11th Transmembrane Research Articles

Enhancing the interpretation of genetic observations in KCNQ1 in unselected populations: relevance to secondary findings.

Rare variants in the KCNQ1 gene are found in the healthy population to a much greater extent than the prevalence of Long QT Syndrome type 1 (LQTS1). This observation creates challenges in the interpretation of KCNQ1 rare variants that may be identified as secondary findings in whole exome sequencing.This study sought to identify missense variants within sub-domains of the KCNQ1-encoded Kv7.1 potassium channel that would be highly predictive of disease in the context of secondary findings. We established a set of KCNQ1 variants reported in over 3700 patients with diagnosed or suspected LQTS sent for clinical genetic testing and compared the domain-specific location of identified variants to those observed in an unselected population of 140 000 individuals. We identified three regions that showed a significant enrichment of KCNQ1 variants associated with LQTS at an odds ratio (OR) >2: the pore region, and the adjacent 5th (S5) and 6th (S6) transmembrane (TM) regions. An additional segment within the carboxyl terminus of Kv7.1, conserved region 2 (CR2), also showed an increased OR of disease association. Furthermore, the TM spanning S5-Pore-S6 region correlated with a significant increase in cardiac events. Rare missense variants with a clear phenotype of LQTS have a high likelihood to be present within the pore and adjacent TM segments (S5-Pore-S6) and a greater tendency to be present within CR2. This data will enhance interpretation of secondary findings within the KCNQ1 gene. Further, our data support a more severe phenotype in LQTS patients with variants within the S5-Pore-S6 region.

OR23-02 Genotype-Phenotype Correlation In Calcium Sensing Receptor (Casr) Gene Gain-Of-Function Mutations: A Case Series And Report Of 2 Novel Mutations

Abstract Disclosure: D.S. Ali: None. F. Marini: None. F. Alsarraf: None. H.H. Alalwani: None. A. Alamri: None. A.A. Khan: Advisory Board Member; Self; Amgen Inc, Takeda. Grant Recipient; Self; Amgen Inc, Alexion Pharmaceuticals, Inc., Takeda, Ultragenyx, Radius Health, Inc. M. Brandi: Consulting Fee; Self; Alexion Pharmaceuticals, Inc. Speaker; Self; Amgen Inc, Alexion Pharmaceuticals, Inc., Eli Lilly & Company. Background: Autosomal dominant hypocalcemia (ADH1) is a genetic disorder characterized by low serum calcium and low or inappropriately normal parathyroid hormone levels. It is caused by a heterozygous activating mutation of the calcium-sensing receptor (CaSR) gene. ADH1 has been linked to 113 unique germline mutations, of which 96% are missense mutations. There is a lack of clear genotype-phenotype correlation in the reported literature. Methods: We described a case series of six unrelated ADH1 probands, each with a gain-of-function CaSR mutation, and two children of one of these cases, comparing clinical, biochemical, and complication profiles and matching our identified mutations to those previously reported in the literature. Results: Of our cases 75% are familial and 25% are secondary to de novo mutation. Our 6 unrelated ADH1 probands showed distinct clinical presentations, while 3 cases within the same pedigree shared similar presentation and clinical characteristics including presentation in infancy, hypocalcemic seizures (2/3; 66.7%), hypomagnesemia, hyperphosphatemia, and hypercalciuria. The biochemical profile differed among the 6 cases in the degree of hypocalcemia, associated hypercalciuria, hypomagnesemia, and/or hypokalemia. Response to therapy also differed. When comparing our cases to previously published cases, we noted that mutations in the 5th transmembrane domain of the CaSR (TM5) carry a more severe form of the disease and may be associated with a higher rate of hypocalcemic seizures, basal ganglia calcifications as well as nephrocalcinosis, nephrolithiasis and renal insufficiency compared to mutations in the extracellular regions. Hypomagnesemia was present in 100% of the cases harboring mutations in TM5. We also describe two novel activating mutations of the CaSR gene; c.2468T>A, p.lle823Asn and c.1756G>A, p.Glu586Lys. Both of these were not reported either on the most common databases of human mutations or in the literature. In silico analysis of p.lle823Asn by PolyPhen-2 predicted the Ile to Asn substitution at position 823 as “probably damaging”, while the p.Glu586Lys substitution was suggested to have a “benign” nature. Our patient with the former mutation had a history of seizure and nephrolithiasis, while our patient with the latter mutation is mildly symptomatic, presented in her 40s, and is on low doses of active vitamin D and calcium supplements. Conclusion: As a result of these genetic and clinical comparisons, we propose that a genotype-phenotype correlation may exist because our cases showed similar presentation, characteristics, and severity, with respect to published cases with the same or similar mutations. We also contended that the severity of the presentation is highly influenced by the specific CaSR variant. These findings, however, require further evaluation and assessment with a systematic review. Presentation: Saturday, June 17, 2023

Negative allosteric modulation of the glucagon receptor by RAMP2

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Tracking the structural dynamics of SERCA regulation in real time.

