An unusual pattern of Na+ and K+ movements across the horse erythrocyte membrane

Marked differences in the activities of three monovalent cation transport systems in horse versus human erythrocytes are reported. Whereas horse erythrocytes exhibit a 6-fold higher sodium-lithium countertransport, the unidirectional flux of potassium through the sodium pump is 3-4 times slower and the sodium-potassium cotransport system cannot be detected. In spite of this, horse and human cells are able to maintain similar Na+ and K+ gradients.     

Acute effects of angiotensin-converting enzyme inhibitor on erythrocyte sodium ion transport in essential hypertension.

The acute effects of angiotensin-converting enzyme inhibitor, captopril, on sodium ion transport systems were investigated in essential hypertensive and normotensive subjects. The passive sodium efflux through the erythrocyte membrane was significantly higher and erythrocyte sodium-potassium cotransport was lower in patients with essential hypertension when compared with normal subjects. However, sodium-potassium pump activity and sodium-lithium countertransport did not differ significantly between the hypertensive patients and the normal subjects. Immediately after captopril administration, erythrocyte passive sodium efflux and sodium-potassium cotransport returned to normal levels in the hypertensive subjects. Although the plasma renin activity and plasma aldosterone concentration were altered by captopril, they did not correlate with changes in any sodium transport system. These results suggest that the changes in sodium transport systems which occur immediately after captopril administration may contribute, at least in part, to its antihypertensive action.     

Ion transport proteins and aquaporin water channels in the kidney of amphibians from different habitats

Amphibians are known to spend part of their life on land and return to water to reproduce. However, some urodeles spend their entire life in water, while others succeed in completely avoiding water even during reproduction. Osmoregulatory mechanisms must therefore be different in the diverse environmental conditions of their respective life histories. The architecture of the kidney is similar in all amphibians; as a consequence the ion-water equilibrium must be regulated in the different environmental conditions. We investigated the immunolocalisation of Na(+)/K(+)/Cl(-) cotransport proteins, sodium pump and water-channel proteins (aquaporins) in aquatic Amphiuma means means, Rana dalmatina, a species that returns to water to reproduce, and Speleomantes genei, a completely terrestrial species. The investigation was carried out with immunohistochemical methods using antibodies to Na(+)/K(+)/Cl(-) cotransport protein NKCC1 T4, Na(+)/K(+)ATPase alpha-subunit, water-channel aquaporin 3 and the inner mitochondrial membrane (AMA). Cotransport proteins and sodium pump, involved in ion reabsorption, are widely distributed in A. means and R. dalmatina and confined to the distal segment in S. genei; conversely water channels, involved in water reabsorption, are limited to the collecting duct in A. means and R. dalmatina and distributed in the proximal and collecting ducts in S. genei.     

Induction of the ATP-dependent proteolytic system in guinea pig reticulocyte lysates by triiodothyronine

The mechanism involved in the decreased numbers of several trans-membrane proteins such as sodium pump sites, sodium-lithium countertransport, sodium potassium cotransport proteins, proteins mediating the passive efflux of sodium and insulin receptors in erythrocytes from patients with hyperthyroidism is not known. The ATP-dependent proteolytic system which is involved in the loss of trans-membrane proteins during the maturation of the reticulocyte may be involved in the accelerated loss of these membrane proteins. Therefore, the effect of thyroid hormones on the ATP-dependent proteolytic activity of reticulocyte lysates was examined in this study. Reticulocytosis was induced in 14 guinea pigs by phenylhydrazine hydrochloride injections for 5 consecutive days followed by 2 days of rest. T3 (10 micrograms/100 g body weight) was injected into 7 animals on day 4 and day 6. Reticulocyte-rich blood was withdrawn on day 8. Oxygen consumption determined 24 hours after injection of T3 was 25% higher (p less than 0.01) and T3 treated animals had a 2.5 fold higher (p less than 0.01) weight loss than control animals. The ATP-dependent proteolytic activity measured in reticulocyte lysates using 125I labelled lysozyme was 3.6 fold higher in the T3 than in the control group of guinea pigs (p less than 0.01). We conclude that thyroid hormones induce the ATP-dependent proteolytic activity of reticulocyte lysates which may be responsible for the reduced number of several trans-membrane proteins found in erythrocytes from patients with hyperthyroidism.     

