New KCNN4 Variants Associated With Anemia: Stomatocytosis Without Erythrocyte Dehydration.

The K+ channel activated by the Ca2+, KCNN4, has been shown to contribute to red blood cell dehydration in the rare hereditary hemolytic anemia, the dehydrated hereditary stomatocytosis. We report two de novo mutations on KCNN4, We reported two de novo mutations on KCNN4, V222L and H340N, characterized at the molecular, cellular and clinical levels. Whereas both mutations were shown to increase the calcium sensitivity of the K+ channel, leading to channel opening for lower calcium concentrations compared to WT KCNN4 channel, there was no obvious red blood cell dehydration in patients carrying one or the other mutation. The clinical phenotype was greatly different between carriers of the mutated gene ranging from severe anemia for one patient to a single episode of anemia for the other patient or no documented sign of anemia for the parents who also carried the mutation. These data compared to already published KCNN4 mutations question the role of KCNN4 gain-of-function mutations in hydration status and viability of red blood cells in bloodstream.

Primary red cell hydration disorders: Pathogenesis and diagnosis.

Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.

Review. Leaky Cl--HCO3- exchangers: cation fluxes via modified AE1.

The abundant membrane protein AE1 normally functions as an obligate anion exchanger, with classical carrier properties, in human red blood cells. Recently, four single point mutations of hAE1 have been identified that have lost the anion exchange function, and act as non-selective monovalent cation channels, as shown in both red cell flux and oocyte expression studies. The red cell transport function shows a paradoxical temperature dependence, and is associated with spherocytic and stomatocytic red cell defects, and haemolytic anaemias. Other forms of AE1, including the native AE1 in trout red cells, and the human mutation R760Q show both channel-like and anion exchange properties. The present results point to membrane domains 9 and 10 being important in the functional modification of AE1 activity.

Evidence for the presence of three different anion exchangers in a red cell. Functional expression studies in Xenopus oocytes.

Anion exchangers (AE) are transmembrane proteins catalyzing electroneutral exchange of Cl(-) for HCO3-. To date, three different genes coding for this protein, AE1, AE2 and AE3, have been identified in many species. AE1 is considered to be the unique anion exchanger expressed in erythrocytes. In this paper we propose the presence of three different AEs in skate erythrocytes, a skAE1, a skAE2 and a skAE3, cloned by RT-PCR (reverse-transcriptase polymerase chain reaction). These three skAE have a similar predicted secondary structure. All three skAE are divided in two main domains: a hydrophilic cytoplasmic N-terminal domain and a C-terminal domain crossing the lipid bilayer at least 12 times. The greatest similarity is found in the membrane-spanning domain of the three skAE. The size as well as the amino-acid sequence of the cytoplasmic domain differ significantly among three anion exchangers. Functional expression studies in Xenopus oocytes led to the conclusion that skAE-1 and -2 share some functional features (Cl-dependence and DIDS sensitivity). The skAE3 could not be expressed in Xenopus oocytes. These data are in agreement with expression data obtained with AEs of different species utilizing the oocyte system. It is highly probable that these three new AE sequences come from three different genes, thus suggesting for the first time the presence of the three AE genes in Chondrichthyes.

Multiple transport functions of a red blood cell anion exchanger, tAE1: its role in cell volume regulation.

1. It was previously shown that expressed in Xenopus oocyte the mouse (mAE1) and the trout (tAE1) anion exchanger behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with a chloride channel activity. The present data, obtained by measurement of Xenopus oocyte membrane permeability and conductance, provide evidence that tAE1 also induces a large increase in Na(+) and K(+) permeability inhibited by several AE1 inhibitors. 2. This inhibition does not result from an effect on the driving force for electrodiffusion but represents a direct effect on the cation pathway. 3. As a control, expression of cystic fibrosis transmembrane conductance regulator (CFTR) having, once stimulated by 3-isobutyl-1-methylxanthine (IBMX), the same anion conductance magnitude as tAE1 did not induce any cation movement. 4. Chloride exchange, channel activity and cation transport induced by anion exchanger expression are inhibited by free or covalently bound H2DIDS as well. This covalent inhibition is reversed by the point mutation of Lys-522, the covalent binding site of H2DIDS to the protein. These data reveal that tAE1 itself acts both as an anion exchanger and as a channel of broad selectivity. 5. All results obtained by expression of AE1 isoforms in Xenopus oocytes and those obtained in erythrocytes are consistent with the proposal that, in nucleated erythrocytes, tAE1 functions as the swelling-activated osmolyte anion channel involved in cell volume regulation. In contrast AE1 from mammalian red cells, which do not regulate their volume, lacks swelling-activated osmolyte channel properties. 6. tAE1 illustrates the ability of a specific transport system to be a multifunctional protein exhibiting other transport functions when submitted to regulation.

