Heterologous Expression In HEK293 Cells Research Articles

Ancestral Sequence Reconstruction of Von Willebrand Factor Reveals Highly Conserved Structure/Function

Von Willebrand factor (VWF) is a large multimeric glycoprotein of critical importance for both primary and secondary hemostasis due to its role in platelet activation and stabilization of coagulation factor VIII (FVIII), respectively. Mutations affecting VWF synthesis, secretion, or function result in one of 6 distinct subtypes of von Willebrand disease (VWD) with mutations inhibiting FVIII binding classified as type 2N. Due to low circulating levels of plasma FVIII, type 2N patients can be misdiagnosed with hemophilia A. Because the clearance of FVIII is dominated by its association with VWF, and given recent evidence demonstrating an immuno-protective role of VWF for FVIII, understanding the molecular interaction between FVIII and VWF remains critically important for both hemophilia A and type 2N VWD therapeutic development. However, limited structural information remains a major limitation for rational design studies. Recently, ancestral sequence reconstruction (ASR) was performed to infer the sequences of FVIII predicted to have existed throughout mammalian evolution. ASR provides a platform for high-resolution mapping of sequential differences within a phylogeny while maintaining a high probability of retaining function. Sequential changes in the amino acid sequence of FVIII appears to have resulted in altered biochemical properties including biosynthesis and stability. In this study, we demonstrate that a previously characterized ancestral (An) FVIII molecule with 95% identity to human FVIII, termed An53-FVIII, exhibits a 2.5 to 5-fold increase in binding to human VWF over commercial recombinant FVIII products, suggesting a dynamic association throughout evolution. We sought to further explore the FVIII-VWF interaction through ASR of VWF and biochemical analysis.A phylogenetic tree was constructed for VWF and the ancestral amino acid sequences predicted through Bayesian inference. A compiled list of 59 extant sequences allowed accurate prediction of the sequences extending beyond the class Mammalia. Of the resulting 50, 5 An-VWF sequences representing significant branch points in primate, rodent, and ungulate evolution were selected for synthesis de novo and subsequent biochemical characterization. All sequences, including an additional human (h) VWF cDNA, were human codon optimized (co). These sequences are predicted to span 105 million years of evolution and contain 81 to 364 amino acid substitutions (Table 1). Heterologous expression in HEK293 cells demonstrated that all An-VWF sequences resulted in secreted antigen as determined by ELISA using anti-human VWF polyclonal antibodies. Furthermore, immunohistochemistry of HEK293 cells expressing An-VWF revealed the formation of pseudo Weibel-Palade bodies by all VWF proteins. Stably transected, clonal producer cell lines were generated and utilized for recombinant VWF production and purification using heparin-sepharose affinity chromatography. Consistent with our IHC studies, multimer analysis of the purified proteins revealed the presence of high molecular weight multimers equivalent to human plasma VWF. To test the functionality of An-VWF, the VWF:GPIbM assay was performed. Compared to an observed specific activity of 45 IU/mg VWF for cohVWF, An-VWF molecules displayed specific activities ranging from 6 to 94 IU/mg. These data suggest that VWF and GPIb may have co-evolved to regulate platelet adhesion and activation. Hydrodynamic injection of An63-, An88-, and An101-VWF plasmid DNA into VWF deficient mice revealed plasma VWF levels of 1.44 ± 0.48, 0.72 ± 0.16, and 2.9 μg/mL at 24 hours after administration, respectively (n = 4, 3, and 1). Additionally, endogenous mouse (m) FVIII levels in these mice were elevated to 61 ± 9, 75 ± 16, and 67% of normal levels from a basal 12.9 ± 2.4%. This data demonstrates that liver synthesized An-VWF retains FVIII association and stabilization. Molar ratios of An88-VWF:mFVIII are 6:1 compared to 14:1 and 25:1 for An63- and An101-VWF, respectively, supporting the coevolution hypothesis for VWF and FVIII. Cumulatively, this study further validates the utility of ASR for the generation of functional sequences for complex proteins with a high propensity for inactivating mutations. Further analysis of ancestral sequences may provide greater resolution of amino acid sequences responsible for biochemical properties including clearance and FVIII association. [Display omitted] DisclosuresGaucher:General Genomics: Equity Ownership.

Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells.

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson’s Disease

In medium-size, spiny striatal neurons of the direct pathway, dopamine D₁- and adenosine A₁-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A₁-receptor (A₁R), A₁R-G279S^7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA–responsive Parkinson’s disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A₁-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A₁R-G279S^7.44 was enhanced by about 2 and 8 K when compared with wild-type A₁-receptor and A₁R-Y288A^7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A₁R-G279S^7.44 (73 ± 23 kJ/mol) than in wild-type A₁R (135 ± 4 kJ/mol) or in A₁R-Y288A^7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A₁R-G279S^7.44 was more effective in promoting guanine nucleotide exchange than wild-type A₁R. We detected similar levels of complexes formed between D₁-receptors and wild-type A₁R or A₁R-G279S^7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D₁-receptor and A₁R-G279S^7.44 than in those coexpressing wild-type A₁R. These observations predict enhanced inhibition of dopaminergic signaling by A₁R-G279S^7.44 in vivo, consistent with a pathogenic role in Parkinson’s disease. <h3>SIGNIFICANCE STATEMENT</h3> Parkinson’s disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A₁-receptor antagonizes responses elicited by dopamine D₁-receptor. We show that this activity is more pronounced in a mutant version of the A₁-receptor (A₁R-G279S^7.44), which was identified in individuals suffering from early-onset Parkinson’s disease.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson's Disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.

Clinical and Functional Characterization of the Recurrent TUBA1A p.(Arg2His) Mutation.

The TUBA1A gene encodes tubulin alpha-1A, a protein that is highly expressed in the fetal brain. Alpha- and beta-tubulin subunits form dimers, which then co-assemble into microtubule polymers: dynamic, scaffold-like structures that perform key functions during neurogenesis, neuronal migration, and cortical organisation. Mutations in TUBA1A have been reported to cause a range of brain malformations. We describe four unrelated patients with the same de novo missense mutation in TUBA1A, c.5G>A, p.(Arg2His), as found by next generation sequencing. Detailed comparison revealed similar brain phenotypes with mild variability. Shared features included developmental delay, microcephaly, hypoplasia of the cerebellar vermis, dysplasia or thinning of the corpus callosum, small pons, and dysmorphic basal ganglia. Two of the patients had bilateral perisylvian polymicrogyria. We examined the effects of the p.(Arg2His) mutation by computer-based protein structure modelling and heterologous expression in HEK-293 cells. The results suggest the mutation subtly impairs microtubule function, potentially by affecting inter-dimer interaction. Based on its sequence context, c.5G>A is likely to be a common recurrent mutation. We propose that the subtle functional effects of p.(Arg2His) may allow for other factors (such as genetic background or environmental conditions) to influence phenotypic outcome, thus explaining the mild variability in clinical manifestations.

Identification of Critical Residues for Anion Conductance in the TMEM16A Channel

TMEM16A (also called DOG-1 and Anoctamin-1) is a transmembrane-localized, homodimeric protein that makes up the pore forming subunit of a calcium-activated chloride channel found in epithelial cells, tumor cells and a subset of DRG neurons. However, the structural and biophysical properties of this channel remain poorly understood. Here, we aimed to identify residues that determine the conductance properties of TMEM16A, by using site-directed mutagenesis and heterologous expression in HEK293 cells followed by patch clamp electrophysiology. Using a recently crystallized fungal orthologue of the TMEM16 family as a model, we identified several residues clustered around the putative pore region that are critical for normal channel conductance of anions. Mutations at these residues also influenced the binding properties of several known channel inhibitors. We conclude that the identified residues are likely to be directly involved in dictating the physical properties of the anion conduction pathway, and may also help to confirm the as yet unknown location of the TMEM16A channel's anion pore.

