Expression Studies In Xenopus Oocytes Research Articles

URAT1 and GLUT9 mutations in Spanish patients with renal hypouricemia

BackgroundRenal hypouricemia (RHUC), a rare inherited disorder characterized by impaired uric acid (UA) reabsorption in the proximal tubule, is caused by mutations in SLC22A12 or SLC2A9. Most mutations have been identified in Japanese patients, and only a few have been detected in Europeans. MethodsWe report clinical, biochemical and genetics findings of fourteen Spanish patients, six Caucasians and eight of Roma ethnia, diagnosed with idiopathic RHUC. Two of the patients presented exercise-induced acute renal failure and another one had several episodes of nephrolithiasis and four of them had progressive deterioration of renal function, while the rest were asymptomatic. ResultsMolecular analysis revealed SLC22A12 mutations in ten of the patients, and SLC2A9 mutations in the other four. A new heterozygous SLC22A12 missense mutation, c.1427C>A (p.A476D), was identified in two affected members of the same family. The rest of the patients presented homozygous, heterozygous or compound heterozygous mutations that have been previously identified in patients with RHUC; SLC22A12 p.T467M and p.L415_G417del, and SLC2A9 p.T125M. Expression studies in Xenopus oocytes revealed that c.1427C>A reduced UA transport but did not alter the location of URAT1 protein on the plasma membrane. ConclusionsThe biochemical and clinical features of our patients together with the genetic analysis results confirmed the diagnosis of RHUC. This is the first report describing SLC22A12 and SLC2A9 mutations in Spanish patients.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome

AbstractThe hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.

Animal models for autosomal dominant frontal lobe epilepsy: on the origin of seizures

Autosomal dominant frontal lobe epilepsy (ADNFLE) can be caused by mutations in either the α4 or β2 subunit of the neuronal nicotinic Ach receptor. In vitro expression studies in Xenopus oocytes or human embryonic kidney cells have been proven to be valuable tools for the characterization of these mutations, but they do not fully resemble the situation in vivo. Compared with them, animal models have the advantage that the functional consequences of a given mutation can be studied in the complex context of an intact living organism. Recent transgenic and knock-in animal models and their valuable contributions to our current understanding of ADNFLE epileptogenesis are discussed in this article. Several of the mouse and rat models support the hypothesis that ADNFLE mutations cause seizures mainly by increasing GABAergic inhibition, and a conditional knock-in mouse model adds early embryonal structural changes as another possible pathogenetic mechanism.

The C-Terminal PDZ-Binding Motif in the Kv1.5 Potassium Channel Governs its Modulation by the Na+/H+ Exchanger Regulatory Factor 2

Kv1.5 belongs to the family of voltage-gated potassium (Kv) channels and contains a N- and a C-terminal PDZ-binding motif that might be recognized by PDZ domains on the scaffold proteins NHERF1 and NHERF2. Expression studies in Xenopus oocytes demonstrated that NHERF1 and NHERF2 activate Kv1.5, an effect requiring the C-terminal PDZ-binding motif on Kv1.5. NHERF2 enhances Kv1.5 activity and cell surface expression as determined by electrophysiology and immunoassays. NHERF2 elevates Kv1.5 abundance at the plasma membrane by decreasing channel internalization as proven by Brefeldin A experiments. Kv1.5 is stimulated by the serum and glucocorticoid inducible kinase SGK1, a kinase known to interact with the second PDZ domain of NHERF2. This study aims to identify if SGK1 and NHERF2 synergize to increase Kv1.5 currents. Expression of NHERF2 potentiated SGK1-mediated Kv1.5 activation, which was significantly attenuated by deletion of the second PDZ domain in NHERF2. Specificity of observed effects was verified by evaluating the influence of NHERFs on Kv1.3, a known SGK1 target that contains an internal PDZ binding motif. In summary, our results suggest that NHERFs might participate in the regulation of electrical excitability in part by controlling Kv1.5 surface abundance and by clustering signal transduction molecules to the channel.

Slow inactivation of the NaV1.4 sodium channel in mammalian cells is impeded by co-expression of the β1 subunit

In response to sustained depolarization or prolonged bursts of activity in spiking cells, sodium channels enter long-lived non-conducting states from which recovery at hyperpolarized potentials occurs over hundreds of milliseconds to seconds. The molecular basis for this slow inactivation remains unknown, although many functional domains of the channel have been implicated. Expression studies in Xenopus oocytes and mammalian cell lines have suggested a role for the accessory beta1 subunit in slow inactivation, but the effects have been variable. We examined the effects of the beta1 subunit on slow inactivation of skeletal muscle (NaV1.4) sodium channels expressed in HEK cells. Co-expression of the beta1 subunit impeded slow inactivation elicited by a 30-s depolarization, such that the voltage dependence was right shifted (depolarized) and recovery was hastened. Mutational studies showed this effect was dependent upon the extracellular Ig-like domain, but was independent of the intracellular C-terminal tail. Furthermore, the beta1 effect on slow inactivation was shown to be independent of the negative coupling between fast and slow inactivation.