Cell volume regulation: the role of taurine loss in maintaining membrane potential and cell pH.

1. In response to a hypo-osmotic stress cells undergo a regulatory volume decrease (RVD) by losing osmotically active solutes and obliged water. During RVD, trout red cells lost taurine, K+ and Cl- but gained Na+ and Cl-. Over the full time course of RVD the chloride concentration in the cell water remained remarkably constant. Thus membrane potential and cell pH, which depends on the ratio of internal to external chloride concentration ([Cl-]i:[Cl-]o), remained fixed. 2. When cell volume decreases it is only possible to keep the chloride concentration in the cell water constant if an equal percentage of the cell chloride pool and of the cell water pool are lost simultaneously. Quantitative analysis of our data showed that this requirement was fulfilled because, over the full time course of RVD, cells lost osmotically active solutes with a constant stoichiometry: 1 Cl-:1 positive charge:2.35 taurine. Any change in taurine permeability, by modifying the stoichiometric relationship, would affect the amount of water lost and consequently cell chloride concentration. 3. Experiments carried out with different cations as substitutes for external Na+ suggest that the constancy of the chloride concentration is not finely tuned by some mechanism able to modulate the channel transport capacity, but results in part from the fact that the swelling-dependent channel constitutively possesses an adequately fixed relative permeability for cations and taurine. However, as a significant fraction of K+ and Cl- loss occurs via a KCl cotransporter, the contribution of the cotransport to the stoichiometric relationship remains to be defined. 4. The large amount of taurine released during RVD (50 % of all solutes) was shown to be transported as an electroneutral zwitterion and not as an anion. How the channel can accommodate the zwitterionic form of taurine, which possesses a high electrical dipole, is considered.

Swelling activation of transport pathways in erythrocytes: effects of Cl-, ionic strength, and volume changes.

If swelling of a cell is induced by a decrease in external medium tonicity, the regulatory response is more complex than if swelling of similar magnitude is due to salt uptake. The present results provide an explanation. In fish erythrocytes, two distinct transport pathways were swelling activated: a channel of broad specificity and a K+-Cl- cotransporter. Each was activated by a specific signal: the channel by a decrease in intracellular ionic strength and the K+-Cl- cotransporter by cell enlargement. A decrease in ionic strength also affected K+-Cl- cotransport activity, but by acting as a negative modulator of the cotransport. Thus cells swollen by salt accumulation respond by activating exclusively the K+-Cl- cotransport, leading to a Cl--dependent K+ loss. By contrast, cells swollen by electrolyte dilution respond by activating both pathways, leading to a reduced loss of electrolytes and a large loss of taurine. Thus two swelling-sensitive pathways, differently regulated, would allow control of the ionic composition of a cell exposed to different volume perturbations.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s).

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Regulation of Na+/H+ antiporter in trout red blood cells.

The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.

A role for the anion exchanger AE1 (band 3 protein) in cell volume regulation.

In response to swelling cells recover their volume by releasing ions (mainly K+, Cl-) and different organic solutes (e.g. taurine) via volume-sensitive pathways. Depending on the cause of swelling (net uptake of electrolytes or decrease in external osmolality) cells use specifically some of these pathways. Previous data indicate that the anion exchanger (AE1) is involved in the choice of the regulatory pattern the cells adopt. Molecular cloning and functional expression of AE1 from the trout erythrocyte shows that this anion exchanger can function as a channel mediating taurine fluxes. In the erythrocyte, the channel activation depends on the conditions as the cell is swollen: when swelling is caused by an accumulation of electrolytes (resulting in an increase of the intracellular ionic strength) the channel is not activated and the regulatory volume decrease occurs exclusively by a release of K and Cl via a KCl cotransporter. When swelling is caused by hypotonic shock (resulting in a decrease in intracellular ionic strength) the KCl cotransporter is then mainly inactivated or even silent; conversely the channel is activated and allows volume recovery by mediating the release of both taurine and probably K and Cl. The possibility that AEs function as volume-activated taurine channels in other cell types and as a malaria-induced channel in malaria-infected human red cells is considered.

