity, the 22 Na ϩ influx assay was conducted as described previously (22) except that acid loading prior to 22 Na ϩ uptake was omitted. Briefly, cells were grown to confluence in 24-well polystyrene tissue culture dishes, washed with isotonic choline chloride solution (130 mM choline chloride, 1 mM MgCl 2 , 2 mM CaCl 2 , 20 mM HEPES-Tris, pH 7.4) and incubated with 2 Ci/ml of 22 Na ϩ in the same solution containing 1 mM ouabain (to inhibit Na ϩ efflux by the Na ϩ /K ϩ -ATPase) in the absence or presence of the specific NHE antagonist EIPA (200 M) at room temperature for 5 min. This concentration of EIPA is sufficient to completely block NHE5 activity (IC 50 ϳ 0.42 M) (22).…”

“…AP-1 cells stably expressing NHE1 and NHE5 (AP-1/NHE1 and AP-5/NHE5, respectively) were generated using an acid challenging selection process as previously described (22,31). All cells were incubated in a humidified atmosphere of 95% air, 5% CO 2 at 37°C.…”

“…Unlike NHE1, NHE5 is more resistant to inhibition by amiloride derivatives and benzyolguanidinium-based (e.g. HOE694) compounds (21,22) and resides predominantly in intracellular vesicles (23,24), although the significance of this distribution is uncertain. However, recent studies (25) found that NHE5 is rapidly recruited to the cell surface of hippocampal neurons in response to NMDA receptor-induced neural activity where it elevates pH i and suppresses growth of postsynaptic dendritic spines.…”