The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific membrane-bound ion pumps [adenosine triphosphatases (ATPases)]. According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important factors for membrane molecular order and herewith a determinant of the degree of non-specific membrane leakiness. Other operative principles, i.e. specific ion channels can be opened and closed according to mechanisms that are specific to the cell. Certain compounds called ionophores can be integrated in the plasma membrane and permit specific inorganic ions to pass. Irrespective of which mechanism ions leak across the plasma membrane the homeostasis may be kept by increasing ion pumping (ATPase activity) in an attempt to restore the physiological ion gradient. The energy source for this work seems to be glycolytically derived ATP formation. Thus an increase in ion pumping is reflected by increased ATP hydrolysis and rate of glycolysis. This can be measured as an accumulation of breakdown products of ATP and end-products of anaerobic glycolysis (lactate). In certain disease entities, the balance between ATP formation and ion pumping may be disordered resulting in a decrease in inter alia (i.a.) cellular energy charge, and an increase in lactate formation and catabolites of adenylates. Cardiac syndrome X is proposed to be due to an excessive leakage of potassium ions, leading to electrocardiographic (ECG) changes, abnormal Tl-scintigraphy of the heart and anginal pain (induced by adenosine). Cocksackie B3 infections, a common agent in myocarditis might also induce an ionophore-like effect. Moreover, Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Perturbation of cellular membranes by the amyloid peptide during the development of Alzheimer's disease is one of several mechanisms proposed to account for the toxicity of this peptide on neuronal membranes. We have studied the effects of the peptide and fragments thereof on 45Ca2+-uptake in human erythrocytes and the energetic consequences. Treatment of erythrocytes with the β1−40 peptide, results in qualitatively similar nucleotide pattern and decrease of energy charge as the treatment with Ca2+-ionophore A23187. Finally, in recent studies we have revealed and published in this journal that a rare condition, Tarui's disease or glycogenosis type VII, primarily associated with a defect M-subunit of phosphofructokinase, demonstrates as a cophenomenon an increased leak of Ca2+ into erythrocytes.
The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific ion pumps. These ion pumps are adenosine triphosphatases (ATPases), which hydrolyse ATP when performing chemiosmotic work, ATP being the chemical energy substrate. These pumps exist as i.a. Ca2+ ATPase , Na+/K+ ATPase , mitochondrial H+ (F1) ATPase (reverse reaction) , and tubulovesicular H+/K+ ATPase . According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. Pro primo there is a diffuse ion leakage across the membrane along the gradient. The ability of the membrane to be ‘ion tight’ is dependent on a number of factors. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important for membrane molecular order and herewith a determinant of the degree of membrane leakiness . Pro secundo other operative principles, i.e. specific ion channels can be opened and closed according to principles that are specific to the cell .
The ATP used for fuelling the ion pumps is believed to be derived from glycolysis via glycolytic enzymes in a membrane compartment. This means that ATP is synthesized in the vicinity of the ion pumps . A high ion pump activity implicates an increased ATP turn over based on glycolysis . This is reflected by an increase in cellular lactate production as well as an increase in the degradation products of ATP (ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and orthophosphate). The expenditure of energy on maintenance of ion gradients is in the range of 30–70% of energy production of different cells .