The Peptide Transporter PEPT2 is Targeted by the Protein Kinase SGK1 and the Scaffold Protein NHERF2

PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na⁺/H⁺ exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of ¹⁸⁵Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2ΔC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.

Modulation of the Voltage-Gated Potassium Channel Kv1.5 by the SGK1 Protein Kinase Involves Inhibition of Channel Ubiquitination

The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.

Essential roles of CFEX-mediated Cl−–oxalate exchange in proximal tubule NaCl transport and prevention of urolithiasis

The majority of the Na(+) and Cl(-) filtered by the kidney is reabsorbed in the proximal tubule. In this nephron segment, a significant fraction of Cl(-) is transported via apical membrane Cl(-)-base exchange: Cl(-)-formate exchange, Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. A search for the transporter responsible for apical membrane Cl(-)-formate exchange in the proximal tubule led to the identification of CFEX (SLC26A6). Functional expression studies in Xenopus oocytes demonstrated that CFEX is capable of mediating not only Cl(-)-formate exchange but also Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. Studies in CFEX-null mice have begun to elucidate which of the anion exchange activities mediated by CFEX is important for renal physiology and pathophysiology in vivo. Measurements of transport in renal brush border vesicles isolated from CFEX-null mice demonstrated that CFEX primarily mediates Cl(-)-oxalate exchange rather than Cl(-)-formate exchange. Microperfusion studies in CFEX-null mice revealed that CFEX plays an essential role in mediating oxalate-dependent NaCl absorption in the proximal tubule. CFEX-null mice were found to have hyperoxaluria and a high incidence of calcium oxalate urolithiasis. The etiology of hyperoxaluria in CFEX-null mice was observed to be a defect in oxalate secretion in the intestine, leading to enhanced net absorption of ingested oxalate and elevation of plasma oxalate. Thus, by virtue of its function as a Cl(-)-oxalate exchanger, CFEX plays essential roles both in proximal tubule NaCl transport and in the prevention of hyperoxaluria and calcium oxalate nephrolithiasis.

Novel β subunit mutation causes a slow-channel syndrome by enhancing activation and decreasing the rate of agonist dissociation

We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V → F substitution in the M1 domain of the β subunit (βV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation βV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the βV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the βV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the βV229F mutation on the miniature endplate currents and thus are direct evidence that the βV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.

Post‐translational regulation of EAAT2 function by co‐expressed ubiquitin ligase Nedd4‐2 is impacted by SGK kinases

The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability.

WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability.

The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion cotransporters: Cl(-) influx is mediated by the Na-K-2Cl cotransporter NKCC1 and Cl(-) efflux via K-Cl cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.

Peter Agre, 2003 Nobel Prize winner in chemistry.

Peter C. Agre, an American Society of Nephrology member, is the recipient of the 2003 Nobel Prize in Chemistry for his discovery of the aquaporin water channels. The function of many cells requires that water move rapidly into and out of them. There was only indirect evidence that proteinaceous channels provide this vital activity until Agre and colleagues purified aquaporin-1 from human erythrocytes and reported its cDNA sequence. They proved that aquaporin-1 is a specific water channel by cRNA expression studies in Xenopus oocytes and by functional reconstitution of transport activity in liposomes after the incorporation of the purified protein. These findings sparked a veritable explosion of work that affects several long-standing areas of investigation such as the biophysics of water permeation across cell membranes, the structural biology of integral membrane proteins, the physiology of fluid transport in the kidney and other organs, and the pathophysiological basis of inherited and acquired disorders of water balance. Agre's discovery of the first water channel has spurred a revolution in animal and plant physiology and in medicine.

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.

Role of C-terminal cytoplasmic domain of the AT2 receptor in ligand binding and signaling

A stop codon at position 322 was introduced to generate a truncated, C-terminal-deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C-terminal-deleted AT2 had reduced affinity to [ 125I]angiotensin II ( K d=1.7 nM) and enhanced binding of the AT2-specific peptidic ligand [ 125I]CGP42112A ( K d=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C-terminal deletion, implying that the C-terminus may have a negative effect on the AT2-mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP-binding domain of the human ErbB3 receptor in yeast two-hybrid assay was abolished by C-terminal deletion. In summary, the C-terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.

Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine.

HCO3(-) secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO3(-) secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl(-)/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl(-)/HCO3(-) exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a approximately 90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl(-)/HCO3(-) exchanger in the small intestine.

Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells.

A key function of the proximal tubule is retrieval of most of the vast quantities of NaCl and water filtered by the kidney. Physiological studies using brush border vesicles and perfused tubules have indicated that a major fraction of Cl(-) reabsorption across the apical membrane of proximal tubule cells occurs via Cl(-)-formate exchange. The molecular identity of the transporter responsible for renal brush border Cl(-)-formate exchange has yet to be elucidated. As a strategy to identify one or more anion exchangers responsible for mediating Cl(-) reabsorption in the proximal tubule, we screened the expressed sequence tag database for homologs of pendrin, a transporter previously shown to mediate Cl(-)-formate exchange. We now report the cDNA cloning of CFEX, a mouse pendrin homolog with expression in the kidney by Northern analysis. Sequence analysis indicated that CFEX very likely represents the mouse ortholog of human SLC26A6. Immunolocalization studies detected expression of CFEX, but not pendrin, on the brush border membrane of proximal tubule cells. Functional expression studies in Xenopus oocytes demonstrated that CFEX mediates Cl(-)-formate exchange. Taken together, these observations identify CFEX as a prime candidate to mediate Cl(-)-formate exchange in the proximal tubule and thereby to contribute importantly to renal NaCl reabsorption. Given its wide tissue distribution, CFEX also may contribute to transcellular Cl(-) transport in additional epithelia such as the pancreas and contribute to transmembrane Cl(-) transport in nonepithelial tissues such as the heart.

