Sixth Transmembrane Domain Research Articles

The L450P mutation in KCND3 brings spinocerebellar ataxia and Brugada syndrome closer together

The autosomal dominant cerebellar ataxias (SCAs) are progressive neurodegenerative disorders caused by atrophy of the cerebellum leading to progressive ataxia of gait and limbs, and speech and eye movement difficulties [1]. The SCAs are genetically heterogeneous and 22 causal genes have already been identified [1, 2].With the introduction of next-generation sequencing, novel SCA genes are being rapidly discovered and many more will certainly follow. Recently, we and others have identified loss-of-function mutations in KCND3 as causing SCA19 and SCA22 (SCA19/22) [3, 4], but gain-of-function mutations in KCND3 were also implicated in Brugada syndrome and atrial fibrillation [5, 6]. KCND3 encodes for the voltage-gated potassium channel Kv4.3, which mediates A-type potassium currents in neurons (IA) and in the heart (I to), although the disease mechanisms underlying these allelic disorders are not yet understood. To determine the disease frequency of SCA19/22 in Europe, we screened 366 familial ataxia cases. Most were of French origin and had tested negative for SCA1–3, SCA6, SCA7, SCA17, SCA23, and DRLPA mutations. The complete coding sequence and exon–intron boundaries ofKCND3 were screened using high-resolution melting (HRM) analysis (LightScanner, Bioke; for primer sequences, see Table S1). We discovered a French man (AAD-722-001) carrying the c.1348C>T, p.L450P mutation, which was reported to be associated with Brugada syndrome and was proven to result in a gain of Kv4.3 channel function [4]. This patient displayed a cerebellar gait ataxia at age 39, associated with pyramidal signs. However, at age 46 years, he has not yet presented with any heart problems. Since this man might be at risk for pathological heart palpitations and lethal ventricular arrhythmias, he should be screened for ECG abnormalities. This result emphasizes that the p.L450P mutation may predispose to both ataxia and ventricular arrhythmias, as might be the case for additional mutations associatedwith heritable heart syndromes such as Brugada syndrome and spinocerebellar ataxia that are located within the sixth transmembrane domain and the C-terminal tail of Kv4.3. Additionally, we identified a heterozygous variant, c. 1840C>T, p.P614S, in a French woman (AAD-114-009), who had developed a cerebellar gait ataxia with decreased reflexes and vibration sense in the ankles at the age of 59 years. This variant was not reported in any genetic database or in 400 Electronic supplementary material The online version of this article (doi:10.1007/s10048-013-0370-0) contains supplementary material, which is available to authorized users. A. Duarri : E. Nibbeling :M. R. Fokkens :D. S. Verbeek (*) Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands e-mail: d.s.verbeek@umcg.nl

Functional analysis of the distal region of the third intracellular loop of PROKR2

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270–272) resulted in undetectable cell surface expression, whereas RKR (264–266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.

Potassium Channels, Renal Fibrosis, and Diabetes

Progressive renal fibrosis is a common outcome of almost all forms of renal damage, including diabetic nephropathy. Fibrosis leads to chronic kidney failure and, eventually, dialysis or transplantation (1–3). Although much is known about the molecular background and mediators that prompt fibroblasts or transdifferentiated kidney cells to release collagen and matrix (4–6), the search for a unique event that initiates the process remains inconclusive. Targeting this putative key signal would enable researchers to “switch off” fibrosis and, perhaps, the progressive loss of renal function that plagues millions of individuals worldwide (1–6). The intermediate/small-conductance Ca2+-activated K+ channel (KCa3.1; KCNN4; SK4) is one intriguing candidate for such function because it promotes fibrogenesis in target tissue by altering the membrane potential of cells, thus enhancing extracellular Ca2+ entry (7,8). Subsequent Smad2/3 or mitogen-activated protein kinase (MEK)–dependent phosphorylation upregulates profibrotic genes and collagens in human and animal fibroblasts (7–9). KCa3.1 Ca2+-activated channels regulate K+ outflow, increasing the driving force for Ca2+ entry through hyperpolarization of the plasma membrane (7,10). In KCa3.1, four identical subunits are gathered as a symmetric homotetramer. Six hydrophobic α-helical domains are inserted into the cell membrane in each subunit (Fig. 1). A five-residue loop between the fifth and sixth transmembrane domain confers K+ selectivity. K+ channels are usually tightly associated with calmodulin, the regulatory protein that accounts for the Ca2+ sensitivity of these channels, usually activated by [Ca2+] …

THU0014 Association between a polymorphism in the fractalkine receptor, CX3CR1, and rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic, autoimmune disease characterized by hyperplasia and inflammatory changes within the synovium. The recruitment and accumulation of monocytes in the synovium contribute to inflammatory response...

Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

Gain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH. The first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca(2+) and to the negative allosteric CASR modulator, NPS2143. Two heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143. The asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

Profiling of the Bcl-2/Bcl-XL-binding sites on type 1 IP3 receptor

Several members of the anti-apoptotic Bcl-2-protein family, including Bcl-2, Bcl-XL and Mcl-1, directly bind and regulate the inositol 1,4,5-trisphosphate receptor (IP3R), one of the two main intracellular Ca2+-release channel types present in the endoplasmic reticulum. However, the molecular determinants underlying their binding to the IP3R remained a matter of debate. One interaction site for Bcl-2 was proposed in the central part of the modulatory domain [Y.P. Rong, A.S. Aromolaran, G. Bultynck, F. Zhong, X. Li, K. McColl, S. Matsuyama, S. Herlitze, H.L. Roderick, M.D. Bootman, G.A. Mignery, J.B. Parys, H. De Smedt, C.W. Distelhorst, Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2’s inhibition of apoptotic calcium signals, Mol. Cell 31 (2008) 255–265] and another site in the C-terminal domain of the IP3R encompassing the sixth transmembrane domain, to which Bcl-2, Bcl-XL and Mcl-1 can bind [E.F. Eckenrode, J. Yang, G.V. Velmurugan, J.K. Foskett, C. White, Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca2+ signaling, J. Biol. Chem. 285 (2010) 13678–13684]. Here, we investigated and compared the binding of Bcl-2 and Bcl-XL to both sites. Two different IP3R domains were used for the C-terminal site: one lacking and one containing the sixth transmembrane domain. Our results show that elements preceding the C-terminal cytosolic tail located at the sixth transmembrane domain of IP3R1 were critical for recruiting both Bcl-2 and Bcl-XL to the C-terminal part of the IP3R. Furthermore, consistent with our previous observations, Bcl-XL bound with higher efficiency to the C-terminal part of the IP3R and to a much lesser extent to the central, modulatory domain, while Bcl-2 targeted both sites with similar efficiencies. In conclusion, IP3R harbors two different binding sites for anti-apoptotic Bcl-2 proteins, one in the central, modulatory domain and one in the C-terminal domain near the Ca2+-channel pore.

Acyl Chain Specificity of Ceramide Synthases Is Determined within a Region of 150 Residues in the Tram-Lag-CLN8 (TLC) Domain

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.

Cell-based and in-silico studies on the high intrinsic activity of two boron-containing salbutamol derivatives at the human β2-adrenoceptor

Salbutamol is a well-known β2 adrenoceptor (β2AR) partial agonist. We synthesized two boron-containing salbutamol derivatives (BCSDs) with greater potency and efficacy, compared to salbutamol, for inducing β2AR-mediated smooth-muscle relaxation in guinea-pig tracheal rings. However, the mechanism involved in this pharmacological effect remains unclear. In order to gain insight, we carried out binding and functional assays for BCSDs in HEK-293T cells transfected with the human β2AR (hβ2AR). The transfected hβ2AR showed similar affinity for BCSDs and salbutamol, but adenosine 3′,5′-cyclic phosphate (cAMP) accumulation induced by both BCSDs was similar to that elicited by isoproterenol and greater than that induced by salbutamol. The boron-containing precursors (boric and phenylboronic acids, 100μM) had no significant effect on salbutamol binding or salbutamol-induced cAMP accumulation. These experimental results are in agreement with theoretical docking simulations on lipid bilayer membrane-embedded hβ2AR structures. These receptors showed slightly higher affinity for BCSDs than for salbutamol. An essential change between putative active and inactive conformational states depended on the interaction of the tested ligands with the fifth, sixth and seventh transmembrane domains. Overall, these data suggest that BCSDs induce and stabilize conformational states of the hβ2AR that are highly capable of stimulating cAMP production.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

The Splice Variant of the V2 Vasopressin Receptor Adopts Alternative Topologies

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.