Cationic concentrations and transmembrane fluxes in erythrocytes of humans during exercise

The effect of exercise on the intraerythrocyte cationic concentrations and transmembrane fluxes such as the Na+-K+-adenosinetriphosphatase (ATPase) pump, the Na+-K+ cotransport, and the Na+-Li+ countertransport system was studied in 11 normal male volunteers. All subjects performed an uninterrupted incremental exercise test on a bicycle ergometer, starting at an initial work load of 20% of the subjects' maximal exercise capacity, as determined in a pretest. The work rate was increased with an additional 20% each 6 min up to a final work load of 80%. Blood samples were taken at rest, at 60 and 80% of maximal exercise capacity, and 1, 2, 3, 4, 5, and 30 min after cessation of exercise. At moderate exercise (60% of maximal exercise capacity) the intraerythrocyte potassium concentration was not changed, but at severe exercise (80% of maximal exercise capacity) it was decreased. After exercise the intraerythrocyte potassium concentration returned to base line within 2 min. Exercise did not affect the intraerythrocyte concentrations of sodium and magnesium. The activity of the Na+-K+-ATPase pump and the Na+-K+ cotransport in the erythrocytes during and after exercise was no different from the resting level. The activity of the Na+-Li+ countertransport system on the contrary tended to decrease during exercise. It is concluded that exercise is accompanied by a leakage of potassium out of the erythrocytes without major alterations in the active red cell cationic fluxes.     

Cotransport of lithium and potassium in human red cells

This paper reports the presence of human red cells of an additional ouabain-insensitive transport pathway for lithium ions, the Li-K cotransport. Several kinds of observations support this conclusion. Cells loaded to contain only K, Na, or Li do not exhibit furosemide- sensitive efflux. Simultaneous presence of K and Li on the same side of the membrane mutually stimulates furosemide-sensitive Li and K fluxes from that side. Cells loaded with both Na and Li exhibit no furosemide- sensitive Li efflux. Thus, Li can apparently replace Na but not K on the outward Na-K cotransport system in human red cells. Furthermore, Lio, like Ko, inhibits outward Na-K cotransport. Additional proof for coupled Li-K cotransport is provided by the observation that an outwardly directed K electrochemical potential gradient can drive net outwardly directed K electrochemical potential gradient can drive net outward Li movement against its gradient. There are several differences between Li-K cotransport and Li-Na countertransport. The cotransport system has an apparent affinity for Li that is about one-half that for Na and 30 times lower than the counter-transport system. Furosemide and chloride replacement inhibit cotransport but do not affect countertransport. The PCMBS loading procedure irreversibly inhibits countertransport but not cotransport. Furthermore, the two systems can apparently function at maximal rates simultaneously. Present evidence, than, indicates that the two pathways can be separated operationally as two different systems.     

Genetic variation in the beta-3-adrenergic receptor in essential hypertension

We investigated the role of the beta-3-adrenergic receptor polymorphism in membrane lipid composition and erythrocyte membrane sodium transport in essential hypertensive patients. We studied 87 essential hypertensive patients determining: The Trp64Arg mutation of the beta-3-adrenergic receptor by PCR, lipoprotein profile by standard laboratory methods, membrane lipid composition by IATROSCAN and erythrocyte sodium lithium countertransport by Canessa technique. Patients with the mutation as compared with those without it showed lower membrane cholesterol, membrane cholesterol phospholipids ratio and erythrocyte sodium lithium countertransport, however blood pressure and the other studied variables were similar in both groups of patients. After adjusting by sex sodium lithium countertransport activity remained significant. These data suggest that although the Trp64Arg mutation of the beta-3-adrenergic receptor is related with a different membrane lipid composition and erythrocyte sodium lithium countertransport values it does not contribute to blood pressure levels in essential hypertensive patients.     

Intracellular sodium concentration and transport in red cells in essential hypertension, hyperthyroidism, pregnancy and hypokalemia

Intracellular sodium content ([Nai]), ouabain-sensitive ('Na-K ATPase') and ouabain-insensitive ('passive permeability') sodium efflux, Na-K cotransport and Na-Li ('Na-Na') countertransport were estimated in erythrocytes in 39 control subjects, 20 patients with essential hypertension, 14 patients with hypokalemia of renal or unknown etiology, 13 hyperthyroid patients and 19 pregnant women. In normokalemic essential hypertension there was only a moderate, but significant elevation of the activity of the Na-Li countertransport system. In the group of patients with hypokalemia, there was a significant increase of [Nai], ouabain-insensitive sodium efflux and Na-Li countertransport. In hyperthyroidism, a marked decrease of Na-Li countertransport was associated with a marked elevation of [Nai], in pregnancy an elevation of the Na-Li countertransport with a [Nai] 43% lower than the control values. The ouabain-sensitive sodium efflux was elevated in hyperthyroidism and hypokalemia, in which [Nai] was increased. In the control subjects there was a positive linear correlation between ouabain-sensitive sodium efflux and [Nai]. The sodium component of the Na-K cotransport was decreased to about one third of the unchanged furosemide-sensitive potassium component during pregnancy. Conclusions: The changes of cellular sodium metabolism in essential hypertension are of minor degree as compared to those in the other conditions studied. Cellular sodium metabolism in blood cells is influenced by thyroid hormones and metabolic disorders. Na-Li countertransport, i.e. Na-Na countertransport, seems to be involved in the regulation of [Nai]: an increase of its activity diminishes [Nai] (pregnancy); a decrease elevates [Nai] (hyperthyroidism). Ouabain-sensitive sodium efflux, i.e. 'Na-K ATPase', is mainly regulated by its substrate, [Nai].     