Calcium (Ca2+) signaling in eukaryotic cells is integral to biological processes such as neuronal activity, transcription, and muscle contraction-relaxation. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) proteins play a vital role in this intricate signaling network by returning Ca2+ to the sarco/endoplasmic reticulum (SR/ER), thereby resetting the signal and restoring SR/ER Ca2+ levels. SERCA activity is optimised for different tissues and developmental stages by expression of different isoforms and by regulatory factors such as regulatory peptides, Ca2+ concentration and internal regulatory domains. Mutations are known to cause Brody's (SERCA1) and Darier's (SERCA2) diseases, and dysregulation of SERCA2a is associated with heart failure. To investigate the kinetic and structural effects of SERCA regulation, we performed time-resolved x-ray solution scattering (TR-XSS) experiments on two SERCA isoforms. In our TR-XSS setup the reaction is initiated by laser-induced ATP release, and the resulting structural dynamics are then monitored in real time using synchrotron x-ray pulses. We investigated the effect of Ca2+ concentration on SERCA1a in native membranes and identified three distinct structural states that developed slower under high-Ca2+ conditions and showed structural differences compared to the low-Ca2+ condition. The SERCA2b isoform has a unique regulatory domain consisting of an 11th transmembrane helix and a luminal extension loop that increases its Ca2+ affinity and lowers the maximal turnover rate. We collected TR-XSS datasets for SERCA2b with and without this regulatory region and observed differences in the reaction-cycle kinetics that indicate a loss of control of transitions between states. In summary, we have developed a synchrotron-based x-ray technique and determined the kinetic and structural basis of regulation of P-type ATPase transport across biological membranes.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrate that RAMP2 enhances local dynamics in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

The Val34Met, Thr164Ile and Ser220Cys Polymorphisms of the β2-Adrenergic Receptor and Their Consequences on the Receptor Conformational Features: A Molecular Dynamics Simulation Study.

The gene encoding the β2-adrenergic receptor (β2-AR) is polymorphic, which results in possible differences in a primary structure of this protein. It has been shown that certain types of polymorphisms are correlated with some clinical features of asthma, including airways reactivity, whereas the influence of other is not yet understood. Among polymorphisms affecting amino acids at positions 16, 27, 34, 164 and 220, the latter three are present in the crystal structure of β2-AR, which facilitates studying them by means of molecular dynamics simulations. The current study was focused on investigating to what extent the three polymorphisms of β2-AR (i.e., Val34Met, Thr164Ile and Ser220Cys) affect the interaction of β2-AR with its natural molecular environment which includes: lipid bilayer (in the case of all three polymorphs) and Gs protein (which participates in β2-AR-mediated signaling; in the case of Ser220Cys). We have designed and carried out a series of molecular dynamics simulations at different level of resolution (i.e., either coarse-grained or atomistic simulations), accompanied by thermodynamic integration protocol, in order to identify potential polymorphism-induced alterations in structural, conformational or energetic features of β2-AR. The results indicate the lack of significant differences in the case of energies involved in the β2-AR-lipid bilayer interactions. Some differences have been observed when considering the polymorphism-induced alterations in β2-AR-Gs protein binding, but their magnitude is also negligible in relation to the absolute free energy difference correlated with the β2-AR-Gs affinity. The Val34Met and Thr164Ile polymorphisms are weakly correlated with alteration of the conformational features of the receptor around polymorphic sites. On the contrary, it has been concluded that the Ser220Cys polymorphism is correlated with several structural alterations located in the intracellular region of β2-AR, which can induce G-protein binding and, subsequently, the polymorphism-correlated therapeutic responses. More precisely, these alterations involve vicinity of intracellular loops and, in part, are the direct consequence of disturbed interactions of Ser/Cys220 sidechain within 5th transmembrane domain. Structurally, the dynamic structure exhibited by the β2-ARSer220 polymorph is closer to the Gs-compatible structure of β2-AR.