Folding and assembly of TMD 6‐related segments of DMT 1 in trifluoroethanol aqueous solution

Divalent metal-ion transporter 1 (DMT1) belongs to a large class of metal-ion transporters that drive the translocation of a wide range of divalent metal substrates across membranes toward the cytosol with couple of protons. Two highly conserved histidines in the sixth transmembrane domain (TMD6) are essential for metal transport activity in DMT1. In the present study, we determine the high-resolution structures of three 25-residue peptides, corresponding to TMD6 of the wildtype DMT1 (the segment 255-279) and its H267A and H272A mutants, in 30% TFE-d(2) aqueous solution by the combined use of circular dichroism (CD) and NMR spectroscopies. The wildtype peptide forms an 'α-helix-extended segment-α-helix' structure with two helices spanning over Gly258-Ala262 and Met265-Lys277 linked by a hinge at residues Val263-Ile264. The H267A mutation reduces the hinge to one residue (Ile264), while the H272A mutation extends the flexible region of the central part from Val263 to His267. Diffusion-ordered spectroscopy (DOSY) study demonstrates that all the peptides are self-assembly as trimer in 30% TFE-d(2) aqueous solution. The H272A substitution decreases the intermolecular interaction whereas the H267A substitution may enhance the intermolecular interaction. The specific structure of the discontinuous helix and the self-assembly feature of DMT1-TMD6 may be crucial for its biological function. The changes in conformation and intermolecular interaction induced by histidine substitution may be correlated with the deficiency of DMT1 in metal-ion permeation.

Molecular defects of the platelet P2 receptors

Human platelets express three types of P2 receptors, which play important roles in platelet function: P2X(1), P2Y(1) and P2Y(12). Only patients with either quantitative or qualitative abnormalities of the platelet P2Y(12) receptor have been well-characterized so far. Deficiencies of P2Y(12) are associated with nucleotide deletions in the open-reading frame, frameshifts, and early truncation of the protein, or with a nucleotide substitution in the transduction initiation codon. Congenital dysfunctions of P2Y(12) are associated with molecular defects involving the sixth trans-membrane domain or the adjacent third extracellular loop of the receptor, which identify a region of the protein whose integrity is necessary for normal receptor function. A mutation, predicting a lysine to glutamate (Lys174Glu) substitution was associated with decreased ligand binding to the receptor, suggesting that it is responsible for disruption of the adenosine diphosphate (ADP)-binding site of the receptor. Patients with P2Y(12) defects display a mild-to-moderate bleeding diathesis, characterized by mucocutaneous bleedings and excessive post-surgical and post-traumatic blood loss. Defects of P2Y(12) should be suspected when ADP, even at high concentrations (≥10μM), is unable to induce full, irreversible platelet aggregation. Tests that evaluate the degree of inhibition of adenylyl cyclase by ADP should be used to confirm the diagnosis.

Functional Characterization of Aquaporin-4 Specific T Cells: Towards a Model for Neuromyelitis Optica

BackgroundAntibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized.Methodology/Principal FindingsUsing overlapping 15-meric peptides of AQP4, we identified the immunogenic T cell epitopes of AQP4 that are restricted to murine major histocompatibility complex (MHC) I-Ab. The N-terminal region of AQP4 was highly immunogenic. More precisely, the intracellular epitope AQP422–36 was detected as major immunogenic determinant. AQP482–108 (located in the second transmembrane domain), AQP4139–153 (located in the second extracellular loop), AQP4211–225 (located in the fifth transmembrane domain), AQP4235–249 (located in the sixth transmembrane domain), as well as AQP4289–306 in the intracellular C-terminal region were also immunogenic epitopes. AQP422–36 and AQP4289–303 specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response.Conclusions and SignificanceAQP4 is able to induce an autoreactive T cell response. The identification of I-Ab restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the α-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the α-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an “α-helix-extended segment-α-helix” structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the α-helical folding. Both histidines are buried in SDS micelles as evidenced by their p K a values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an α-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific “α-helix-extended segment-α-helix” structure of TM6 may have an important implication for the binding of the transporter to H + and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.

Identification and Functional Characterization of Two Novel Activating Thyrotropin Receptor Mutants in Toxic Thyroid Follicular Adenomas