Larvae of the small white butterfly, Pieris rapae, express a novel serotonin receptor.

The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G-protein-coupled receptors. Five 5-HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT7 receptors. We isolated a cDNA (Pr5-HT8 ) from larval Pieris rapae, which shares relatively low similarity to the known 5-HT receptor classes. After heterologous expression in HEK293 cells, Pr5-HT8 mediated increased [Ca(2+)]i in response to low concentrations (< 10 nM) of 5-HT. The receptor did not affect [cAMP]i even at high concentrations (> 10 μM) of 5-HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5-HT8 was also activated by various 5-HT receptor agonists including 5-methoxytryptamine, (±)-8-Hydroxy-2-(dipropylamino) tetralin, and 5-carboxamidotryptamine. Methiothepin, a non-selective 5-HT receptor antagonist, activated Pr5-HT8 . WAY 10635, a 5-HT1A antagonist, but not SB-269970, SB-216641, or RS-127445, inhibited 5-HT-induced [Ca(2+)]i increases. We infer that Pr5-HT8 represents the first recognized member of a novel 5-HT receptor class with a unique pharmacological profile. We found orthologs of Pr5-HT8 in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate-specific receptor because there were no similar receptors in mammals. We isolated a cDNA (Pr5-HT8) from larval Pieris rapae, which shares relatively low similarity to the known GPCRs. After heterologous expression in HEK293 cells, Pr5-HT8 mediated increased [Ca(2+)]i in response to low concentrations (< 10 nM) of 5-HT and various 5-HT receptor agonists. We found orthologs of Pr5-HT8 in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee, parasitoid wasps, or mammals.

TMEM16C facilitates Na+-activated K+ currents in rat sensory neurons and regulates pain processing

TMEM16C belongs to the TMEM16 family, which includes the Ca2+-activated Cl– channels (CaCCs) TMEM16A and TMEM16B and a small conductance Ca2+-activated, non-selective cation channel (SCAN), TMEM16F. Here we report that in rat dorsal root ganglia (DRG) TMEM16C is expressed mainly in the IB4 positive, non-peptidergic nociceptors that also express the sodium-activated potassium (KNa) channel Slack. Together these channel proteins promote KNa channel activity and dampen neuronal excitability. DRG from TMEM16C knock out rats have reduced Slack expression, broadened action potential and increased excitability. Moreover, the TMEM16C knock out rats as well as rats with Slack knockdown via intrathecal injection of siRNA exhibit increased thermal and mechanical sensitivity. Experiments involving heterologous expression in HEK293 cells further show that TMEM16C modulates the single channel activity of Slack channels and increases its sodium sensitivity. Our study thus reveals that TMEM16C enhances KNa channel activity in DRG neurons and regulate the processing of pain messages.

Human melanopsin forms a pigment maximally sensitive to blue light ( λ max ≈ 479 nm) supporting activation of G q /11 and G i/o signalling cascades

A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both Gq and Gi/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some Gs activity in addition to a strong Gq/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca(2+) channel (CaV2.1α1). Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Functional analysis of the K1336E mutation revealed a reduction of Ca(2+) current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca(2+) channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

A Cytoplasmic Inter-Subunit Salt Bridge, Kir6.1R347/SUR2AE1318, Contributes to Allosteric Information Transmission in Kir6.14/SUR2A4 Channel Complexes