Sodium leak pathway and substrate binding order in the Na+-glucose cotransporter.

The Na+-glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes was shown to generate a phlorizin-sensitive sodium leak in the absence of sugars. Using the current model for SGLT1, where the sodium leak was presumed to occur after two sodium ions are bound to the free carrier before glucose binding, a characteristic concentration constant (Kc) was introduced to describe the relative importance of the sodium leak versus Na+-glucose cotransport currents. Kc represents the glucose concentration at which the Na+-glucose cotransport current is equal to the sodium leak. As both the sodium leak and the Na+-glucose cotransport current are predicted to occur after the binding of two sodium ions, the model predicted that Kc should be sodium-independent. However, by using a two-microelectrode voltage-clamp technique, the observed Kc was shown to depend strongly on the external sodium concentration ([Na+]o): it was four times higher at 5 mM [Na+]o than at 20 mM [Na+]o. In addition, the magnitude of the sodium leak varied as a function of [Na+]o in a Michaelian fashion, and the sodium affinity constant for the sodium leak was 2-4 times lower than that for cotransport in the presence of low external glucose concentrations (50 or 100 microM), whereas the current model predicted a sigmoidal sodium dependence of the sodium leak and identical sodium affinities for the sodium leak and the Na+-glucose cotransport. These observations indicate that the sodium leak occurs after one sodium ion is associated with the carrier and agree with predictions from a model with the binding order sodium-glucose-sodium. This conclusion was also supported by experiments performed where protons replaced Na+ as a "driving cation."     

Characteristics of membrane transport losses during reticulocyte maturation

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+-glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+-glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.     

交感神经损毁术对自发性高血压大鼠血压和红细胞Na~＋外流动力学的影响

本实验研究了皮下注射６—羟多巴胺（６—ＯＨＤＡ）施行交感神经损毁术对成年自发性高血压大鼠（ＳＨＲ）血压和红细胞Ｎａ＋外流动力学的影响。结果表明,在幼年期施行交感神经损毁术的ＳＨＲ血压显著低于未损毁组,同时红细胞Ｎａ泵驱动的的Ｎａ＋外流最大速率显著下降、Ｎａ＋－Ｋ＋外向协同转运系统的单位活性升高。三者均接近ＷＫＹ大鼠的测定值。相反,损毁成年ＳＨＲ交感神经不影响上述两个动力学参数,血压也未见明显改变。此外,不论幼年或成年期注射６—ＯＨＤＡ均可降低Ｎａ＋—Ｌｉ＋对向转运系统驱动的Ｎａ＋外流最大速率。上述结果提示,在ＳＨＲ早期发育过程中,交感神经营养因子可能降低Ｎａ＋—Ｋ＋外向协同转运活性,继而刺激Ｎａ泵代偿功能增强。这种现象可能同时存在于ＳＨＲ动脉平滑肌,因而是高血压产生的一个原因。关于交感神经损毁术后ＳＨＲ红细胞Ｎａ＋—Ｌｉ＋对向转运最大速率下降的机制尚不清楚,但与交感神经早期营养作用的消除无关。     

Characterization of the Erythrocyte Sodium-Lithium Countertransporter: Limitations and Assumptions of Traditional and Kinetic Methodologies