The erythrocyte Na+/H+ exchangers of eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss): a comparative study

Trout and eel red blood cell Na+/H+ exchangers show widely different regulatory properties. Catecholamines, cyclic AMP and phorbol esters, which activate the trout red cell antiporter, do not affect the eel exchanger. Unlike the trout red cell exchanger, the eel red cell exchanger is strongly activated by cell shrinkage, allowing a remarkable cell volume recovery. These different regulatory properties probably indicate the existence of different isoforms of the exchangers in nucleated erythrocytes, since sensitivity to catecholamines is known to be dependent upon the presence of protein kinase A consensus sites on the cytoplasmic domain of the antiporter. After shrinkage of eel erythrocytes, the Na+/H+ exchange rate gradually increases to reach a maximum value after about 10 min. The magnitude of activation is a graded function of cell shrinkage. Deactivation, like activation, is induced by a volume change and occurs after some delay (lag time). The response of the trout antiporter (betaNHE) to cell shrinkage is much reduced compared with that of the eel antiporter. In addition, the antiporter is deactivated prior to restoration of the normal control volume, leaving cell volume regulation notably defective. The trout red cell antiporter, which is desensitized and enters a refractory state following hormonal activation, is only deactivated (it can be reversibly reactivated) after shrinkage-induced activation. This dual control may occur by both phosphorylation-dependent and phosphorylation-independent mechanisms. In view of the similarities in the regulatory properties of eel and salamander (Amphiuma sp.) Na+/H+ exchangers, the expression of a putative K+/H+ exchange mediated by the N+/H+ exchanger was sought in eel erythrocytes. However, neither osmotic swelling nor calyculin-A-dependent phosphorylation revealed such a K+/H+ exchange.

Regulation of Na+/H+ exchange activity by recruitment of new Na+/H+ antiporters: effect of calyculin A.

The Na+/H+ antiporter of trout red blood cells, beta-NHE, is activated by agonists of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) and by those of protein kinase C (PKC). beta-NHE, once activated, shifts into a refractory state, accounting for its desensitization. It had previously been shown that desensitization is blocked and reversed by the protein phosphatase inhibitor okadaic acid (OA). In this study we examined the effect of another protein phosphatase inhibitor, calyculin A (CIA). CIA was at least 10 times more potent than OA in blocking beta-NHE desensitization, suggesting that desensitization is controlled by phosphatase-1. Furthermore, CIA alone induced a large Na+/H+ exchange in unstimulated red blood cells, a property not shared by OA. The characteristics of ClA-induced Na+/H+ exchange are very different from those of the exchange triggered by activation of beta-NHE by PKA or PKC agonists, i.e., a flat pH dependence and total insensitivity to PKA and PKC inhibitors. Simultaneous addition of maximal concentrations of ClA and catecholamine produced an additive stimulation of the Na+/H+ exchange, consistent with the interpretation that these agents act on two distinct pools of exchangers. Screening of different cDNA libraries suggested that only one isoform of antiporter exists in the trout red blood cell; it therefore seems likely that regulation of the Na+/H+ antiporter beta-NHE involves a recycling mechanism. The reasons why intracellular beta-NHE show different properties from membrane beta-NHE are discussed.

Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent Na+/H+ antiport.

The Na+/H+ antiporter of trout erythrocytes is activated by agents raising intracellular cAMP, whereas other Na+/H+ exchangers are insensitive to or inhibited by cAMP. Cloning of the beta agonist-activated exchanger (beta NHE) reveals the presence of two consensus sites for phosphorylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cytoplasmic loop. Transfected to fibroblasts, beta NHE can no longer be activated by cAMP when these consensus sites are removed, indicating regulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase and phosphatase inhibitors on the antiporter activation and desensitization in trout red cells. Na+/H+ exchange was not induced by strong acidification, indicating that beta NHE is normally in a nonfunctional state, whereas cAMP did activate the system by forcing beta NHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confirming the role of cAMP-PKA in the activation process. The protein phosphatase inhibitor okadaic acid (OA) neither activated the exchange when added on unstimulated cells nor prevented deactivation of beta agonist-activated beta NHE by propranolol. Hence, the cAMP-dependent phosphorylation involved in the activating process is controlled by an OA-insensitive phosphatase. beta NHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously described desensitization. Desensitization was blocked and reversed by OA, indicating a control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitizing process. Phosphorylation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAMP concentration and kinase inhibitor H89 on the Na+/H+ exchange activity. Based on these data, we proposed that beta NHE can exist in three different states (inactive I, activated A, and desensitized D). Conversion of I to A needs the simultaneous phosphorylation by cAMP-PKA of sites 1 and 2. These two sites might constitute the two neighboring cAMP-PKA sites located on the cytoplasmic loop as deduced from the oligonucleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a K(+)-H+ exchange in trout red blood cells.

