The S4–­­S5 linker – gearbox of TRP channel gating

Transient receptor potential (TRP) channels are cation channels which participate in a wide variety of physiological processes in organisms ranging from fungi to humans. They fulfill roles in body homeostasis, are sensors for noxious chemicals and temperature in the mammalian somatosensory system and are activated by light stimulated phospholipase C activity in Drosophila or by hypertonicity in yeast. The transmembrane topology of TRP channels is similar to that of voltage-gated cation channels. TRP proteins assemble as tetramers with each subunit containing six transmembrane helices (S1–S6) and intracellular N- and C-termini. Here we focus on the emerging functions of the cytosolic S4–S5 linker on TRP channel gating. Most of this knowledge comes from pathogenic mutations within the S4–S5 linker that alter TRP channel activities. This knowledge has stimulated forward genetic approaches to identify additional residues around this region which are essential for channel gating and is supported, in part, by recent structures obtained for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2.     

Electrostatic interaction between inactivation ball and T1–S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a “ball and chain” inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the “ball and chain” inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1–S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1–S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.     

Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

The Ca²⁺-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca²⁺ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca²⁺ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca²⁺-dependent gating of KCa3.1 originates from the binding of Ca²⁺ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1^−/− embryos. Inactivating β-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→β-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.     

Distinct properties of Ca2+–calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca²⁺–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca²⁺–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca²⁺–CaM, respectively. This structure is similar to canonical Ca²⁺–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca²⁺–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca²⁺–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca²⁺–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.     

Synthesis and Anti-HBV Activity of Thiouracils Linked via S and N-1 to the 5-Position of Methyl β-D-Ribofuranoside

Abstract

Reverse nucleoside derivatives of 2-(methylsulfanyl)uracils 6a-d were prepared by treating of the sodium salt of 2-(methylsulfanyl)uracils (5a-d) with methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-β-D-ribofuranoside (2). The alkylation of 2-thiouracils 4a-d with methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-ribofuranoside (3) afforded the corresponding S-ribofuranoside derivatives 8a-d. Deisopropylidenation of 6a-d and 8a-d afforded the corresponding deprotected derivatives 7a-d and 9a-d, respectively. The Anti-HBV activity of selected compounds was studied.     

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel's closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.     

A modified rinsing method for the determination of the S,W–S and D + U fraction of protein and starch in feedstuff within the in situ technique

A modified rinsing method for the in situ technique was developed to separate, isolate and characterise the soluble (S), the insoluble washout (W–S) and the non-washout fractions (D + U) within one procedure. For non-incubated bags (t = 0 h), this method was compared with the conventional, Combined Fractionation (CF) method that measures the D + U and S fractions in separate steps and subsequently calculates the W–S fraction. The modified method was based on rinsing of nylon bags in a closed vessel containing a buffer solution (pH 6.2) during 1 h, where shaking speeds of 40, 100, and 160 strokes per minutes (spm) were evaluated, and tested for six feed ingredients (faba beans, maize, oats, peas, soya beans and wheat) and four forages (two ryegrass silages and two maize silages). The average recoveries as the sum of all fractions were 0.972 ± 0.041 for N and 0.990 ± 0.050 for starch (mean ± s.d.). The mean W–S fraction increased with increasing shaking speed and varied between 0.017 (N) and 0.083 (starch) at 40 spm and 0.078 (N) and 0.303 (starch) at 160 spm, respectively. For ryegrass silages, the W–S fraction was absent at all shaking speeds, but was present in the CF method. The modified method, in particular at 40 and 100 spm, reduced the loss of small particles during rinsing, resulting in lower W–S and higher D + U fractions for N and starch compared with the CF method. For soya beans and ryegrass silage, the modified method reduced the S fraction of N compared with the CF method. The results obtained at 160 spm showed the best comparison with those from the CF method. The W–S fraction of the feedstuff obtained at 160 spm contained mainly particles smaller than 40 μm (0.908 ± 0.086). In most feedstuff, starch was the most abundant chemical component in the W–S fraction and its content (726 ± 75 g/kg DM) was higher than in the D + U fraction (405 ± 177 g/kg DM). Alkaline-soluble proteins were the dominant N-containing components in the W–S fraction of dry feed ingredients and its relative content (0.79 ± 0.18 of total N in W–S) was higher than in the D + U fraction (0.59 ± 0.07 of total N in D + U) for all feedstuff except maize. The molecular weight distribution of the alkaline-soluble proteins differed between the W–S and the D + U fractions of all dry feed ingredients, except soya beans and wheat.     

Do sires and juvenile male mice (C57BL/6) contribute to the rearing of the offspring?

Copulation and/or cohabitation with a pregnant female facilitate paternal behavior in male mice. However, their contribution to the rearing of the offspring is still not well understood. Our aims were to investigate the behavior of sires toward own or alien pups; the immediate consequences of the presence of fathers on the offspring and the behavior of the mother; and whether the exposure of juvenile males to newborn siblings, in an overlapping litters context, facilitates paternal behavior in C57BL/6 mice. We found that sires behaved paternally toward alien pups at both postpartum days 3 and 7; did not affect the behavior of the mother (e.g., licking and grooming, retrieval behavior, time in the nest, and crouching postures); and reduced the time offspring stayed alone in the nest. The exposure to newborn siblings did not promote paternal behavior in juvenile males. Therefore, sires are more paternal than usually described in the literature for laboratory mice, suggesting a facultative role in the rearing of the offspring. However, juvenile male mice, in contrast to juvenile females, could be adapted to leave the nest earlier without major contribution to the offspring.     

