A transport metabolon. Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.     

Chloride--bicarbonate exchange in red blood cells: physiology of transport and chemical modification of binding sites

About 80% of the CO2 formed by metabolism is transported from tissues to lungs as bicarbonate ions in the water phases of red cells and plasma. The catalysed hydration of CO2 to bicarbonate takes place in the erythrocytes but most of the bicarbonate thus formed must be exchanged with extracellular chloride to make full use of the carbon dioxide transporting capacity of the blood. The anion transport capacity of the red cell membrane is among the largest ionic transport capacities of any biological membrane. Exchange diffusion of chloride and bicarbonate is nevertheless a rate-limiting step for the transfer of CO2 from tissues to lungs. Measurements of chloride and bicarbonate self-exchange form the basis for calculations that demonstrate that the ionic exchange processes cannot run to complete equilibration at capillary transit times less than 0.5 s. The anion exchange diffusion is mediated by a large transmembrane protein constituting almost 30% of the total membrane protein. The kinetics of exchange diffusion must depend on conformational changes of the protein molecule, associated with the binding and subsequent translocation of the transported anion. We have characterized the nature of anion-binding sites facing the extracellular medium by acid-base titration of the transport function and modification of the transport protein in situ with group-specific amino acid reagents. Anion binding and translocation depend on the integrity and the degree of protonation of two sets of exofacial groups with apparent pK values of 12 and 5, respectively. From the chemical reactivities towards amino acid reagents it appears that the groups whose pK = 12 are guanidino groups of arginyl residues, while the groups whose pK = 5 are likely to be carboxylates of glutamic or aspartic acid. Our studies suggest that the characteristics of anion recognition sites in water-soluble proteins and in the integral transport proteins are closely related.     

Plasma membrane Cl-/HCO3-exchangers: Structure,mechanism and physiology

Anion exchanger proteins facilitate the exchange of bicarbonate for chloride across the plasma membrane. When bicarbonate combines with a proton it undergoes conversion into CO2, either spontaneously, or catalyzed by carbonic anhydrase enzymes. The CO2/HCO3- equilibrium is the body’s central pH buffering system. Rapid bicarbonate transport across the plasma membrane is essential to maintain cellular and whole body pH, to dispose of metabolic waste CO2, and to control fluid movement in our bodies. Cl-/HCO3- exchangers are found in two distinct gene families: SLC4A and SLC26A. Differences in the tissue distribution, electrogenicity, and regulation of the specific anion exchanger proteins allow for precise regulation of bicarbonate transport throughout the human body. This review provides a look into the structural and functional features that make this family of proteins unique, as well as the physiological significance of the different anion exchangers.     

Effects of darbepoetin injections on erythrocyte membrane transport protein expressions in humans.

The present study investigated the effects of injected darbepoetin [novel erythropoietin stimulating protein (NESP)] on the density of three erythrocyte membrane transport proteins: the lactate-H+ cotransporter (monocarboxylate transporter 1), the chloride/bicarbonate exchanger 1 (anion exchanger 1), and the water channel aquaporin 1. Thirteen subjects were injected with NESP once a week for 4 wk. Blood samples were obtained before, during, and after the injection period, and the erythrocyte transport proteins were determined by Western blotting. The NESP injections induced a transient increase in hematocrit, red cell volume, and reticulocyte fraction. The density of aquaporin 1 protein was higher (maximal increase +59%) (P < 0.01) during the injection period compared with the preinjection value and lower (P < 0.01) after the injection period. The density of anion exchanger 1 protein was higher (maximal increase +15%) (P < 0.05) during the injection period compared with the preinjection value and tended (P = 0.06) to be lower after the injection period than before the injection period. The density of the erythrocyte monocarboxylate transporter 1 protein was higher (maximal increase +43%) (P < 0.05) during the injection period than in the preinjection period. Age separation experiments using self-creating Percoll gradients demonstrated a higher density of membrane transport proteins in young red blood cells. These data suggest that the NESP-induced increase in membrane transport proteins is caused by a higher fraction of newly formed erythrocytes (and reticulocytes), which have a higher density of membrane transport proteins. However, increased incorporation of membrane proteins during erythrocyte formation may also be involved. We suggest that NESP improves the quality of erythrocyte membrane transport through these mechanisms.     

Slc4-like anion transporters of the larval mosquito alimentary canal

Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH>10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na(+)-independent chloride/bicarbonate anion exchanger (AE) as well as a Na(+)-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.     

