Peter M. Cala

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na+/H+ exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716–19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na+/H+ antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg425, which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

NHE1 Inhibition by Amiloride- and Benzoylguanidine-type Compounds INHIBITOR BINDING LOCI DEDUCED FROM CHIMERAS OF NHE1 HOMOLOGUES WITH ENDOGENOUS DIFFERENCES IN INHIBITOR SENSITIVITY

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.

Cloning and expression of the Na+/H+exchanger from Amphiuma RBCs: resemblance to mammalian NHE1

The cDNA encoding the Na+/H+exchanger (NHE) from Amphiumaerythrocytes was cloned, sequenced, and found to be highly homologous to the human NHE1 isoform (hNHE1), with 79% identity and 89% similarity at the amino acid level. Sequence comparisons with other NHEs indicate that the Amphiuma tridactylum NHE isoform 1 (atNHE1) is likely to be a phylogenetic progenitor of mammalian NHE1. The atNHE1 protein, when stably transfected into the NHE-deficient AP-1 cell line (37), demonstrates robust Na+-dependent proton transport that is sensitive to amiloride but not to the potent NHE1 inhibitor HOE-694. Interestingly, chimeric NHE proteins constructed by exchanging the amino and carboxy termini between atNHE1 and hNHE1 exhibited drug sensitivities similar to atNHE1. Based on kinetic, sequence, and functional similarities between atNHE1 and mammalian NHE1, we propose that the Amphiuma exchanger should prove to be a valuable model for studying the control of pH and volume regulation of mammalian NHE1. However, low sensitivity of atNHE1 to the NHE inhibitor HOE-694 in both native Amphiuma red blood cells (RBCs) and in transfected mammalian cells distinguishes this transporter from its mammalian homologue.

Na/H exchange-dependent cell volume and pH regulation and disturbances

1. The role of Na/H exchange in cell volume and pH regulation is discussed. In addition the roles of Cl/HCO3 exchange and system buffers are evaluated as they relate to Na/H exchange-dependent changes in cell salt and water content and intracellular pH. 2. Data obtained from studies of Amphiuma red blood cells showed that in addition to previously reported Na/H exchange dependent volume regulation the pathway is also involved in regulating cell pH. 3. These data showed that in contrast to volume activated Na/H exchange, when the pathway is pH activated it does not deactivate as a function of cell volume. 4. Given what appeared to be mutually exclusive volume and pH regulatory functions of the Na/H exchange, we hypothesized that the pathway might play a role in hypoxic cell swelling (cytotoxic edema). 5. In studies performed on perfused rabbit hearts employing 23Na NMR we were able to observe that relative to normoxic controls hypoxic hearts exhibited a five-fold increase in intracellular Na content when the Na-K pump was inhibited by ouabain and/or K-free perfusate. 6. These studies lead us to conclude that hypoxia-induced Na uptake is the result of an increased inward Na leak as opposed to decreased Na pumping. 7. Based upon studies with a variety of inhibitors of dissipative Na transport, we conclude that the increased inward Na leak in hypoxic hearts is via Na/H exchange.

Chapter 5 Volume-Sensitive Alkali Metal-H Transport in Amphiuma Red Blood Cells

Publisher Summary Many cells exhibit volume-sensitive ion flux pathways that are activated by osmotic swelling or shrinkage. Because of activating such pathways, the net ion and osmotically obliged H 2 O fluxes proceed until cell volume is returned toward or to normal levels. These pathways can function in a cell volume regulatory role. The Amphiuma red blood cell exhibits volume-sensitive, net alkali metal and Cl fluxes. The volume-sensitive flux pathways are quiescent in the steady state; yet on activation, they can mediate net fluxes that have greater magnitude than the pathways that function at normal volume. The chapter illustrates changes in cell K and Na content in response to osmotic swelling and shrinkage, respectively. Ca 2+ can activate K loss in a manner similar to osmotic swelling. The diacylglycerol analogs activate both Na–H and K–H exchange, suggests that if diacylglycerol through kinase C is important in volume-dependent activation of alkali metal–H exchange, then other events must be involved in determining alkali metal ion selectivity. Diacylglycerol through protein kinase C appears to play an important role as an activator of a variety of cellular processes. The Amphiuma red blood cell has volume-sensitive ion flux pathways that mediate net Na–H and K–H exchange. When osmotically swollen, the K–H exchange is activated, while osmotic shrinkage activates only Na–H exchange.

Effects of the plant alkaloid sanguinarine on cation transport by human red blood cells and lipid bilayer membranes

SummaryThe plant alkaloid, sanguinarine, inhibits the ouabain-sensitive K−Na pump and increases the downhill, ouabain-insensitive movements of K and Na in human red cells. These two effects have different temporal and concentration dependencies and are mediated by two different chemical forms of sanguinarine. The oxidized, charged form (5×10−5m) promptly inhibits the pump but does not affect leakage of K and Na. The reduced, uncharged form of sanguinarine causes lysis of red cells but does not inhibited the pump. Sanguinarine also increases the conductance of bilayers formed from sheep red cell lipids. The effect is produced by the uncharged but not by the charged form of sanguinarine. Bilayer conductance increases as the fourth power of sanguinarine concentration when the compound is present on both sides of the membrane and as the second power of concentration when present on only one side. Conductance also increasee-fold for each 34 mV increase in the potential difference imposed across the membrane. The results suggest that the uncharged forms of sanguinarine produce voltage-dependent channels in bilayers.