A novel mutation in the SLCO2A1 gene, encoding a prostaglandin transporter, induces chronic enteropathy.

Chronic enteropathy associated with SLCO2A1 gene (CEAS) is caused by loss-of-function mutations in SLCO2A1, which encodes a prostaglandin (PG) transporter. In this study, we report a sibling case of CEAS with a novel pathogenic variant of the SLCO2A1 gene. Compound heterozygous variants in SLCO2A1 were identified in an 8-year-old boy and 12-year-old girl, and multiple chronic nonspecific ulcers were observed in the patients using capsule endoscopy. The splice site mutation (c.940 + 1G>A) of the paternal allele was previously reported to be pathogenic, whereas the missense variant (c.1688T>C) of the maternal allele was novel and had not yet been reported. The affected residue (p.Leu563Pro) is located in the 11th transmembrane domain (helix 11) of SLCO2A1. Because SLCO2A1 mediates the uptake and clearance of PGs, the urinary PG metabolites were measured by liquid chromatography coupled to tandem mass spectrometry. The urinary tetranor-prostaglandin E metabolite levels in the patients were significantly higher than those in unaffected individuals. We established cell lines with doxycycline-inducible expression of wild type SLCO2A1 (WT-SLCO2A1) and the L563P mutant. Immunofluorescence staining showed that WT-SLCO2A1 and the L563P mutant were dominantly expressed on the plasma membranes of these cells. Cells expressing WT-SLCO2A1 exhibited time- and dose-dependent uptake of PGE2, while the mutant did not show any uptake activity. Residue L563 is very close to the putative substrate-binding site in SLCO2A1, R561 in helix 11. However, in a molecular model of SLCO2A1, the side chain of L563 projected outside of helix 11, indicating that L563 is likely not directly involved in substrate binding. Instead, the substitution of Pro may twist the helix and impair the transporter function. In summary, we identified a novel pathogenic variant of SLCO2A1 that caused loss-of-function and induced CEAS.

SAT-043 A Trafficking Defective Mutant PROKR2, Identified In Patients With Hypogonadotropic Hypogonadism, Cycles Between Endoplasmic Reticulum (ER) And Golgi To Attenuate ER Stress

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins and are a major class of therapeutic drug targets. Mutations in GPCRs resulting in loss of function or alteration of signaling can cause inherited or acquired diseases. Such mutations frequently cause impaired cell surface expression, resulting from protein misfolding and intracellular trapping due to careful monitoring by the cellular protein surveillance system. However, protein quality control mechanisms and intracellular trafficking of misfolded GPCRs remain incompletely understood. Here, we investigated the effects of mutations in prokineticin receptor 2 (PROKR2), identified in patients with hypogonadotropic hypogonadism or Kallmann syndrome, characterized by GnRH deficiency, as a model of the GPCR trafficking process. We first confirmed the impairment in cellular localization and downstream signaling of these PROKR2 mutants. To better understand the mechanisms underlying differences in cellular pathway targeting between wild type (WT) PROKR2 and P290S PROKR2, a trafficking defective mutant in which the mutation is in one of the most highly conserved amino acids among GPCRs, located in the 6th transmembrane domain, we performed a comparative interactome study using immunoprecipitation followed by mass spectrometry analysis. This revealed that P290S PROKR2 interacts strongly with proteins involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, transport to the Golgi, and retrograde transport from the Golgi to the ER. Guided by these interactome findings, we demonstrated that both the trafficking defective mutant, P290S PROKR2, as well as WT PROKR2 were degraded by the ERAD pathway, with the P290S mutant entering the ERAD pathway to a greater extent instead of being transported to the plasma membrane. Surprisingly, we found that P290S PROKR2 enters the Golgi, even though it is degraded by the ERAD pathway from the ER. When misfolded proteins accumulate in the ER, they evoke ER stress, which activates the unfolded protein response and can cause cell death. We found that misfolded P290S PROKR2 cycles to the ER-Golgi interface to reduce ER stress and to promote efficient degradation via the ERAD pathway. Our studies demonstrate that misfolded GPCRs are controlled in the early secretory pathway to maintain cellular homeostasis, thereby providing a fundamental strategy for restoration of defective cell surface transport caused by GPCR mutations as well as protection from the initiation and progression of human disease caused by ER stress. Our findings offer insights which could lead to development of potential therapeutic targets for diseases caused by human GPCR mutations.