F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo

The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα–GPR–LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα–GPR–LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3–p21.2 between D1S2896 and D1S457 but outside ABCA4

A severe form of autosomal recessive retinitis pigmentosa (arRP) was identified in a large Pakistani family ascertained in the Punjab province of Pakistan. All affected individuals in the family had night blindness in early childhood, early complete loss of useful vision, and typical RP fundus changes plus macular degeneration. After exclusion of known arRP loci, a genome-wide scan was performed using microsatellite markers at about 10 cM intervals and calculating two-point lod scores. PCR cycle dideoxynucleotide sequencing was used to sequence candidate genes inside the linked region for mutations. RP in this family shows linkage to markers in a 10.5 cM (8.9 Mbp) region of chromosome 1p13.3-p21.2 between D1S2896 and D1S457. D1S485 yields the highest lod score of 6.54 at theta=0. Sequencing the exons and intron-exon boundaries of five candidate genes and six ESTs in this region, OLFM3, GNAI3, LOC126987, FLJ25070, DKFZp586G0123, AV729694, BU662869, BU656110, BU171991, BQ953690, and CA397743, did not identify any causative mutations. This novel locus lies approximately 4.9 cM (7.1 Mbp) from ABCA4, which is excluded from the linked region. Identification and study of this gene may help to elucidate the phenotypic diversity of arRP mapping to this region.     

Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5–SMG7 and SMG6

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14–3–3-like proteins. In contrast, the SMG6 14–3–3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14–3–3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6–UPF1 interaction is mediated by a low-complexity region bordering the 14–3–3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5–SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.     

Partial 3q duplication syndrome and assignment of D3S5 to 3q25–3q28

Summary We report a girl with a de novo duplication of the distal part of the long arm of chromosome 3 and review the literature. Our patient had the facial characteristics and many other anomalies of the partial 3q duplication syndrome. As a hitherto undescribed symptom in partial 3q trisomy syndrome, she had microphthalmia. The karyotype of this girl was interpreted as an inverse duplication of the terminal portion of chromosome 3: 46,XX,inv dup (3)(pter-q28::q28–q25::q28-qter). Quantitative hybridisation studies with 3p and 3q probes gave a consistent 32 ratio of the relative intensities of the q bands in relation to the p bands between patient and control. This confirmed the presence of a 3q duplication and delineated the location of D3S5 to 3q25–3q28.     

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S–5.8S–26S and 5S ribosomal RNA gene families,and the two tandemly repeated DNA sequences

In the genus Carthamus (2n = 20, 22, 24, 44, 64; x = 10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S–26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S–26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n = 20, and the two botanical varieties of B genome C. tinctorius (2n = 24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S–26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n = 20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n = 44), and C. lanatus is one of the progenitors for the hexaploid (2n = 64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2–10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.     

Mapping of the interleukin 5 receptor gene to human Chromosome 3 p25–p26 and to mouse Chromosome 6 close to the Raf-1 locus with polymorphic tandem repeat sequences

Simple-sequence tandem repeat sequences in the 3 UTR of interleukin 5 (IL5)-receptor gene of human and mouse are polymorphic in their length among humans and different strains of mice. In 20 different human Epstein-Barr virus (EBV)-transformed cell lines, six alleles of IL5R could be distinguished. In the mouse, three different alleles are found. With the human-specific IL5R tandem repeat marker in human-rodent somatic cell hybrids, the IL5R gene was mapped to human Chromosome (Chr) 3 p25–p26. With the mouse-specific IL5R tandem repeat sequence in recombinant inbred strains of mice, the Il5r gene was mapped to the distal part of mouse Chr 6 close to the Raf-1 locus.     

Polarity-Dependent Asymmetric Distribution and MEX-5/6–Mediated Translational Activation of the Era-1 mRNA in C. elegans Embryos

The early C. elegans embryo is an attractive model system to investigate fundamental developmental processes. With the exception of mex-3 mRNA, maternally contributed mRNAs are thought to be distributed uniformly in the one-cell embryo. Here, we report and characterize the striking distribution of the mRNA encoding the novel protein ERA-1. We found that era-1 mRNA is enriched in the anterior of the one-cell embryo and present solely in anterior blastomeres thereafter. Although era-1 is not an essential gene, we uncovered that era-1 null mutant embryos are sensitive to slight impairment of embryonic polarity. We found that the asymmetric distribution of era-1 mRNA depends on anterior-posterior polarity cues and on the era-1 3’UTR. Similarly to the era-1 mRNA, the YFP-ERA-1 protein is enriched in anterior blastomeres. Interestingly, we found that the RNA-binding protein MEX-5 is required for era-1 mRNA asymmetry. Furthermore, we show that MEX-5, together with its partially redundant partner MEX-6, are needed to activate era-1 mRNA translation in anterior blastomeres. These findings lead us to propose that MEX-5/6–mediated regulation of era-1 mRNA contributes to robust embryonic development.     

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits

The pore-forming component of voltage-gated calcium channels, α₁ subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca_v1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca_v1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca_v1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly¹⁸² in domain I is involved in the membrane trafficking or surface expression of α₁ subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca_v1.2, although G579Q showed a normal VDI, implying that Gly⁵⁷⁹ in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α₁ subunit to become functional.