KATP channels are hetero-octamers composed of pore forming Kir6 (Kir6.1 or Kir6.2) and a regulatory sulphonylurea receptor (SUR1, SUR2A or SUR2B) subunits. Previous studies have revealed a salt bridge between Kir6.2K338 and SUR2AE1318 that is involved in inter-subunit transmission of allosteric information (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether Kir6.1R347, which corresponds to Kir6.2K338, plays a similar role in Kir6.1-containing channels.Whole-cell patch clamp recording was employed to assess channel sensitivity to pinacidil and glibenclamide following mutation of single residues or reinstatement of the proposed salt bridge by paired charge reversals in full length Kir6.1/SUR2A channel subunits after heterologous expression in HEK-293 cells. A single point mutation, Kir6.1R347E expressed with wild type SUR2A increased significantly (p < 0.006) the sensitivity to activation by pinacidil of Kir6.1R347E/SUR2AWT channels (EC50 = 0.71 ± 1.21 µM) versus wild type Kir6.1/SUR2A channels (EC50 = 43.90 ± 1.28 µM). Reinstatement of the cytoplasmic electrostatic interaction in the Kir6.1R347E/SUR2AE1318R subunit combination reversed the sensitivity to pinacidil to near wild type (EC50 = 23.5 ± 1.3 µM, p < 0.028). Furthermore, glibenclamide sensitivity was reduced significantly in the Kir6.1R374E/SUR2AWT channel (IC50 = 241 ± 1.09 nM, p < 0.015) and restored in Kir6.1R347E/SUR2AE1318R (IC50 = 13.75 ± 1.11 nM, p < 0.080) versus wild type Kir6.1/SUR2A channel (IC50 = 6.14 ± 1.13 nM). These data indicate that, like Kir6.2K338, Kir6.1R347 makes a crucial contribution to allosteric information transmission from SUR2A to the channel pore through inter-subunit salt bridge formation with SUR2A1318.

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA(A) receptor, as with other cys-loop ligand-gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β(2) Tyr97, β(2) Tyr157, and β(2) Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK-293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA(A) receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β(2) Tyr157 and β(2) Tyr205 are more detrimental than β(2) Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.

The characterization of a concentration-sensitive α-adrenergic-like octopamine receptor found on insect immune cells and its possible role in mediating stress hormone effects on immune function

Octopamine (OA), the insect equivalent of norepinephrine, links the nervous system and immune system in insects. This study examines the underlying molecular mechanisms (i.e. second messenger systems) mediating OA effects on insect immune cells. At low concentrations (<1μM), OA stimulatedhemocyte spreading and phagocytosis in the larval Lepidopteran (caterpillar) Chilo suppressalis, whereas at high concentrations (>10 μM), OA inhibited hemocyte spreading and phagocytosis. Similarly, OA concentration had differential effects on two intracellular signaling pathways, Ca(2+) and cAMP. Low concentrations of OA increased intracellular Ca(2+), but only high concentrations of OA (>1 μM) led to an increase in both Ca(2+) and cAMP. We identified an α-adrenergic-like octopamine receptor in this species (CsOA1) and confirmed that it is expressed in hemocytes. After heterologous expression in HEK-293 cells, the CsOA1 receptor produced the same OA concentration-dependent responses on intracellular Ca(2+) and cAMP as had been observed in hemocytes. These findings support earlier work showing that OA has both stimulatory and suppressive effects on immune responses, depending on the OA concentration. Our evidence suggests that these biphasic effects are mediated by an octopamine receptor signaling through intracellular Ca(2+) and cAMP second messenger pathways. Stress hormones/neuromodulators have complex effects on immune function in animals across phyla. This complexity may be mediated, in part, by conserved connections between adrenergic-like G-coupled protein receptors and second messenger systems.