Structural Basis of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2b Regulation via Transmembrane Helix Interplay

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and theN-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions.

A Darier disease mutation relieves kinetic constraints imposed by the tail of sarco(endo)plasmic reticulum Ca2+-ATPase 2b

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2+-pump reaction cycle. Here, we report that a Darier disease (DD) mutation of SERCA2b that changes a glutamate to a lysine in the cytoplasmic loop between TM8 and TM9 (E917K) relieves these kinetic constraints. We analyzed the effects of this mutation on the overall reaction and the individual partial reactions of the Ca2+ pump compared with the corresponding mutations of the SERCA2a and SERCA1a isoforms, lacking the SERCA2b tail. In addition to a reduced affinity for Ca2+, caused by the mutation in all three isoforms examined, we observed a unique enhancing effect on the turnover rates of ATPase activity and Ca2+ transport for the SERCA2b E917K mutation. This relief of kinetic constraints contrasted with inhibitory effects observed for the corresponding SERCA2a and SERCA1a (E918K) mutations. These observations indicated that the E917K/E918K mutations affect the rate-limiting conformational change in isoform-specific ways and that the SERCA2b mutation perturbs the interactions of TM11 with other SERCA2b regions. Mutational analysis of an arginine in TM7 that interacts with the glutamate in SERCA1a crystal structures suggested that in wildtype SERCA2b, the corresponding arginine (Arg-835) may be involved in mediating the conformational restriction by TM11. Moreover, the E917K mutation may disturb TM11 through the cytoplasmic loop between TM10 and TM11. In conclusion, our findings have identified structural elements of importance for the kinetic constraints imposed by TM11.

Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

AbstractHereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx Vmax of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates. The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or both.

In silico and functional evaluation of PTH1R mutations found in patients with primary failure of eruption (PFE).

The genetic basis of PFE (OMIM ID: 125350) was interrogated using molecular functional studies. PFE is a disorder that results in a poor prognosis in the eruption of teeth and by extension, in treatment with a continuous archwire. We tested the hypothesis that PTH1R mutations result in loss of function due to altered protein structure to determine (i) the fate of a functional PTH1R mutation and (ii) the resulting PTH1R protein structure of each functional mutation. We used immunofluorescence assay of COS7 cells that were transfected with either the WT or 1092delG PTH1R mutation sequence to compare the fate of the expressed protein. We also performed in silico analysis of the WT PTH1R and four different functional PTH1R mutations RESULTS: Functional studies (IFA) showed a variation in expression between the WT and mutant PTH1R. Further, in silico analysis showed structural differences between WT and mutant PTH1R proteins, particularly in the regions of the 3rd intracellular loop and the 6th transmembrane domain required for efficient PTH1R function. PTH1R mutations identified in PFE likely result from diminished function due to truncation of the protein, lack of efficient G-protein interactions and putatively attenuated signal transduction. By identifying the mode of protein dysfunction, scientist-clinicians are better prepared to recognize and thereby develop improved methods of treatment, starting at the molecular level.

Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction.

Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesis in vivo Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.

Pungent and Non-Pungent General Anesthetics Interact with TRPA1 via Distinct Binding Pocket in the Pore Domain Region

General anesthetics (GAs) suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile and intravenous GAs can excite peripheral sensory nerves to cause pain and irritation upon administration. Noxious GAs activate Transient Receptor Potential Ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms remain unknown. Here we show that in addition to activation by pungent GAs, a broad variety of noxious and non-noxious anesthetics inhibit agonist-evoked activation of mammalian but not drosophila TRPA1. Analysis of chimeric drosophila and mouse TRPA1 channels reveal that the 5th transmembrane domain (S5) is critical for anesthetic sensitivity. We identify two amino acids at the N-terminal S5 domain required for the inhibitory effects of GAs and introduction of these residues into dTRPA1 confers GA-induced block in these channels. Further, we show that activation by noxious GAs requires several amino acids in the S5, S6 and the first pore helix previously implicated in binding of the high-affinity antagonist, A-967079. Notably, isoflurane relieves A-967079-mediated inhibition consistent with a competitive interaction and in silico modeling predicts that pungent GAs occupy the A-967079 binding pocket. Thus inhibition and activation by GAs are governed by distinct amino acids in the pore domain region. These data reveal the mechanism by which TRPA1 discriminates between pungent and non-pungent GAs, and provide structural requirements for designing antagonists to counter the noxious properties of GAs.