Glycolytic enzyme associations in other cells
Studies on other cells and tissues indicate that glycolytic enzymes form reversible associations with the plasma membrane and the cytoskeleton and that these interactions modulate enzyme activities . The association of these enzymes with the plasma membrane containing the ‘pump’ ATPases may therefore be similar to that of the red blood cell. This juxtaposition to the ATPases is important for rapid generation of ATP through glycolysis. Hence, glucose metabolism in membrane-associated compartments of other cellular systems has been demonstrated which is related functionally to the activity of membrane ion pumps or channels . There is strong evidence that in vascular smooth muscle, ATP is synthesized by a plasma membrane-associated glycolytic cascade which is employed specifically to drive the Na+- and K+-dependent ATPase . Particularly notable to this end is the report by Weiss and Lamp , who claimed that ATP-dependent K+-channels in cardiac myocytes were more strongly linked to glycolytic than oxidative metabolism. Isolated skeletal muscle was found to contain a compartmentalized glycolytic reaction sequence leading to synthesis of ATP . What was more, the ATP formation occurred transiently and appeared to be kinetically compartmentalized, i.e. the synthesized ATP was not in equilibrium with the bulk ATP . Similarly, a correlation is evident between glycolysis and function in the plasma membrane of cells from the brain  and the kidney . A compartmentalized energy metabolic pool has also been claimed to exist in cultured cells [17, 18]. By applying radiolabelled inorganic phosphate to the incubation medium of intact metabolizing erythrocytes, it was discovered that intracellular ATP became radiolabelled before intracellular inorganic phosphate [19, 20]. This labelling pattern occurred because extracellular phosphate was channelled from the extracellular medium via the anion transporter (Band 3) to glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase for substrate-level generation of ATP, thus demonstrating a functional interaction between Band 3 and glyceraldehyde 3-phosphate dehydrogenase . These observations strengthen the argument of a membrane-compartmentalized ATP pool which is not in equilibrium with the major cellular ATP pool.
Membrane ion channels
Ion channels are membrane spanning proteins which allow an ion flux across the cell membrane by forming internal water-filled pores . Ion channels may be formed from the assembly of short peptides, but long enough to span the lipid bilayer. However, even shorter peptides can be involved after dimerization to fully transverse the bilayer. These ion channels constitute a class of proteins that is ultimately responsible for generating and arranging the electrical signals passing through the activated brain, the beating heart and the contracting muscle. Rojas et al.  reported that patients were periodically paralysed by eating bananas, which are rich in potassium, due to mutations in the sodium channels of muscle. Subsequently, other inherited disorders caused by defects in voltage-sensitive channels were described, including those affecting quarter horses with hyperkalaemic periodic paralysis [23, 24] because of substitution of a single amino acid in the sodium channel. In pigs, a single amino acid alteration in a calcium channel in muscle resulted in a high incidence of malignant hyperthermia . Hence, the occurrence of inherited disorders as a result of defects in voltage-sensitive channels could be used as a tool in efforts to increase the knowledge about ion fluxes and cell energy metabolism.
Experimental testing of clinical disease
Characteristics of ionophores
Ionophores can be used as an experimental tool to gain further knowledge about the effects of permeability changes of membranes to specific ions. They are small hydrophobic molecules that dissolve in lipid bilayers and increase their permeability to specific inorganic ions. Most are synthesized by microorganisms and some have been used as antibiotics. There are two types of ionophores, mobile ion carriers and channel formers. Both of them work by shielding the charge of the transported ion so that it can penetrate the hydrophobic barrier of the lipid bilayer. The ionophores are not primarily coupled to energy metabolism and therefore, they permit net movement of ions only down their electrochemical or chemical gradients. The ionophore A23187 is a representative example of a mobile ion carrier transporting divalent cations such as Ca2+ and Mg2+. The ionophore acts as an ion-exchange shuttle, carrying two H+ out of the cell for every divalent cation that is translocated into the cell. Accordingly, when cells are exposed to the ionophore A23187, Ca2+ enters the cell interior along a steep chemical gradient . Valinomycin is another example of a mobile ion carrier. It is a ring-shaped polymer that increases the permeability of a biological membrane to K+. Valinomycin transports K+ down its electrochemical gradient by picking up K+ on one side of the membrane, diffusing across the lipid bilayer, and releasing K+ on the other side . Gramicidin is a representative of a channel-forming ionophore. It is a polypeptide of 15 amino acids. Unlike the polypeptides of proteins, gramicidin consists of l- and d-amino acids in strict alternation. When inserted into a membrane, the gramicidin polypeptide assumes a helical shape, with hydrophobic groups on the outside in contact with membrane phospholipids. Polar groups are on the inside of the helix, forming a 0.4-nm channel down the centre of the helix. A single gramicidin helix spans only one-half of the membrane. When a helix in one monolayer lines up with another such helix in the opposite monolayer, the two polypeptides associate end to end, forming a continuous channel that spans the membrane .