1. Exposure of trout red blood cells to beta-adrenergic agonist isoprenaline activates a cAMP-dependent Na(+)-H+ antiport, the movements of protons being compensated by a Cl(-)-OH- (or HCO3-) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl- entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)-H+ exchanger by a beta-antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume-independent K+ efflux, which is thus independent of the Na(+)-H+ exchanger, was not accompanied by a Cl- efflux but was associated with large internal and external pH changes consistent with K(+)-H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)-anion co-transporter in trout red cells, i.e. 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (Dids) and niflumic acid.

Volume-activated Cl(-)-independent and Cl(-)-dependent K+ pathways in trout red blood cells.

1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta-adrenergic-stimulated Na(+)-H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling). In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume-activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl- dependent; after hypotonic swelling the K+ loss is essentially Cl- independent. 2. In order to determine the nature of these volume regulatory pathways (i.e. whether the net K+ loss was conductive or was by electroneutral K(+)-H+ exchange or KCl co-transport), studies were performed to analyse ion fluxes and associated electrical phenomena. The cell membrane potential and intracellular ionic activities of volume-regulating and volume-static cells were measured by impalement with conventional microelectrodes and double-barrelled ion-sensitive microelectrodes. 3. The information gained from the electrical and ion flux studies leads to the conclusion that both Cl(-)-independent and Cl(-)-dependent K+ loss proceed via electrically silent pathways. 4. Experiments were designed to distinguish between electroneutral K(+)-H+ exchange or KCl co-transport. These were based upon the inhibition of Cl(-)-OH- exchange to evaluate the degree of coupling between K+ and Cl- (KCl stoichiometry, pH change). The experimental observations are consistent with the fact that both Cl(-)-independent and Cl(-)-dependent K+ loss are mediated by coupled K(+)-anion co-transport and not by K(+)-H+ exchange. 5. On the basis of previous data, we suggest that only one type of K(+)-anion co-transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.

Arrestin from nucleated red blood cells binds to bovine rhodopsin in a light-dependent manner.

Using a panel of monoclonal antibodies, it has previously been demonstrated that the cytosol of nucleated red cells (trout and turkey) contains a protein similar to arrestin, a soluble protein found so far only in the photosensitive cells and which, by binding to photoexcited rhodopsin, inhibits the phototransduction process. The role of this arrestin-like protein in non-photosensitive cells is questionable. In this report we present evidence that partially purified red blood cell arrestin (RBC arrestin) behaves functionally like bovine retinal arrestin: it binds to phosphorylated bovine rhodopsin only when this receptor has been photoactivated. Thus RBC arrestin and bovine retinal arrestin are closely related both structurally and functionally. By analogy with the function of retinal arrestin, it is proposed that RBC arrestin is involved in desensitization of membrane transport proteins and/or adrenergic receptors.

Effects of anions on the Na(+)-H+ exchange of trout red blood cells.

1. Replacement of chloride by foreign anions in the suspending medium of trout erythrocytes can affect in a complex manner both the activation by catecholamines of the latent Na(+)-H+ exchanger and its subsequent desensitization. These changes are discussed in relation to other cellular modifications (distribution of permeant anions and accumulation of cyclic AMP) induced by foreign anions. 2. The transfer of trout erythrocytes from a chloride-containing medium to media containing lyophilic permeable anions, NO3- or SCN-, immediately induces a decrease of distribution ratios of permeable anions across the red cell membrane (i.e. Donnan ratios). It is probable that the binding of lyophilic anions to haemoglobin, by altering the amount of negative fixed charges, results in changes of distribution of permeant anions across the membrane. 3. The effectiveness of anions in decreasing both the activation of the Na(+)-H+ exchanger and the Donnan ratio follows the same sequence in both cases, i.e., SCN- greater than NO3- greater than Cl- = propionate. It was demonstrated that a change in Donnan ratio affects antiport activity possibly through a shift in intracellular pH; such a mechanism however cannot account for all the effects of foreign anions on antiport activity. 4. The present results show that lyophilic anions do not modify the affinity of the antiporter for sodium ions but greatly decrease the transport capacity of the exchange system. This is interpreted as indicating that the binding of lyophilic anions to some component of the transport system prevents antiporters from establishing their activated configuration once stimulated. Since the inhibitory effect of anions on Na(+)-H+ exchange has been demonstrated in all erythrocytes studied but in no other cell, the crucial substance involved in this inhibition could well be haemoglobin, which appears to control antiport activity in erythrocytes. 5. Some anions affect desensitization of the exchanger. This effect is not related to the lyophilic character of the anion and is not mediated by a change in intracellular cyclic AMP. 6. Propionate and acetate drastically reduce the intracellular level of cyclic AMP and seem to facilitate the activated configuration of the exchanger.