Regulation of the Pleuronectes americanus Na+/H+ exchanger by osmotic shrinkage, β-adrenergic stimuli, and inhibition of Ser/Thr protein phosphatases

The ubiquitous Na+/H+ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, β-adrenergic stimuli, and the Ser/Thr protein phosphatase PP1 and PP2A inhibitor calyculin A (CLA) (Pedersen et al. [2003] Am. J. Physiol.284, C1561–C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by osmotic cell swelling. Activation by the β-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic shrinkage or CLA. IP-mediated, but neither shrinkage-nor CLA-mediated paNHE1 activation were associated with an increase in cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A (PKA) inhibitor H89 (10μM), wherease shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected by inhibitors of protein kinase C (calphostin C, 5 μM) and protein kinase G (KT5823, 10 μM) as well as of myosin light chain kinase (ML-7, 10 μM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially convergent signaling pathways, whereas activation of paNHE1 by β-adrenergic stimuli is mediated by a separate pathway.

Effects of hypoxia and acidification on myocardial Na and Ca: Role of Na-H and Na-Ca exchange

Until recently, increases in cell Na content during ischemic and hypoxic episodes were thought to result from impaired ATP production causing decreased Na-K ATPase activity. We report the results of testing the alternate hypothesis that hypoxia-induced Na uptake is (1) the result of increased entry, as opposed to decreased extrusion, and (2) via Na-H exchange operating in a pH regulatory capacity and that cell Ca accumulation occurs via Na-Ca exchange secondary to collapse of the Na gradient.

Cell volume and pH regulation by the Amphiuma red blood cell : a model for hypoxia-induced cell injury

The Amphiuma red blood cell is one of the model systems employed early in the study of vertebrate cell volume regulation. Following both cell swelling and shrinkage the Amphiuma red blood cell demonstrates volume regulation to virtual completion in 90-120 min. When swollen the Amphiuma red blood cell loses K, Cl and osmotically obliged water, while following shrinkage volume regulation is the result of Na, Cl and therefore water uptake. The main contribution of the Amphiuma red cell as a model is that it was the first cell in which volume regulation was demonstrated to be electroneutral and more specifically that K/H and Na/H exchangers were responsible for regulation following cell swelling and shrinkage, respectively. Additionally, the Amphiuma red blood cell K/H and Na/H exchangers have been demonstrated to function in a pH regulatory capacity. The latter observation in turn led to the demonstration of the mutually exclusive and contradictory nature of volume and pH regulation predicted upon Na/H exchanger activity. These observations prompted our recent investigations of the Na/H exchanger as a contributor to hypoxia-induced cell damage, using the rabbit heart as a model. These studies illustrated that Na, and Ca imbalances characteristic of hypoxia-induced cell damage are ultimately referable to the Na/H exchangers function in a pH regulatory capacity, which contributes fundamentally to cell volume and Ca derangement and ultimately cell injury.

Na/H exchange inhibition protects newborn heart from ischemia/reperfusion injury by limiting Na+-dependent Ca2+ overload

The results of the Guardian/Expedition trials demonstrate the need for more precisely controlled studies to inhibit Na/H exchange (NHE1) during ischemia/reperfusion. This is because overwhelming evidence is consistent with the hypothesis that myocardial ischemic injury results in part from increases in intracellular Na (Nai) mediated by NHE1 that in turn promote Na/Ca exchanger-mediated increases in intracellular Ca ([Ca]i) and Ca-dependent cell damage. We used a more potent and specific NHE1 inhibitor HOE 694 (HOE) to test whether inhibition of NHE1 during ischemia limits increases in Nai and [Ca]i in newborns. NMR was used to measure pHi, Nai, [Ca]i, and ATP in isolated newborn rabbit hearts. Perfusion pressure, left ventricular developed pressure, and creatine kinase were measured. HOE was added before global ischemia. Results are reported as mean ± SE. Nai (mEq/kg dry weight) rose from 11.6 ± 0.9 before ischemia to 114.0 ± 16.1 at the end of ischemia and recovered to 55.2 ± 11.8 in the control group. During ischemia and reperfusion, the corresponding values for Nai in the HOE group (63.1 ± 8.4 and 15.9 ± 2.5, respectively, P < 0.05) were lower than control. In the control group [Ca]i (nM/L) rose from 331 ± 41 to 1069 ± 71 and recovered to 814 ± 51, whereas in the HOE group [Ca]i rose less (P < 0.05): 359 ± 50, 607 ± 85, and 413 ± 40, respectively. Total creatine kinase release was significantly reduced in the HOE group. Perfusion pressure and left ventricular developed pressure also recovered significantly better in the HOE group than in the control. In conclusion, NHE1 inhibition diminishes ischemia-induced increases in Nai and therefore [Ca], and thus diminishes myocardial injury in neonatal hearts.