The leak and pump phenomenon of interest was supposed to be confined to the plasma membrane. Therefore, we chose mainly a unicellular, simplified system for our ionophoric studies on cellular energy metabolism, viz. the human red blood cell. This cell is devoid of other organelles except the plasma membrane, the organelle to be studied in its whole context, i.e. the intact cell. The representativity of the erythrocyte plasma membrane has proven useful in other situations, e.g. the sarcolemma defect in Duchenne's muscle dystrophy is also found in the erythrocyte membrane of these patients [27, 28].
Ionophore A23187 in incubation medium within 40 min induced a decrease by about 75% of ATP content and energy charge (EC) and a concomitant rise in adenosine diphosphate (ADP) and adenosine monophosphate (AMP) contents in a dose-dependent fashion. A transition was noted for the ionophore concentration at 2–3 μmol L−1, where the concentration of ADP exceeded that of ATP . Calmidazolium, an inhibitor of the Ca2+-dependent ATPase of red blood cells  counteracted the effects on ADP and AMP concentrations but with only marginal improvement of ATP and EC-values .
An improving effect on energy metabolism of red blood cells subjected to the ionophore was obtained by inclusion of 5 mmol L−1 glucose in incubation medium illustrating the association between the ionophore action and energy metabolism. Adding ethylene glycol-bis N,N,N′,N′-tetraacetic acid (EGTA) (a chelator of Ca2+) to the incubation medium abrogated the effect of A23187 on energy status pointing to the fact that Ca2+-movement was the working principle. The ionophore was influential on lactate production of red blood cells only in presence of added glucose to the medium resulting in an hyperbolic curve profile of lactate over time in contrast to linearity which was obtained in the absence of the ionophore. Hence, the hyperbolic curve profile reflecting lactate production in presence of ionophore and glucose indicated an enhanced glycolytic flux compatible with the view that an increased calcium ion pumping was the underlying cause. However, the increased intracellular concentration of Ca2+ may have different actions besides those on the Ca2+-stimulated ATPase.
Several enzymes, especially the kinases of glycolysis are Mg2+-dependent and inhibited by Ca2+, e.g. pyruvate kinase. Therefore, at a certain intracellular Ca2+-load, glycolysis will diminish and thereby lactate production. Accordingly, supplementing incubation medium with 5 mmol L−1 KCl and 3 mmol L−1 MgCl2 together with glucose and ionophore will preserve energy state over a prolonged time period . Further, the lactate production was maintained linearly with time and clearly augmented . The presence of ouabain in such an incubation system led to continued stable conditions regarding energy state (as ouabain is a specific inhibitor of Na+/K+ ATPase and not involved in the operation of Ca2+ ATPase) and an effect on lactate production was hardly discernible during the first 90 min of incubation demonstrating neutrality of the Na+- and K+-dependent ATPase under these conditions .
The monovalent cationophore gramicidin D, in a concentration of 10 mg L−1 in phosphate-buffered saline with 5 mmol L−1 KCl and 3 mmol L−1 MgCl2 resulted in a lowered EC to about 0.50 and ATP content was reduced by more than 50%. A concomitant increase of AMP and ADP values was seen as well . Gramicidin D in as low a concentration as 5 mg L−1 resulted in an almost linear decrease of EC and ATP content together with a linear increase of AMP content during the first 60 min of incubation whilst ouabain inclusion distinctly blunted the ionophoric effects on EC value and adenine nucleotide contents . Hence, the gramicidin D effect was exerted via an enhanced Na+- and K+-dependent ATPase activity resulting in an increased decay of ATP which was typically kept prevented by ouabain . The addition of 5 mmol L−1 glucose abrogated the ionophoric effect on energy metabolism by about 80% whilst at the same time the lactate production was heavily increased . Accordingly, ion fluxes, ATPase activation and glycolysis are tightly coupled to each other . However, it is not elucidated whether the Na+- and K+-dependent ATPase is the controlling principle of glycolysis or whether the latter determines the rate of ion pumping.
