Two epilepsy-associated variants in KCNA2 (KV 1.2) at position H310 oppositely affect channel functional expression.

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV 1.2 by KV ß1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of KV 1.2 voltage-activated, K+ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels. The p.H310Y variant caused 'dual gain of function', increasing both KV 1.2-channel activity and the number of KV 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of KV 1.2 by KV ß1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of KV 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.

An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding.

KEY POINTS: Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel CaV 1.2. Arrhythmia mutations in CaM can lead to loss of Ca2+ binding, uncoupling of Ca2+ binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease. ABSTRACT: Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca2+ channel CaV 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the CaV 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca2+ binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca2+ -saturating conditions when unbound to the IQ domain. Ca2+ binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca2+ ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.

Adsorption of divalent cadmium by calcified iron-embedded carbon beads.

A novel iron-embedded carbon bead was prepared by the calcination of a calcium alginate gel bead mixed with iron nanoparticles coated by polydopamine. The prepared iron-embedded carbon bead was characterized by infrared spectrum analysis, X-ray diffraction, Raman spectroscopy, vibrating sample magnetometry, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron microscopy. It was discovered that the novel structure efficaciously prevented the agglomeration of iron nanoparticles. Additionally, the effects of dose, pH, exposure time, temperature and initial concentration on the adsorption of Cd(ii) were studied, and the reusability of the material was analyzed. Fe/SA-C showed high Cd(ii) removal capability (220.3, 225.7, 240.8 mg g-1 at 288, 298, 308 K), easy recoverability and high stability. In addition, some slightly different interpretations of the adsorption mechanism are given. This study clearly revealed that Fe/SA-C has potential application in the removal of Cd(ii).

Efficient removal of aqueous hexavalent chromium by activated carbon derived from Bermuda grass.

In this study, a novel raw material, Bermuda grass had been devised for the synthesis of activated carbon (BGAC) and enhanced the pore volume by potassium hydroxide. The effects of different factors on activated carbon products by orthogonal experiment was optimized. The synthesized BGAC was characterized by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET) analysis, energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS) and then, Cr(VI) removal batch experiments were conducted to investigate the Cr(VI) removal performance. Kinetic model and Weber-Morris diffusion model were fitted to the Cr(VI) removal process indicated that the chemisorption was the predominant removal mechanism and intraparticle diffusion was the sole rate-controlling mechanism. Langmuir isotherms could fit the experimental date well, which revealed that the adsorption of Cr(VI) ions was monolayer adsorption and the maximum adsorption capacity could be reached at 403.23 mg g-1. The results also promulgated that BGAC had an excellent potential on Cr(VI) removal. The removal processes were considered to comprise adsorption, reduction, precipitation and other ways through the study of the removal mechanism.

A highly selective H/D exchange reaction of 1,4-dihydropyridines.

Herein, we report a simple, economical, and effective acid-mediated method for the in situ deuteration of Hantzsch esters and their 4-substituted derivatives, including some drugs that constitute important calcium channel blockers which are effective for hypertension treatment. Hydrogen isotope exchange occurred selectively at α-alkyl C-H bonds in the 2,6-substituents.

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel.

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the CALM genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (CaV1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the CaV1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca2+ by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca2+-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca2+-dependent inactivation of the voltage-gated calcium channel.

Influence of type and proportion of lyoprotectants on lyophilized ginsenoside Rg3 liposomes.

OBJECTIVES: To improve stability and shelf life, lyophilized formulations of 20(R)-Ginsenoside Rg3 liposomes (G-Rg3-Ls) were prepared. METHODS: Glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-ß-cyclodextrin and polyethylene glycol were used as single lyoprotectant and then compared in terms of their ability to protect lyophilized G-Rg3-Ls. Further, a glucose-mannitol complex was used to determine the optimal lyophilized preparation. The analysis of lyophilized liposomes or lyoprotectant was further investigated by scanning electron microscopy, thermogravimetry-differential thermal analysis, X-ray diffractometry and Fourier transform infrared spectroscopy. Cytotoxicity assay was used to assess the cyto-inhibition of freshly prepared and lyophilized liposomes. KEY FINDINGS: When the ratio of glucose-mannitol to phospholipids was 4 : 2 : 1 (w/w) the lyophilized G-Rg3-Ls exhibited good appearance, high DRR (86.52% ± 5.02%), small change in particle size (45.83 ± 0.50%) and short rehydration reconstruction time (8.3 ± 1.5 s). All indices were considerably better than those of each single protective agent. Results indicated that when the two lyoprotectants were combined, the stabilizing effect of glucose and shaping effect of mannitol were well maintained. The cyto-inhibition of freshly prepared and lyophilized G-Rg3 liposomes showed that lyophilization did not affect the bioactivity of G-Rg3. CONCLUSIONS: The application of glucose-mannitol composite lyoprotectants can obtain a good G-Rg3 lyophilized preparation.

Ginsenoside Rg3 bile salt-phosphatidylcholine-based mixed micelles: design, characterization, and evaluation.

20(R)-Ginsenoside Rg3 (G-Rg3) has good inhibition of tumor angiogenesis and anti-tumor effect. However, its poor aqueous solubility and liposolubility are not ideal for clinical applications. In this study, a G-Rg3 bile salt-phosphatidylcholine-based mixed micelle system (BS-PC-MMS) was prepared. The optimization of G-Rg3 BS-PC-MMS was carried out using response surface methodology based on a central composite design. The encapsulation efficiency (EE) and light transmission (LT) of the optimized formulation were 90.69±2.54% and 99.10±3.12%, respectively. The average particle size of micelles was 20 nm. To increase the stability of G-Rg3 BS-PC-MMS, the lyophilized formulation of micelles was prepared. The G-Rg3 BS-PC-MMS did not produce hemolysis of erythrocytes within a certain concentration range and exhibited a good inhibition of tumor cells. The chick embryo chorioallantoic membrane assay results showed that the G-Rg3 BS-PC-MMS significantly inhibited angiogenesis. The G-Rg3 BS-PC-MMS is thus shown to be a safe, stable, and promising drug delivery system.

A role for the budding yeast separase, Esp1, in Ty1 element retrotransposition.

Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition - one to remove cohesin and the second to target Ty1-IN to chromatin.

Preparation and characterization of stable pH-sensitive vesicles composed of α-tocopherol hemisuccinate.

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of -74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca(2+)-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t(1/2)) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.

[Recent advances in the study of accelerated blood clearance phenomenon of PEGylated liposomes].

It is generally believed that liposomes modified with polyethylene glycol (PEG) have no or lower immunogenicity. However, based on many recent literatures, when the PEGylated liposomes were repeatedly applied to the same animal, the immune responses occurred. The first injection of PEGylated liposomes resulted in a reduction in the circulation time and an increase in hepatic and splenic accumulation of the second dose of PEGylated liposomes in a time-interval, which was called "accelerated blood clearance (ABC)" phenomenon. Such immunogenicity of PEGylated liposomes presents a barrier in the research of liposomal formulations and their use in the clinics. This review focused on the definition, the method of verification, the development of the reason for ABC phenomenon, influencing factors of ABC phenomenon, and discussed if other PEGylated nanocarriers also induce ABC phenomenon.