Abstract 9741: A Novel Mutation Identified in a Large Multi-Generational Family With Long QT Syndrome Reveals Distinct, Disease-Specific Mutation Hot Spots within the CACNA1C- Encoded L-type Calcium Channel

Background: Mutations in the CACNA1C-encoded L-type calcium channel have been associated with Timothy Syndrome (TS) with severe QT prolongation, cardiac hypertrophy, syndactyly, facial dysmorphisms, developmental delay, and sudden death by impaired channel inactivation and increase peak current. Recently, a small number of patients with CACNA1C mutations with only long QT syndrome (LQTS) have been described. Objective: To explore whether CACNA1C mutations associated with TS were mechanistically and clinically distinct from CACNA1C-mediated LQTS. Methods/Results: Illumina HiSeq 2000 platform-based whole exome sequencing with variant alignment using HGSC Mercury analysis and Atlas2 revealed a novel variant CACNA1C-L762F that co-segregated with affected patients of a large, multi-generational family with LQTS and sudden death. The proband presented at 3 years of age with out-of-hospital cardiac arrest and demonstrated QTc of 530. The mutation localized to the DII/DIII intracellular linker domain of the channel in a highly conserved region in close proximity to the 6th DI transmembrane domain. Whole cell patch clamp of heterologously expressed CACNA1C-L762F in TSA201 cells demonstrated slower inactivation tau and increased late current indicating its gain-of-function cellular electrophysiological phenotype. Comprehensive review and topological mapping of all described CACNA1C mutations associated with TS or LQTS revealed disease-specific hotspots. TS-associated mutations localized to cytoplasmic aspects of transmembrane domains while LQTS-associated mutations localized to cytoplasmic N- and C-terminal domains and linker regions. Probands hosting TS mutations demonstrated markedly prolonged QTc values compared to LQTS (618 vs 486 ms, p<0.001) and were younger at presentation (2.7 vs 20.1 years, p<0.002). Supporting these associations, the L762F mutation localized closer to the CACNA1C transmembrane domain than any other LQTS mutation and are associated with a QTc and age of diagnosis reflective of TS-associated mutations. Conclusions: The CACNA1C-L762F mutation is associated with biochemical and clinical features which highlight a molecular and clinical distinction between TS- and LQTS-associated CACNA1C mutations.

Vigna subterranea ammonium transporter gene (VsAMT1): Some bioinformatics insights

Ammonium transporters (AMTs) play a role in the uptake of ammonium, the form in which nitrogen is preferentially absorbed by plants. Vigna subterranea (VsAMT1) and Solanum tuberosum (StAMT1) AMT1s were characterized using molecular biology and bioinformatics methods. AMT1-specific primers were designed and used to amplify the AMT1 internal regions. Nucleotide sequencing, alignment and phylogenetic analysis assigned VsAMT1 and StAMT1 to the AMT1 family. The deduced amino acid sequences showed that VsAMT1 is 92% and 89% similar to Phaseolus vulgaris PvAMT1.1 and Glycine max AMT1 respectively, while StAMT1 is 92% similar to Solanum lycopersicum LeAMT1.1, and correspond to the 5th–10th trans-membrane domains. Residues VsAMT1 D23 and StAMT1 D15 are predicted to be essential for ammonium transport, while mutations of VsAMT1 W1A-L and S87A and StAMT1 S76A may further enhance ammonium transport. In addition to nitrogen uptake from the roots, VsAMT1 may also contribute to interactions with rhizobia.

Eukaryotic G protein-coupled receptors as descendants of prokaryotic sodium-translocating rhodopsins.

Microbial rhodopsins and G-protein coupled receptors (GPCRs, which include animal rhodopsins) are two distinct (super) families of heptahelical (7TM) membrane proteins that share obvious structural similarities but no significant sequence similarity. Comparison of the recently solved high-resolution structures of the sodium-translocating bacterial rhodopsin and various Na+-binding GPCRs revealed striking similarity of their sodium-binding sites. This similarity allowed us to construct a structure-guided sequence alignment for the two (super)families, which highlighted their evolutionary relatedness. Our analysis supports a common underlying molecular mechanism for both families that involves a highly conserved aromatic residue playing a pivotal role in rotation of the 6th transmembrane helix.ReviewersThis article was reviewed by Oded Beja, G. P. S. Raghava and L. Aravind.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0091-4) contains supplementary material, which is available to authorized users.