To increase the complexity even further, there are reasons to believe that a membrane metabolic pool exists that may display a high metabolic turnover in close association to the Na+- and K+-dependent ATPase [7, 32]. Glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase are erythrocyte membrane-bound enzymes that may be involved in membraneous ATP formation . Moreover, the Na+- and K+-dependent ATPase of human erythrocytes has likewise been implicated in either direct or indirect interactions with glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase individually [34, 35].
In addition to direct association between ATP synthesizing and hydrolysing enzymes, there is also evidence for enzyme–substrate–enzyme complexes where the substrate is the link between two enzymes. Kinetic evaluation has demonstrated that the complex between glyceraldehyde 3-phosphate dehydrogenase and 1,3 bisphosphoglycerate is formed during catalysis of high energy bonds . Likewise, a lactate dehydrogenase–[nicotinamide adenine dinucleotide (reduced form) (NADH)]–glyceraldehyde 3-phosphate dehydrogenase interaction can be demonstrated . These types of associations between enzymes have substantial implications for the metabolic efficiency of the cell, both through compartmentation by steric proximity in a membraneous context and by substrate bridging between enzymes. Hence, a coupling may exist between ATP formation and ATP hydrolysis regardless of what type of ion pump being involved in the human erythrocyte membrane . This concept is envisaged in Fig. 1. Based on these in vitro results from experiments on erythrocytes we carried out ex vivo experiments using isolated perfused rat hearts to demonstrate ionophoric effects of valinomycin on myocardial metabolism as well as in vivo experiments on Coxsackie B infected mice [39, 40]. In both situations, we found signs of an increased glycolytic flux combined with decreased EC in accordance with the hypothesis.
Microcalorimetry has proved suitable for studying overall metabolism in healthy and diseased conditions [41, 42]. The technique allows the use of experimental conditions close to physiological ones, with freshly prepared and intact cells suspended in their own plasma or in a defined medium. Because of the high sensitivity of the technique, it is possible to quantify small changes in metabolic rate accurately. Using this technique the impact of the Ca2+-ionophore A23187 on red blood cell energy metabolism has been studied . In typical microcalorimetric experiments the time–power curve profiles of erythrocytes exposed to ionophore A23187 were different from those not exposed to the ionophore . When the ionophore was added, the first event was assumed to be an inward Ca2+-current leading to a dissipation of the Ca2+-gradient. This event was in fact mirrored by an early peak of heat production . The descending part of the peak may be interpreted as a retardation of the influx rate of calcium ions (Fig. 2). This conforms to the idea that the calcium ion uptake in human erythrocytes consists of two components, a fast component followed by a slow one . The subsequent levelling off, reaching a steady state, is consistent with heat production mainly due to glycolytic metabolism and increased ion pumping in order to compensate for the dissipation of the ion gradient . Hence, we may here recognize an uncoupling of the ATPase function leading to heat production without any gain in ATP formation . Such an uncoupling mechanism may be behind the phenomenon earlier described in pigs with a single amino acid alteration in a calcium channel of myocytes ending up also in a high incidence of malignant hyperthermia .
