Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V₂ receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication.     

Appropriate polarization following pharmacological rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation

To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, Delta V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of Delta V278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells.     

Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone

Cell-surface expression and biological functions of several intracellular-retained G protein-coupled receptors are restored by membrane-permeable ligands called pharmacological chaperones. We have previously demonstrated that a mutation of the hydrophobic motif 341FNX2LLX3L350 in the C terminus of the human pituitary vasopressin V3 receptor (MUT V3R) led to it being retained in the endoplasmic reticulum (ER). Here, we establish the precise role of this motif and investigate whether SSR149415, a non-peptide V3R antagonist, behaves as a pharmacological chaperone for the ER-retained MUT V3R. The absence of the mutated receptor in the plasma membrane is linked to its prolonged association with the molecular chaperone calnexin in the ER and to its intensive degradation by the ubiquitin-proteasomal machinery. However, this is not because of a lack of oligomerization, as demonstrated by the presence of MUT V3R homodimers in the ER. Treatment with SSR149415 restores expression of the mutated receptor on the cell surface and its correct maturation, resulting into the functional recovery of its signaling properties. SSR149415 acts by stabilizing a native-like conformation of the V3R, reducing its association with calnexin and, thus, favoring a secretory pathway rather than the proteasomal degradation pathway. In conclusion, the FN(X)2LL(X)3L sequence is an important motif for the V3R conformation, and the misfolding resulting from its mutation alters the receptor export but can be reverted by SSR149415.     

Disease-causing V(2) vasopressin receptors are retained in different compartments of the early secretory pathway

The G protein-coupled V(2) vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V(2) vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease-causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V(2) vasopressin receptor mutants. We show that mutants L62P, DeltaL62-R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease-causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.     

Novel vasopressin type 2 (AVPR2) gene mutations in Brazilian nephrogenic diabetes insipidus patients

Nephrogenic diabetes insipidus (NDI) is an inherited disorder characterized by renal resistance to the antidiuretic effect of arginine vasopressin (AVP), resulting in polyuria, polydipsia, and hypoosmolar urine. In the vast majority of cases, NDI is associated with germ-line mutations in the vasopressin receptor type 2 gene (AVPR2) and in about 8% of the cases with the water channel aquaporin-2 gene (AQP-2) mutations. To date, approximately 277 families with 185 germ-line mutations in the AVPR2 gene have been described worldwide. In the present study, the AVPR2 gene was genotyped in eight unrelated Brazilian kindred with NDI. In five of these NDI families, novel mutations were noted (S54R, I130L, S187R, 219delT, and R230P), whereas three seemingly unrelated probands were found to harbor previously described AVPR2 gene mutations (R106C, R137H, R337X). Additionally a novel polymorphism (V281V) was detected. In conclusion, although NDI is a rare disease, the findings of mutations scattered over the entire coding region of the AVPR2 gene are a valuable model to determine structure function relationship in G-protein-coupled receptor related diseases. Furthermore, our data indicate that in Brazil the spectrum of AVPR2 gene mutations is "family specific".     

Intracellular mislocalization of mutant podocin and correction by chemical chaperones

The NPHS2 gene encoding the podocin protein was causally linked to the autosomal recessive type of steroid-resistant nephrotic syndrome. In this study, we investigated the consequence of the R138Q mutation of podocin, one of the most common missense mutations in the NPHS2 gene, by examining the expression of the wild-type and R138Q mutant podocins in mammalian cells. Either myc- or FLAG-tagged wild-type podocin was strongly stained in plasma membrane, particularly in the fine processes wherein the protein was colocalized with actin stress fibers. On the other hand, the R138Q mutant podocin was completely retained intracellularly and colocalized with the endoplasmic reticulum (ER) marker, calnexin. These results suggest that the R138Q mutation affected podocin protein folding, thereby interfering with the mutant protein's departure from the ER. To determine if the ER retention of R138Q mutant is correctable, cells were incubated with the chemical chaperones glycerol, trimethylamine-N-oxide, and DMSO. Using these two methods, namely, cell surface labeling with sulfo-NHS-S-S-biotin and Alexa 488-streptavidin, and immunostaining to detect the podocin protein close to the plasma membrane, we confirmed that these chemical chaperone treatments elicit a cellular redistribution of R138Q podocin. Our results reveal defective cellular processing of the mutant podocin, and provide evidence for pharmacological correction of the processing defect.     

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.     

Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.     

Retention of mutant low density lipoprotein receptor in endoplasmic reticulum (ER) leads to ER stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.     

Defective protein folding and intracellular retention of thyroglobulin-R19K mutant as a cause of human congenital goiter

It has been suggested that a thyroglobulin (Tg)-R19K missense mutation may be a newly identified cause of human congenital goiter, which is surprising for this seemingly conservative substitution. Here, we have examined the intracellular fate of recombinant mutant Tg expressed in COS-7 cells. Incorporation of the R19K mutation largely blocked Tg secretion, and this mutant was approximately 90% degraded intracellularly over a 24-h period after synthesis. Before its degradation, the Tg-R19K mutant exhibited abnormally increased association with molecular chaperones BiP, calnexin, and protein disulfide isomerase, and was unable to undergo anterograde advance from the endoplasmic reticulum (ER) through the Golgi complex. Inhibitors of proteasomal proteolysis and ER mannosidase-I both prevented ER-associated degradation of the Tg-R19K mutant and increased its association with ER molecular chaperones. ER quality control around Tg residue 19 is not dependent upon charge but upon side-chain packing, because Tg-R19Q was efficiently secreted. Whereas a Tg mutant truncated after residue 174 folds sufficiently well to escape ER quality control, introduction of the R19K point mutation blocked its secretion. The data indicate that the R19K mutation induces local misfolding in the amino-terminal domain of Tg that has global effects on Tg transport and thyroid hormonogenesis.     

Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, Delta F508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of Delta F508 CFTR with calnexin, an ER chaperone, results in the ERAD of Delta F508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of Delta F508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the Delta F508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated Delta F508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of Delta F508 CFTR in the ER.     

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer’s disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1ΔR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1ΔR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.     

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neurons.     

Evidence of the importance of the first intracellular loop of prokineticin receptor 2 in receptor function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2.     

Structural insight into the role of the second intracellular loop of the bradykinin 2 receptor in signaling and internalization.

The second cytoplasmic loop (IC2) of the bradykinin B2 receptor plays a vital role in its dynamic life cycle including the activation, internalization, desensitization, and resensitization of this receptor. Here, we probe the structure and function of the IC2, with particular emphasis on threonine-137, which is crucial for signal transduction and internalization. Mutation of this threonine to proline (T137P) produces wild type (WT) signaling and complete inhibition of internalization. Incorporation of aspartate (T137D) leads to a marked reduction in receptor signaling but with WT receptor uptake. The T137D mutation coupled with serine to alanine substitution of S335 and S341 within the distal C-terminus recovers signaling, leading to an actually enhanced arachidonic acid release and phosphoinositide turnover compared to WT bradykinin B2 receptor (BKB2R). To provide a structural basis for the actions of this mutant, the conformational features of IC2 (both WT and mutant) were investigated by high-resolution NMR. The NMR analysis illustrated two prominent alpha-helices at the N- (L123-M138) and C-termini (A149-I156) of the IC2 receptor domain. Incorporating these structural characteristics into a model of BKB2R, we determined that the entire N-terminal helix of IC2 is incorporated as TM3, placing Y131 1.5 helical turns into TM3 and T137 at the membrane surface. The NMR data indicated no structural changes upon substitution of T137D. These results suggest that the altered signaling of the T137D mutant can be attributed to the introduction of a negative charge, indicating that phosphorylation of this residue takes place and participates in the life cycle of this receptor. Additionally, the return to WT signal capacity of the mutation T137D/S335A/S341A, to overcome the deleterious T137D substitution points to a functional interaction between the IC2 and the C-terminus.     

A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane

Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.     

Intracellular accumulation of antithrombin Morioka (C95R), a novel mutation causing type I antithrombin deficiency

Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds, and its deficiency is associated with increased venous thrombosis. Recently, we found a novel missense mutation named AT Morioka (C95R), which causes the loss of one of the three disulfide bonds. In this study, we prepared Chinese hamster ovary cells stably overexpressing wild type or mutant AT and examined the intracellular fate of the ATs. In pulse-chase experiments, newly synthesized wild type AT was secreted into the medium with a half-life of approximately 1.5 h. In contrast, most of the mutant type AT was not secreted during the chase period of 9 h and, surprisingly, was not degraded in the cells. The kinetics of the secretion suggests that the mutant was secreted about 50 times more slowly into the medium. Most of the mutant AT in the cells had high mannose type oligosaccharides, suggesting that it was retained in the endoplasmic reticulum (ER). In addition, half of the mutant AT existed in a dimeric form with an intermolecular disulfide bond. On immunoelectron microscopy, the mutant AT was found to have accumulated in variously sized structures surrounded by a single membrane in the cytoplasm. Immunogold particles exhibiting calnexin immunoreactivity were detected on the membranes. Ribosomes were attached to some of the small structures that had accumulated the mutant AT. Further, we prepared Chinese hamster ovary cells stably overexpressing another mutant AT in which two cysteine residues at 21 and 95, responsible for disulfide bond formation, were substituted for arginines. In pulse-chase experiments, the mutant AT (C21C,C95R) was secreted faster than that of AT Morioka (C95R) into the medium. These results suggest that AT Morioka remained for a long time in ER without being degraded and accumulated in newly formed membrane structures derived from the ER. The dimerization of AT Morioka (C95R) through Cys-21 seems to be critical for its intracellular accumulation.     

Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1 induce distinct trafficking defects in MDCK cells

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl-/HCO3- anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of alpha-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of alpha-intercalated cells.     

Defective potassium channel Kir2.1 trafficking underlies Andersen-Tawil syndrome

Andersen-Tawil syndrome is a skeletal and cardiac muscle disease with developmental features caused by mutations in the inward rectifier K+ channel gene KCNJ2. Patients harboring these mutations exhibit extremely variable expressivities. To explore whether these mutations can be correlated with a specific patient phenotype, we expressed both wild-type (WT) and mutant genes cloned into a bi-cistronic vector. Functional expression in human embryonic kidney 293 cells showed that none of the mutant channels express current when present alone. When co-expressed with WT channels, only construct V302M-WT yields inward current. Confocal microscopy fluorescence revealed three patterns of channel expression in the cell: 1) mutations D71V, N216H, R218Q, and pore mutations co-assemble and co-localize to the membrane with the WT and exert a dominant-negative effect on the WT channels; 2) mutation V302M leads to channels that lose their ability to co-assemble with WT and traffic to the cell surface; 3) deletions Delta 95-98 and Delta 314-315 lead to channels that do not traffic to the membrane but retain their ability to co-assemble with WT channels. These data show that the Andersen-Tawil syndrome phenotype may occur through a dominant-negative effect as well as through haplo-insufficiency and reveal amino acids critical in trafficking and conductance of the inward rectifier K+ channels.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.