DMT1 gene expression and cadmium absorption in human absorptive enterocytes

Divalent metal transporter 1 (DMT1) is a transmembrane, proton-coupled metal ion transporter that is upregulated in the duodenum of iron-deficient rodents and in hereditary hemochromatosis patients, suggesting that it may constitute a key factor in the uptake of dietary iron. Functional expression studies in Xenopus oocytes have shown that DMT1 not only mediates transport of iron but also other divalent metal ions, including the toxic metal cadmium. In the present study, the correlation between the cadmium absorption process and gene expression of DMT1 was investigated in an experimental model of human absorptive enterocytes. Fully differentiated Caco-2 cells were grown in monolayers and treated with iron supplemented medium or control medium for 1, 3 or 7 days. At each time point, cadmium transport experiments across the Caco-2 cell monolayers were performed and gene expression of DMT1 measured. Iron treatment for 3 and 7 days resulted in a 50% reduction in the cadmium uptake and a 75% reduction in the transport of the metal across the basolateral membrane. No effects were observed at 24 h. Gene expression of DMT1 in the iron-treated Caco-2 cells was reduced by about 50% at 3 and 7 days and thus, correlated well with the uptake of cadmium. In summary, our results indicate that the uptake of cadmium into human absorptive enterocytes may be mediated by DMT1.

Molecular heterogeneity of the Jknull phenotype: expression analysis of the Jk(S291P) mutation found in Finns

AbstractPolymerase chain reaction genotyping of 32 unrelated Jknull individuals originating predominantly from Polynesia and Finland indicated that all were homozygous for the JK*Bpolymorphism and that 17 of 32, including the 14 Polynesians, carried a 3′-acceptor splice site mutation of intron 5 that resulted in the skipping of exon 6 (called mutation JkΔ6). The remaining 15 Jknull donors from Finland were homozygous for a new T871C transition resulting in a S291P amino acid substitution at a consensusN-glycosylation site of the Jk polypeptide. Transcription-translation assays revealed that the Jk(S291P) mutant was translated into a glycosylated component as efficiently as the wild-type Jk polypeptide (wt Jk)] in the presence of microsomes, thus indicating that the S291P mutation has no effect on theN-glycosylation pattern of the Jk protein. Expression studies in Xenopus oocytes revealed that the Jk(S291P) polypeptide functions as a urea transporter, but the transport activity and the membrane expression level of the mutant protein was reduced to a similar extent. A substantial fraction of the mutant protein was retained intracellularly suggesting that the transit to the plasma membrane was reduced, presumably because of the S→P mutation. After transfection in erythroleukemia K562 cells the wild-type, but not the mutant, protein was efficiently expressed at the cell surface. Because the Jk(S291P) mutant polypeptide was not present in human red cells from Jknull individuals, expression data in the erythroid context clearly indicates that the S→P mutation is the molecular basis of the Finnish Jknull phenotype.

Molecular heterogeneity of the Jknull phenotype: expression analysis of the Jk(S291P) mutation found in Finns

Polymerase chain reaction genotyping of 32 unrelated Jknull individuals originating predominantly from Polynesia and Finland indicated that all were homozygous for the JK*Bpolymorphism and that 17 of 32, including the 14 Polynesians, carried a 3′-acceptor splice site mutation of intron 5 that resulted in the skipping of exon 6 (called mutation JkΔ6). The remaining 15 Jknull donors from Finland were homozygous for a new T871C transition resulting in a S291P amino acid substitution at a consensusN-glycosylation site of the Jk polypeptide. Transcription-translation assays revealed that the Jk(S291P) mutant was translated into a glycosylated component as efficiently as the wild-type Jk polypeptide (wt Jk)] in the presence of microsomes, thus indicating that the S291P mutation has no effect on theN-glycosylation pattern of the Jk protein. Expression studies in Xenopus oocytes revealed that the Jk(S291P) polypeptide functions as a urea transporter, but the transport activity and the membrane expression level of the mutant protein was reduced to a similar extent. A substantial fraction of the mutant protein was retained intracellularly suggesting that the transit to the plasma membrane was reduced, presumably because of the S→P mutation. After transfection in erythroleukemia K562 cells the wild-type, but not the mutant, protein was efficiently expressed at the cell surface. Because the Jk(S291P) mutant polypeptide was not present in human red cells from Jknull individuals, expression data in the erythroid context clearly indicates that the S→P mutation is the molecular basis of the Finnish Jknull phenotype.