Relation to pathophysiology
During the process of virus infection, alteration of the membrane takes place in that several properties in membrane permeability are modified during virus entry. This results in the appearance of new surface antigens on the plasma membrane and changes in the physical state and composition of the lipid bilayer, that normally lead to increased membrane fluidity [45, 46]. Hence, a change in K+ permeability occurs within minutes after addition of Sendai virus to cells [47, 48]. It seems reasonable that K+ leakage is accompanied by an increase in sodium permeability, leading to an influx of Na+ into the cell . Increased permeability of divalent cations has also been observed early during animal virus infection , and permeability to Ca2+ in Sendai virus-infected cells changes at about the same time as the modifications in monovalent cations are detected . The plasma membrane is a key structure for the maintenance and regulation of cellular processes. A corollary is that a change in its function would have repercussions on cellular metabolism.
Some patients with symptoms of angina pectoris show normal coronary angiograms despite ECG findings and myocardial perfusion scintigrams indicative of ischaemic heart disease. The negative angiography has not been given a conclusive explanation by some investigators, whilst others claim that the negative findings would be due to ‘small vessel disease’ that can not be demonstrated by conventional techniques. Whether this proposed perfusion disturbance is also combined with ischaemic metabolism in causing the chest pains is not at all clear. This syndrome denoted syndrome X  (not to be confused with the metabolic syndrome with insulin resistance) has been known for the last 30 years. Published studies so far are based on conventional assumptions that there is indeed a latent perfusion defect at hand with reduced dilatative properties of coronary arteries (reduced coronary reserve) and theories on pathological microperfusion [51, 52].
Tissue ischaemia results in depressed cellular ATP synthesis. A possible underlying ischaemic cause for the angina pectoris-like chest pain in syndrome X would accordingly lead to diminished cellular ATP production and thereby generate the well-known imbalance between energy supply and energy demand. In this contradictory situation with scintigram and ECG findings favouring the diagnosis of myocardial ischaemia despite a normal coronary angiogram, it might be wise to question the specificity of the analytical methods without preconceived ideas. The depressed ST-segment of the ECG during ischaemia is considered to be due to an altered potassium homeostasis. The same mechanism could well be the explanation for the ‘ischaemic’ perfusion scan when thallium is used as a tracer, as monovalent thallium compounds share many properties with potassium compounds and thallium can accordingly distribute as a function of the concentration of potassium ions. The ion gradients across the plasma membrane are normally maintained by ion pumps fuelled by ATP. Because the ATP-producing ability of the cell is reduced during ischaemia, this results in an acute lack of substrate for the ion pumps which in turn results in increased net fluxes of Na+- and K+ along their gradients, thereby increasing intracellular Na+ and extracellular K+ concentrations.
In the absence of any proof of ischaemia being the cause of syndrome X, we have tried to find alternative explanations of the symptoms and of the results of the clinical examinations. The presence of extracellular K+ and intracellular Na+ is necessary for ion pump activity and increased amounts of intracellular Na+ will activate the Na+- and K+-dependent ATPase further. Theoretically, an excessive leak of monovalent cations in the absence of ischaemia and with a normal cellular production of ATP could lead to a situation whereby the cell reaches the point where Na+- and K+-dependent ATPase activity will be stimulated to such an extent that the ATP regeneration machinery would not catch up with the rate of ATP decay. Accordingly, the existence of an ionophore- or ionophore-like mechanism may be the underlying cause of syndrome X .
Moreover, some of the patients eventually developing the typical features of chronic fatigue syndrome, will start with an acute and severe chest pain, so severe that the diagnosis of myocardial infarction, myocarditis, or Bornholm disease will be considered . Hence, these patients appear to fall into the category of those with syndrome X. Abnormalities in cardiac thallium scans of these patients with chronic fatigue syndrome have been identified to be similar to those found in syndrome X  and a common aetiological basis for the two conditions seems reasonable.
The interaction of peptides with lipids involves a number of non-covalent interactions . Hydrophobic interactions occur between lipid acyl chains and hydrophobic residues of the peptide, whilst electrostatic interactions take place between the polar residues of the peptide, the phospholipid head groups and the solvent molecules. As shown by infrared spectroscopic measurements , most lipid-associating peptides adopt an α-helical conformation when associated with the lipids, although some peptides can exist as β-sheets , especially under a self-associated or aggregated form. Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Genetic causes of Alzheimer's disease include mutations in the amyloid precursor protein, presenilin 1 and presenilin 2 genes .