Anion exchanger 1: Protean function and associations

Anion exchanger 1 (AE1) is the most abundant protein on the erythrocyte membrane and is also present on the basolateral surface of the alpha intercalated cell in the distal nephron. Mutations can cause either hereditary haemolytic red cell diseases, or hereditary distal renal tubular acidosis. Classically it mediates the electroneutral exchange of chloride for bicarbonate, as well as comprising an important mechanical component of the red cell membrane. It is increasingly recognised that it plays many other roles too: alternative anion transport, such as sulphate transport and proton and sulphate symport, associations with other erythrocyte membrane proteins as part of the AE1 macrocomplex, regulation of glycolysis and more recently cation transport through the so-called ‘leak’ pathway. These new functions and associations are reviewed in health and disease, and the role of AE1 as a putative regulator of cell volume is discussed.     

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

Anion exchangers in the red cell and beyond.

The anion exchanger genes (AE1-3) encode a family of transport proteins responsible for the electroneutral exchange of bicarbonate and chloride across membranes. These transporters are important in processes such as pH regulation and bicarbonate metabolism. This article reviews recent progress in this field based on presentations made at a satellite workshop on anion exchangers held in conjunction with the 8th Fisher Winternational Symposium on Cellular and Molecular Biology entitled "Membrane proteins in health and disease." The transmembrane topology of AE1 has been refined using various combinations of protein chemistry and site-directed mutagenesis. The use of specific inhibitors and novel expression systems continues to reveal fundamental features of the anion exchanger mechanism and its regulation. The importance of anion exchangers in blood and kidney diseases is underscored by the identification and characterization of a plethora of novel mutations in the AE1 gene. Investigations of anion exchanger structure and function have moved beyond studies of the red cell protein into the larger arenas of cellular and molecular biology.     

Is CFTR an exchanger?: Regulation of HCO3−Transport and extracellular pH by CFTR

The disease, cystic fibrosis, is caused by the malfunction of the cystic fibrosis transmembrane conductance regulator. Expression of functional CFTR may normally regulate extracellular pH via control of bicarbonate efflux. Reports also suggest that the CFTR may be a Cl-/HCO3- exchanger. If true, this could be very important for treatment of CF given the airway host defense system is quite sensitive to pH, and acidic pH been found to increase mucus viscosity. We compared evidentiary support of four possible models of CFTR's role in the transport of bicarbonate: 1) CFTR as a Cl-channel that permits bicarbonate conductance, 2) CFTR as an anion Cl-/HCO3- exchanger (AE), 3.) CFTR as both a Cl-channel and an AE, and 4.) CFTR as a Cl-channel that allows for transport of bicarbonate and regulates an independent AE. The effect of stimulators and inhibitors of CFTR and AEs were evaluated via iodide efflux and studies of extracellular pH. This data, as well as that published by others, suggest that while CFTR may support and regulate bicarbonate flux it is unlikely it directly performs Cl-/HCO3- anion exchange.     

A new member of the HCO3(-) transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (alpha-type) and bicarbonate-secreting (beta-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H(+)-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the alpha- and beta-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of beta-intercalated cells is the band 3 protein localized to the basolateral membrane of alpha-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of beta-intercalated cells. Cloned cDNA from the beta-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of beta-intercalated cells, although some beta-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the beta-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.     

A Substrate Access Tunnel in the Cytosolic Domain Is Not an Essential Feature of the Solute Carrier 4 (SLC4) Family of Bicarbonate Transporters

Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na⁺-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg²⁸³) and at an interacting glutamate (Glu⁸⁵) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-Å resolution crystal structure of cdΔ54AE1 (residues 55–356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg²⁸³ and Glu⁸⁵. Mutations at these residues affected the pH-dependent conformational changes and stability of cdΔ54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.     

Mini Review

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane [Formula: See Text] anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 [Formula: See Text] transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and [Formula: See Text] co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) [Formula: See Text] exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."     

The bicarbonate transport metabolon

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."     

Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.     

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell type-specific and developmentally regulated expression of the AE1 anion exchanger in the chicken chorioallantoic membrane

Antibodies specific for the chicken AE1 anion exchanger have been used to determine the cell-type specific pattern of expression of this electroneutral transporter in the chick chorioallantoic membrane (CAM) during embryonic development. Immunolocalisation analyses demonstrated that the AE1 anion exchanger accumulated in the basolateral membrane of a subset of cells in both the chorionic and allantoic epithelial layers. Double immunostaining indicated that the AE1-positive cells in the chorionic and allantoic epithelia were also positive for the carbonic anhydrase isoform, CAII, which serves as a marker for the villus cavity (VC) cells of the chorionic epithelium and the mitochondria-rich cells of the allantoic epithelium. Immunoelectron microscopy revealed that AE1 accumulated in extensive projections that extended from the lateral membrane of VC cells towards the adjacent capillary covering cells. These results represent the first demonstration of anion exchanger expression in the chick CAM, and they suggest a role for basolateral AE1 in bicarbonate reabsorption that is required in the embryo for maintaining acid-base balance during development.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.