The present work examined the key elements featuring in the various methods used to characterize the erythrocyte sodium-lithium countertransport. Effects of medium composition on lithium efflux were investigated in 20 subjects. Mean lithium efflux (mmol Li/l RBC.h) into a 150 mm sodium medium was significantly higher than efflux into a revised sodium-rich medium (149 mm) containing 1 mm Mg (0.335 ± 0.100 vs. 0.298 ± 0.085 respectively; P < 0.03). Mean lithium efflux into sodium-free media where sodium had been entirely replaced by magnesium, was significantly lower than efflux into a choline-based medium containing only 1 mm magnesium (0.088 ± 0.027 vs. 0.109 ± 0.034 respectively; P= 0.03). Sodium-lithium countertransport activity and the transporter's kinetic profile were measured simultaneously in 35 subjects using traditional choline-based and kinetic methodologies. There was a significant correlation between countertransport activity and maximal rate of turnover (V _max) (r= 0.62; P < 0.001); V _max values were consistently greater than their corresponding countertransport activities (P < 0.001). On subdividing the subject group into tertiles based on the Michaelis-Menten constant (k _m ) values (mm), <75, 75 − 150 and >150, the slopes of the regression lines for each group diminished progressively (0.64, 0.49 and 0.23 respectively), correlations within each group remained significant (P < 0.001, P < 0.001 and P < 0.02). No significant correlation was found between k _m values and countertransport activity (r= 0.035; P=ns). Increasing the number of points representing sodium concentrations within the range 0–150 mm, improved the confidence in the emerging estimates of V _max and k _m obtained by linear transformation. Comparison of kinetic data derived using four different analytical methods (two linear transformations, a nonlinear regression and a statistical method), showed no significant differences between the estimates yielded for either V _max (P= 0.88, ns) or k _m (P= 0.92, ns). This study has highlighted the critical roles of assay conditions and derivation techniques used when measuring sodium-lithium countertransport, emphasizing the need for standardization of the methodology. Received: 10 December 1996/Revised: 2 October 1997     

Internal magnesium, 2,3-diphosphoglycerate, and the regulation of the steady-state volume of human red blood cells by the Na/K/2Cl cotransport system.

This study is concerned with the relationship between the Na/K/Cl cotransport system and the steady-state volume (MCV) of red blood cells. Cotransport rate was determined in unfractionated and density-separated red cells of different MCV from different donors to see whether cotransport differences contribute to the difference in the distribution of MCVs. Cotransport, studied in cells at their original MCVs, was determined as the bumetanide (10 microM)-sensitive 22Na efflux in the presence of ouabain (50 microM) after adjusting cellular Na (Nai) and Ki to achieve near maximal transport rates. This condition was chosen to rule out MCV-related differences in Nai and Ki that might contribute to differences in the net chemical driving force for cotransport. We found that in both unfractionated and density-separated red cells the cotransport rate was inversely correlated with MCV. MCV was correlated directly with red cell 2,3-diphosphoglycerate (DPG), whereas total red cell Mg was only slightly elevated in cells with high MCV. Thus intracellular free Mg (Mgifree) is evidently lower in red cells with high 2,3-DPG (i.e., high MCV) and vice versa. Results from flux measurements at their original MCVs, after altering Mgifree with the ionophore A23187, indicated a high Mgi sensitivity of cotransport: depletion of Mgifree inhibited and an elevation of Mgifree increased the cotransport rate. The apparent K0.5 for Mgifree was approximately 0.4 mM. Maximizing Mgifree at optimum Nai and Ki minimized the differences in cotransport rates among the different donors. It is concluded that the relative cotransport rate is regulated for cells in the steady state at their original cell volume, not by the number of copies of the cotransporter but by differences in Mgifree. The interindividual differences in Mgifree, determined primarily by differences in the 2,3-DPG content, are responsible for the differences in the relative cotransport activity that results in an inverse relationship with in vivo differences in MCV. Indirect evidence indicates that the relative cotransport rate, as indexed by Mgifree, is determined by the phosphorylated level of the cotransport system.     

Trans-membrane cationic flow and hemodialysis]

Abnormalities of intracellular ion concentrations and transmembrane fluxes were reported in uremia. In RBC from 12 chronically hemodialyzed patients (age 41 + 12, 7 men, 5 women; mean dialysis duration 31 + 24 months), we evaluated the acute effects of hemodialysis on intracellular Na and K concentrations, ouabain sensitive Na/K pump, furosemide sensitive Na/K cotransport, Na/Li countertransport, and passive permeability to Na. Six patients were normotensive and 6 were taking antihypertensive drugs which were withdrawn before the study. When compared to our normal reference group, uremic patients showed a significant increase in intracellular K concentration and a significant decrease in ouabain-sensitive Na/K pump. Intracellular sodium was not increased. No correlation was found between the activity of sodium-potassium pump and the duration of hemodialysis. The other transport systems were comparable to normal. No significant change was observed between the values measured before and after dialysis. Ouabain sensitive Na/K pump was lower in hypertensive as compared to normotensive patients, but this difference was not significant. Our data support the existence of ion transport derangements in uremia, which are not acutely affected by hemodialysis.     