The major component of brain amyloid plaques is a 38–42 residue peptide termed amyloid β-protein , which is a proteolytic product of the amyloid precursor protein . Data have been provided giving further support to the concept that cell death in Alzheimer's disease may be due to amyloid ion-channel activity . Deposits are formed not only in Alzheimer's disease but also in prion diseases, and there is homology between certain protein sequence domains that are believed to be involved in the two processes [62, 63]. Perturbation of cellular membranes by the amyloid peptide during the development of Alzheimer's disease is one of several mechanisms proposed to account for the toxicity of this peptide on neuronal membranes . The amyloid β-protein nucleates rapidly into amyloid fibrils .
In order to obtain further insight into the mode of interaction of amyloid β-protein with the plasma membrane, we have studied the effects of the β-protein and fragments thereof on 45Ca2+-uptake in human erythrocytes and the energetic consequences. Treatment of erythrocytes with the β1−40 peptide, which is derived from a larger amyloid precursor protein [65, 66], results in an increased Ca2+-leakage and qualitatively similar nucleotide pattern and decrease of EC as the treatment with ionophore A23187. It means significant reductions in EC-value and in ATP concentration as well as an increase in AMP concentration.
Similar alterations are also possible to bring about by the two fragments β1−28 and β25−35 . Also, an effect of the β1−40 and its two fragments is obvious on 45Ca-uptake in human erythrocytes and the effect is similar to the one obtained by the ionophore A23187 (Fig. 3). There is thus evidence that the Alzheimer β-peptide interacts with the plasma membrane of human erythrocytes. By doing so, the ionophoric properties may be exposed, leading to an increase of the intracellular Ca2+-concentration in turn resulting in enhanced Ca2+-dependent ATPase activity with influences on intracellular adenylates and decrease in EC . Such an ionophoric action by Alzheimer-related amyloid peptides has also been demonstrated in human hNT-cells displaying neurone-like calcium channel activation . Hence, this ionophoric action of the Alzheimer β-peptide may be valid for neuronal plasma membranes and may accordingly be an etiological factor in Alzheimer's disease. Hence, a mutant amyloid protein associated with Alzheimer's disease will form morphologically indistinguishable annular protofibrils that resemble a class of pore forming bacterial toxins, suggesting that inappropriate membrane permeabilization might be the cause of cell dysfunction and even cell death in Alzheimer's disease.
Inherited deficiency of the M-subunit of phosphofructokinase (PFK) in man is associated with myopathy and/or haemolysis or an asymptomatic state . The most common type glycogenosis VII or Tarui's disease is characterized by the co-existence of muscle disease and moderate haemolysis . We discovered different lines of evidence that the disease not only consisted of the well-described PFK-M deficiency manifested by muscle fatigue and haemolysis but also of an additional defect localized in the plasma membrane of erythrocytes. Findings of an enhanced hydrolysis of ATP, combined with an increased glycolytic flux suggested an accelerated ATP-dependent Ca2+-extrusion machinery due to an increased membrane leakiness of Ca2+ in these cells. This membrane leakiness should be understood as a co-phenomenon to the PFK-M deficiency [71, 72]. This co-phenomenon, leading to Ca2+-induced membrane stiffness, i.e. decreased deformability results in enhanced haemolysis in reticuloendothelial system (RES). Accordingly, the enhanced erythrocyte haemolysis in Tarui's disease is not the result of the modest decrease of PFK activitiy but rather due to enhanced leakage of Ca2+.
Excess leakage of ions across the plasma membrane seems to be a major component of the pathophysiology of certain diseases such as syndrome X, Tarui's disease and Alzheimer's disease (and possibly also prion disease). The properties and function of the plasma membrane in this respect have hitherto been little discussed as a basis for disease.