Two previously unreported thyrotropin (TSH) receptor mutations, A623F and I635V, were identified in toxic follicular thyroid adenoma specimens from two patients with hyperthyroidism. Our aim was to characterize both novel mutants in terms of the following: cAMP basal constitutive activity, cAMP response to TSH, plasma membrane expression levels, and TSH binding properties. We performed DNA extraction for TSHR gene sequencing. COS-7 cells were transiently transfected with wild-type and mutated TSH receptor constructs for determination of basal cAMP constitutive activity and dose-response accumulation of cAMP using recombinant human TSH. Flow cytometry analysis was performed to evaluate plasma membrane expression. Binding studies using bovine TSH as a ligand were performed to compare the affinities of wild-type and mutated TSH receptors for TSH. Both mutants, A623F and I635V, had higher cAMP basal constitutive activities than the wild-type TSH receptor. A623F but not I635V showed lower plasma membrane expression than the wild-type receptor. IC50, an indirect measurement of ligand-receptor affinity, was lower in A623F and higher in I635V than in the wild-type TSH receptor, although no statistically significant differences were observed. No differences were observed in EC50 and although the absolute values of maximal stimulation achieved with both mutants were higher than the wild type, the differences did not achieve statistical significance. A623F and I635V are two naturally occurring TSH receptor mutations that increase basal cAMP accumulation and consequently promote the development of toxic follicular thyroid adenoma. cAMP response to increasing TSH dose is retained by A623F and I635V mutated receptors and the maximal stimulation obtained is not different from that of the wild-type receptor. Substitution of alanine 623 by phenylalanine 623 at the third intracellular loop of the TSH receptor decreases its plasma membrane expression, indicating that alanine 623 is important in directing the TSH receptor to the cell surface or in down-regulating the constitutive receptor. By contrast, isoleucine 635, located in the sixth transmembrane domain, is important in regulating TSH receptor basal activity but does not modify its plasma membrane expression.

Engineering a G protein‐coupled receptor for structural studies: Stabilization of the BLT1 receptor ground state

Structural characterization of membrane proteins is hampered by their instability in detergent solutions. We modified here a G protein-coupled receptor, the BLT1 receptor of leukotriene B(4), to stabilize it in vitro. For this, we introduced a metal-binding site connecting the third and sixth transmembrane domains of the receptor. This modification was intended to restrain the activation-associated relative movement of these helices that results in a less stable packing in the isolated receptor. The modified receptor binds its agonist with low-affinity and can no longer trigger G protein activation, indicating that it is stabilized in its ground state conformation. Of importance, the modified BLT1 receptor displays an increased temperature-, detergent-, and time-dependent stability compared with the wild-type receptor. These data indicate that stabilizing the ground state of this GPCR by limiting the activation-associated movements of the transmembrane helices is a way to increase its stability in detergent solutions; this could represent a forward step on the way of its crystallization.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

A Somatic Gain-of-Function Mutation in the Thyrotropin Receptor Gene Producing a Toxic Adenoma in an Infant

Activating mutations of the thyroid stimulating hormone receptor gene (TSHR) are rare in the neonate and in the pediatric population. They are usually present in the germline, and are either inherited or occur de novo. Somatic mutations in TSHR are unusual in the pediatric population. We describe a nine-month-old infant with thyrotoxicosis who harbored an activating somatic mutation in TSHR that was not present in the germline. As genomic DNA analysis failed to show a TSHR gene mutation, a radioiodide scan was performed to reveal a unilateral localization of uptake suppressing the remaining thyroid tissue. Genomic and complementary DNA analyses of the active thyroid tissue, removed surgically, identified a missense mutation (D633Y) located in the sixth transmembrane domain of the TSHR. The absence of this TSHR mutation in circulating mononuclear cells and in unaffected thyroid tissue confirmed the somatic nature of this genetic alteration. To the authors' knowledge, this is the youngest patient to receive definitive treatment for hyperthyroidism due to an activating mutation of TSHR.

A novel mutation in the ATP2C1 gene is associated with Hailey–Hailey disease in a Chinese family

A three-generation Chinese family with Hailey-Hailey disease (HHD) was identified and characterized. The proband developed HHD with severe recurrent blisters and crusted erosions involving the body folds. Skin biopsy studies showed epidermal hyperkeratosis and defects in cell-to-cell adhesion. Three other members in the family were also affected with HHD and had the same clinical manifestations. The purpose of this study was to identify the pathogenic gene or mutation in the family. All exons and exon-intron boundaries of ATP2C1 were polymerase chain reaction (PCR) amplified and sequenced with DNA samples from the proband. Restriction fragment length polymorphism (RFLP) analysis for the intron 23-exon 24 boundary of ATP2C1 was performed in all family members and in 100 normal control subjects. A novel 2-bp deletion (c.2251delGT) was detected in exon 24 of the ATP2C1 gene. The mutation was present in the three other affected family members and in two asymptomatic young carriers, but not in the other normal family members or the 100 normal controls. The mutation resulted in a frameshift change and led to the formation of a premature termination codon (PTC) four amino acid residues downstream from the sixth transmembrane domain. Our results indicate that the novel c.2251delGT (p.V751fs) mutation in the ATP2C1 gene is responsible for HHD in this Chinese family. This study expands the spectrum of ATP2C1 mutations associated with HHD.