Erythrocyte sodium-lithium countertransport and blood pressure in identical twin pairs discordant for insulin dependent diabetes.

OBJECTIVE--To investigate whether insulin dependent diabetes is responsible for the abnormal behaviour of the carrier in sodium-lithium countertransport and whether the diabetic state is associated with rise in blood pressure. DESIGN--Case-control study. SETTING--London teaching hospital. SUBJECTS--44 twin pairs discordant for insulin dependent diabetes living in United Kingdom and 44 healthy control subjects matched for age, sex, and body mass index. None of the twin pairs or the controls had evidence of microalbuminuria. MAIN OUTCOME MEASURES--Sodium-lithium countertransport activity in erythrocytes and arterial blood pressure. RESULTS--The mean (95% confidence interval) sodium-lithium countertransport activity (mmol Li per litre of red blood cells per h) of the diabetic twins (0.291 (0.244 to 0.338)) was similar to that of their non-diabetic cotwins (0.247 (0.204 to 0.290)); both values were significantly higher than that of the controls (0.187 (0.157 to 0.216); p < 0.05). In addition, systolic blood pressure was higher in those twins with diabetes (127 (122 to 133) mm Hg) than in the non-diabetic cotwins (122 (117 to 127) mm Hg; p < 0.01). There were no significant differences in mean diastolic blood pressure between any of the groups studied. CONCLUSIONS--The raised erythrocyte sodium-lithium countertransport activity in the diabetic twins compared with the controls seems to be inherited rather than a consequence of overt diabetes. The higher systolic blood pressure in diabetic twins than non-diabetic cotwins indicates that insulin dependent diabetes does exert a small influence on systolic blood pressure.     

Erythrocytic cation transport receptor numbers and activity in pregnancies complicated by essential hypertension and pre-eclampsia.

Various functions of erythrocytic cation transport were studied in normotensive and hypertensive pregnancy (women with pre-eclampsia and essential hypertension). The results showed that in pregnancy there is an increase in the number of erythrocytic glycoside binding sites accompanied by a proportional increase in the active inward transport of rubidium (used as a substitute for potassium). There was no evidence of an effect of pregnancy on intraerythrocytic sodium concentrations. These changes were apparently entirely attributable to pregnancy and not affected by pre-eclampsia or essential hypertension. It is suggested that these alterations indicate an adaptive increase in sodium pump numbers and activity secondary to a tendency for the intraerythrocytic sodium concentration to rise during pregnancy and compensating for that tendency.     

Genetic and environmental explanations for the distribution of sodium-lithium countertransport in pedigrees from Rochester, MN.

An elevated level of erythrocyte sodium-lithium (Na-Li) countertransport has been suggested as a predictor of predisposition to essential hypertension. In order to evaluate whether a single genetic or environmental factor with large effects explains the mixture of distributions in Na-Li countertransport in the general population, complex segregation analyses were conducted by using 1,273 individuals more than age 20 years from 276 pedigrees selected without respect to disease risk factors or health status. Either a single genetic locus or a single environmental factor with large gender-specific effects explained the mixture of distributions for Na-Li countertransport in this sample equally well. In the subsample of pedigrees supporting a single-locus etiology, the single genetic locus explained 29.0% of the variability in adjusted Na-Li countertransport in males and 16.6% of that in females. In a subsample of pedigrees supporting an environmental factor etiology, the environmental factor explained 35.2% of the adjusted Na-Li countertransport in males and 20.5% of that in females. These results suggest that there are at least two different explanations for the mixture of distributions in Na-Li countertransport in the general population. Attempts to relate genetic variation in Na-Li countertransport to risk of essential hypertension must consider that the factor with large phenotypic effects on this trait is gender specific and may not be a single major locus in all pedigrees.     

Sodium-substrate cotransport in bacteria

A variety of sodium-substrate cotransport systems are known in bacteria. Sodium enters the cell down an electrochemical concentration gradient. There is obligatory coupling between the entry of the ion and the entry of substrate with a stoichiometry (in the cases studied) of 1:1. Thus, the downhill movement of sodium ion into the cell leads to the accumulation of substrate within the cell. The melibiose carrier of Escherichia coli is perhaps the most carefully studied of the sodium cotransport systems in bacteria. This carrier is of special interest because it can also use protons or lithium ions for cotransport. Other sodium cotransport carriers that have been studied recently are for proline, glutamate, serine-threonine, citrate and branched chain amino acids.     

Cysteine mutagenesis reveals novel structure-function features within the predicted third extracellular loop of the type IIa Na(+)/P(i) cotransporter

The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.