Molecular cloning and characterization of dog TRPA1 and AITC stimulate the gastrointestinal motility through TRPA1 in conscious dogs

Transient receptor potential ankyrin1 (TRPA1) is a non-selective cation channel activated by cold stimuli under 17 °C, mechanosensation, and pungent irritants such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA). In this study, we cloned the dog orthologue of TRPA1 for the first time and induced its heterologous expression in HEK293 cells to investigate its functional properties using a fluorescence imaging plate reader-based Ca 2+ influx assay. Moreover, we examined the effect of AITC on gastrointestinal motility in dogs. At the amino acid level, the sequence of dog TRPA1 was 82–83% identical to that of human, mouse, and rat orthologues. TRPA1 is strongly expressed in the brain, cerebellum, stomach, pancreas, and small and large intestine of dogs. Like other mammalian orthologues, TRPA1 agonists, including AITC, CA, allicin, and diallyl disulfide, evoked a concentration-dependent increase in intracellular Ca 2+ influx in dog TRPA1-expressing cells. AITC stimulated gastric antrum and jejunum motility and induced the occurrence of giant migrating contractions in the colon of fasted dogs. The effects of AITC were inhibited by ruthenium red, a TRPA1 antagonist. These results indicate that AITC stimulated the gastrointestinal motility through TRPA1 in conscious dogs.

Effects of divalent cations and spermine on the K+ channel TASK‐3 and on the outward current in thalamic neurons

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca2+ or Mg2+. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.

Gγ13 Interacts with PDZ Domain-containing Proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c).

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Dissection of the Functional Differences between Human Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 Isoenzymes by Steady-state and Transient Kinetic Analyses

Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.

Olfactory specific chymotrypsin-like serine protease from the aesthetasc tegumental gland of the lobster, Homarus americanus

Numerous proteases and protease inhibitors are expressed in the lobster olfactory organ. One of these proteases, olfactory enriched transcript 03 (OET-03), is particularly interesting because its mRNA is expressed only in one cell type of the olfactory organ of the American lobster, Homarus americanus. We have obtained a full-length cDNA clone of OET-03. The predicted amino acid sequence is equally divided between a novel N-terminal domain and a conserved serine protease catalytic domain at the C-terminus. Heterologous expression in HEK293 cells allowed protease assays demonstrating that OET-03 cleaved a specific serine protease substrate, N-alpha benzoyl-L-arginine p-nitroanilide, but did not cleave a substrate of metalloproteases and cysteine proteases. OET-03 protease activity was significantly inhibited by the chymotrypsin-like protease inhibitor, tosyl-L-phenylalanine chloromethyl ketone, but not by the general protease inhibitor, phenylmethylsulfonyl fluoride. Immunoreactivity for OET-03 was detected only in the cells previously shown to contain OET-03 mRNA. The cytoplasm of these cells was filled with enlarged smooth endoplasmic reticulum (a characteristic of secretory cells) that appeared to expand into large electron-translucent areas at the ventral end of the cell. The ventral ends of these secretory cells were apposed to phalloidin-labeled triangular structures reminiscent of the beginnings of the ducts of crustacean tegumental glands. This putative gland was found only in association with the aesthetasc sensory units of the olfactory organ, hence the name, aesthetasc tegumental gland.

Glycosylation of asparagines 136 and 184 is necessary for the α 2δ subunit-mediated regulation of voltage-gated Ca 2+ channels

The Ca Vα 2δ auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage-gated Ca 2+ channels. One of the most prominent roles of Ca Vα 2δ is to increase whole-cell Ca 2+ current amplitude. Using N-glycosidase F and truncated forms of Ca Vα 2δ, earlier studies suggested an important role for N-linked glycosylation in current stimulation. Here, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to examine the impact of individual glycosylation sites within the Ca Vα 2δ subunit on the regulation of Ba 2+ currents through recombinant Ca 2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular α 2 domain of the protein that are functional. Substitution of asparagines for glutamines at amino acid positions 136 and 184 rendered these sites non-functional as shown by patch-clamp experiments. These results corroborate that N-glycosylation is required for the Ca Vα 2δ subunit-induced current stimulation and suggest that sites N136 and N184 are directly involved in this action. Likewise, N136Q and N184Q mutations prevented whole-cell current stimulation without altering its kinetic properties, suggesting a regulation on the number of functional channels at the plasma membrane.