A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression

Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions

The Different Microbial Etiology of Prosthetic Joint Infections According to Route of Acquisition and Time After Prosthesis Implantation, Including the Role of Multidrug-Resistant Organisms

The aim of our study was to characterize the etiology of prosthetic joint infections (PJIs)-including multidrug-resistant organisms (MDRO)-by category of infection. A multicenter study of 2544 patients with PJIs was performed. We analyzed the causative microorganisms according to the Tsukayama's scheme (early postoperative, late chronic, and acute hematogenous infections (EPI, LCI, AHI) and "positive intraoperative cultures" (PIC)). Non-hematogenous PJIs were also evaluated according to time since surgery: 12 months. AHIs were mostly caused by Staphylococcus aureus (39.2%) and streptococci (30.2%). EPIs were characterized by a preponderance of virulent microorganisms (S. aureus, Gram-negative bacilli (GNB), enterococci), MDROs (24%) and polymicrobial infections (27.4%). Conversely, coagulase-negative staphylococci (CoNS) and Cutibacterium species were predominant in LCIs (54.5% and 6.1%, respectively) and PICs (57.1% and 15.1%). The percentage of MDROs isolated in EPIs was more than three times the percentage isolated in LCIs (7.8%) and more than twice the proportion found in AHI (10.9%). There was a significant decreasing linear trend over the four time intervals post-surgery for virulent microorganisms, MDROs, and polymicrobial infections, and a rising trend for CoNS, streptococci and Cutibacterium spp. The observed differences have important implications for the empirical antimicrobial treatment of PJIs.Acknowledgments: This work was supported by the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness (grant number PI15/1026) (Co-funded by European Regional Development Fund/European Social Fund "Investing in your future"). REIPI (Spanish Network for Research in Infectious Disease) is supported by the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness, and by the European Development Regional Fund “A way to achieve Europe”

Cryo-EM structures and functional properties of CALHM channels of the human placenta

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we observed that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences

Cryo-EM structures and functional properties of CALHM channels of the human placenta.

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we found that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences

Stereochemical significance of O to N atom interchanges within cationic helicenes: experimental and computational evidence of near racemization to remarkable enantiospecificity

Oxygen atoms of cationic dioxa and azaoxa [6]helicenes can be exchanged by amino groups to form azaoxa and diaza [6]helicenes respectively. The mild reaction conditions developed herein allow the construction of libraries of derivatives with sensitive and/or functionalized side chains. Using enantioenriched dioxa or azaoxa helicene precursors, these exchanges lead to either near racemization (es 3%) or to a remarkable enantiospecificity (es up to 97%). This unusual behavior is fully characterized via experimental and computational mechanistic evidence. Based on these investigations, the enantiospecificity of the first transformation can be improved to 57–61%

Anion Transport with Pnictogen Bonds in Direct Comparison with Chalcogen and Halogen Bonds

In this Communication, we introduce transmembrane anion transport with pnictogen-bonding compounds and compare their characteristics with chalcogen- and halogen-bonding analogues. Tellurium-centered chalcogen bonds are at least as active as antimony-centered pnictogen bonds, whereas iodine-centered halogen bonds are 3 orders of magnitude less active. Irregular voltage-dependent single-channel currents, high gating charges, and efficient dye leakage support for the formation of bulky, membrane-disruptive supramolecular amphiphiles due to “too strong” binding of anions to tris(perfluorophenyl)stibanes. In contrast, the chalcogen-bonding bis(perfluorophenyl)tellanes do not cause leakage and excel as carriers with nanomolar activity, with P(Cl/Na) = 10.4 for anion/cation selectivity and P(Cl/NO3) = 4.5 for anion selectivity. The selectivities are lower with pnictogen-bonding carriers because their membrane-disturbing 3D structure also affects weaker binders (P(Cl/Na) = 2.1, P(Cl/NO3) = 2.5). Their 2D structure, directionality, hydrophobicity, and support from proximal anion−π interactions are suggested to contribute to the unique power of chalcogen bonds to transport anions across lipid bilayer membranes

A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of Ca2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca 2+ Influx

Mutations in the CACNA1A gene, encoding the pore-forming Ca2.1 (P/Q-type) channel α subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α domain III (ΔF1502). Functional studies of ΔF1502 Ca2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba 2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α mutation, detected by whole-exome sequencing, and analyze its functional consequences on Ca2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca 2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca 2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca 2+ current density through ΔF1502 Ca2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of Ca2.1 within a wide range of voltage depolarization. ΔF1502 also slowed Ca2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on Ca2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca 2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function Ca2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A -associated phenotypic spectru

ΔF1502 effects on Ca²⁺ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).

<p>(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) Ca_V2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca²⁺ current density through Ca_V2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s <i>t</i> test) and the significant left-shift induced by ΔF1502 on the Ca_V2.1 voltage-dependent activation is also noticed (right panel: WT V_{1/2 act} = 2.32 ± 1.18 mV (n = 10) <i>versus</i> ΔF1502 V_{1/2 act} = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s <i>t</i> test). (B) Representative Ca²⁺ current traces evoked by every 200^th pulse of a 42 Hz train of medium (2 ms) APWs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#sec002" target="_blank">Materials and Methods</a> for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) Ca_V2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca²⁺ current density through WT and ΔF1502 Ca_V2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V_{1/2 act} values for WT and ΔF1502 Ca_V2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca²⁺ influx normalized by cell size (Q_Ca²⁺) in response to every 5^th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) Ca_V2.1 channels.</p

Evolutionary conservation of the F1502 residue and predicted location at the channel pore.

<p>(A) Sequence alignment of individual S6 segments at domains I to IV (DI-DIV) of human Ca_V2.x channel α₁ subunits (P/Q type Ca_V2.1; N-type Ca_V2.2; R-type Ca_V2.3), human Ca_V1.x (L-type) channel α₁ subunits, and the bacterial sodium channel Na_VAb (top); sequence alignment of S6-DIII of Ca_V2.1 channels from different species (as indicated). The three Phenylalanine’s group (in red) is conserved in the human Ca_V2.1 channel α_1A subunit, where F1502 is located at the third position. This particular amino acid residue is only conserved in S6-DIII of Ca_V2 type channels. The phenylalanine’s group is totally conserved in S6-DIII of Ca_V2.1 channels from different species. The alignments were performed with T-Coffee (T-Coffee). (B,C,D) Location of the F1502 homologous methionine residue (M209), using the Na_VAb structure as a model (PDB 4EKW). A methionine residue is also present at the F1502 position in L-type channels. The side view (B) show a red highlighted M209 residue in Na_VAb, which lines the inner pore vestibule of the channel. A view from the cytoplasm looking up through the channel pore show the arrangement of M209 residue in the four Na_VAb subunits (C), and a zoom of the pore region from the same view is shown in (D). Images were generated using UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#pone.0146035.ref079" target="_blank">79</a>].</p