Expression of temperature-sensitive ion channel TRPM8 in sperm cells correlates with vertebrate evolution.

Transient Receptor Potential cation channel, subfamily Melastatin, member 8 (TRPM8) is involved in detection of cold temperature, different noxious compounds and in execution of thermo- as well as chemo-sensitive responses at cellular levels. Here we explored the molecular evolution of TRPM8 by analyzing sequences from various species. We elucidate that several regions of TRPM8 had different levels of selection pressure but the 4th–5th transmembrane regions remain highly conserved. Analysis of synteny suggests that since vertebrate origin, TRPM8 gene is linked with SPP2, a bone morphogen. TRPM8, especially the N-terminal region of it, seems to be highly variable in human population. We found 16,656 TRPM8 variants in 1092 human genomes with top variations being SNPs, insertions and deletions. A total of 692 missense mutations are also mapped to human TRPM8 protein of which 509 seem to be delateroiours in nature as supported by Polyphen V2, SIFT and Grantham deviation score. Using a highly specific antibody, we demonstrate that TRPM8 is expressed endogenously in the testis of rat and sperm cells of different vertebrates ranging from fish to higher mammals. We hypothesize that TRPM8 had emerged during vertebrate evolution (ca 450 MYA). We propose that expression of TRPM8 in sperm cell and its role in regulating sperm function are important factors that have guided its molecular evolution, and that these understandings may have medical importance.

A Missense Mutation in SLC45A2 Is Associated with Albinism in Several Small Long Haired Dog Breeds.

Homozygosity for a large deletion in the solute carrier family 45, member 2 (SLC45A2) gene causes oculocutaneous albinism (OCA) in the Doberman Pinscher breed. An albino Lhasa Apso did not have this g.27141_31223del (CanFam2) deletion in her SLC45A2 sequence. Therefore, SLC45A2 was investigated in this female Lhasa Apso to search for other possible variants that caused her albinism. The albino Lhasa Apso was homozygous for a nonsynonymous substitution in the seventh exon, a c.1478G>A base change that resulted in a glycine to aspartic acid substitution (p.G493D). This mutation was not found in a wolf, a coyote, or any of the 15 other Lhasa Apso dogs or 32 other dogs of breeds related to the Lhasa Apso. However, an albino Pekingese, 2 albino Pomeranians, and an albino mixed breed dog that was small and long haired were also homozygous for the 493D allele. The colored puppies of the albino Lhasa Apso and the colored dam of the 2 albino Pomeranians were heterozygous for this allele. However, an albino Pug was homozygous for the 493G allele and therefore although we suggest the 493D allele causes albinism when homozygous in several small, long haired dog breeds, it does not explain all albinism in dogs. A variant effect prediction for the albino Lhasa Apso confirms that p.G493D is a deleterious substitution, and a topology prediction for SLC45A2 suggests that the 11th transmembrane domain where the 493rd amino acid was located, has an altered structure.

A novel splicing site mutation of the GPR143 gene in a Chinese X-linked ocular albinism pedigree

Ocular albinism is an X-linked inherited disease characterized by hypopigmentation of the iris and nystagmus. To identify a new disease-causing mutation of ocular albinism, we collected a Han Chinese pedigree with 7 male congenital nystagmus patients over 3 generations. Slit-lamp photography and optical coherence tomography were performed for the proband. Genomic DNA was extracted from a whole blood sample from the proband using the high-salt method. Polymerase chain reaction (PCR) sequencing was carried out for GPR143 and FRMD7 genes. The three-dimensional structures of the wild-type and mutant GPR143 proteins were determined using SWISS-MODEL. The transmission of the disease in the pedigree clearly followed an X-linked pattern. The proband had significant iris and fundus hypopigmentation. Optical coherence tomography showed severe foveal hypoplasias in both eyes of the proband. A novel splicing site (G/C) mutation was found on the boundary of the 6th intron and the 7th exon of the GPR143 gene, resulting in a 9-amino-acid deletion (codons 257-265) in the 6th transmembrane domain of the GPR143 protein. In conclusion, a novel splicing site mutation of the GPR143 gene was found in a Han Chinese congenital ocular albinism pedigree.