Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains.

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hHV1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hHV1, mutation of this motif did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the HV1 protein. However, the data are fully explainable if HV1 preferentially associates with cholesterol-dependent lipid domains, or "rafts." The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that HV1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hHV1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hHV1 binding to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

Voltage-Gated Proton Channels in the Tree of Life.

With a single gene encoding HV1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian HV1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of HV1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of HV channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding HV channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different HV channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.

Proton channels in molluscs: A new bivalvian-specific minimal HV 4 channel.

Recently, three proton channels (HV ) have been identified and characterized in Aplysia californica (AcHV 1-3). Focusing on AcHV 1 and AcHV 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV 1 and to AcHV 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV 4. This new family has high homology to AcHV 1 and AcHV 2 and was identified only in bivalvian molluscs (13 species, 12 full-length sequences). Typically, these channels possess an extracellular S1-S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV 1 channels (~ 13 amino acids) and the much larger loops of molluscan HV 2 channels (> 65 amino acids). The characteristic voltage-sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV 4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV 4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH- and voltage-regulated gating. This work is the first description of a HV 4 type channel, adding a new member to the recently expanded family of proton channels.

Trapped Pore Waters in the Open Proton Channel HV 1.

The voltage-gated proton channel, HV 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10-12 cm3 s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the HV 1 wild-type channel and the D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while interrupting the water wire across the membrane.

Guided Access Cavity Preparation Using a New Simplified Digital Workflow.

INTRODUCTION: This study evaluated the precision of a simplified workflow using only preoperative cone-beam computed tomographic (CBCT) scans to gain guided access to root canals of extracted mandibular molars. A workflow using CBCT scanning associated with 3-dimensional oral scanning was used as a reference for comparison. The influence of the presence of coronal restoration in the simplified workflow was also evaluated. METHODS: Forty-five mandibular molars were randomized into 3 groups: a control group in which digital planning was performed with CBCT and oral scanning and 2 experimental groups in which digital planning was performed only with CBCT examination. In experimental group 1, teeth had no coronal restorations, whereas in group 2 teeth presented with coronal composite restorations. After digital planning, the teeth were accessed using the guides, and a new CBCT scan was made to overlap the pre- and postoperative examinations. Precision was measured by calculating the deviation between the planned and prepared cavities in millimeters and angle. Data were compared using 1-way analysis of variance (P < .05). RESULTS: All root canals were accessible after access preparation in all tested groups. Deviations of the planned and prepared access cavities were low, with a mean value of 0.55, 0.58, and 0.47 mm and 1.98°, 2.45°, and 1.43° for the control group, group 1, and group 2, respectively. No significant differences in millimeters or angle were observed among the 3 tested groups (P > .05). CONCLUSIONS: The simplified digital workflow using only CBCT examination allowed a high level of precision in obtaining access in extracted molars with and without coronal restoration, presenting similar results compared with the digital workflow using CBCT and 3-dimensional oral scanning.

Unexpected expansion of the voltage-gated proton channel family.

Voltage-gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage-gated proton channels (HV ) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV 1, AcHV 2, and AcHV 3) are distributed throughout the whole animal. Patch-clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV 1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV 3, which differs from AcHV 1 and AcHV 2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species.

Editorial for 'Issue focus on 2nd Costa Rica biophysics symposium - March 11th-12th, 2021'.

This Editorial describes both the motivation for, and the five articles appearing in, the Issue Focus dedicated to the 2nd Costa Rica Biophysics Symposium which was held in March 2021. Some recent history about both the symposium and developments in science occurring within Costa Rica is described.

The pH-dependent gating of the human voltage-gated proton channel from computational simulations.

Gating of the voltage-gated proton channel HV1 is strongly controlled by pH. There is evidence that this involves the sidechains of titratable amino acids that change their protonation state with changes of the pH. Despite experimental investigations to identify the amino acids involved in pH sensing only few progress has been made, including one histidine at the cytoplasmic side of the channel that is involved in sensing cellular pH. We have used constant pH molecular dynamics simulations in symmetrical and asymmetrical pH conditions across the membrane to investigate the pH- and ΔpH-dependent gating of the human HV1 channel. Therefore, the pKa of every titratable amino acids has been assessed in single simulations. Our simulations captured initial conformational changes between a deactivated and an activated state of the channel induced solely by changes of the pH. The pH-dependent gating is accompanied by an outward displacement of the three S4 voltage sensing arginines that moves the second arginine past the hydrophobic gasket (HG) which separates the inner and outer pores of the channel. HV1 activation, when outer pH increases, involves amino acids at the extracellular entrance of the channel that extend the network of interactions from the external solution down to the HG. Whereas, amino acids at the cytoplasmic entrance of the channel are involved in activation, when inner pH decreases, and in a network of interactions that extend from the cytoplasm up to the HG.

A Novel Simplified Workflow for Guided Endodontic Surgery in Mandibular Molars With a Thick Buccal Bone Plate: A Case Report.

One of the challenges in apical surgery in mandibular molars is the thick buccal cortical bone plate, which might lead to access errors, damage to neighboring teeth, and injury to healthy tissue. Surgical guide templates with 3-dimensional (3D) impressions have been suggested for use in such cases, allowing greater precision. Usually, this technique involves a cone-beam computed tomography (CBCT) examination related to a 3D oral scanning to generate surgical templates printed from 3D imaging data. This study reports a novel workflow possibility in which apical surgery is performed with the aid of a surgical guide planned only with CBCT, excluding the need for 3D oral scanning. A 32-year-old woman presented with asymptomatic apical periodontitis and external root resorption on the mesial root of tooth #19. A surgical template was planned using only the patient's CBCT scan to provide precise access to the lesion and remove a mandibular cortical bone block. The procedure was carried out with apicoectomy, root-end preparation, and retrograde filling, and the bone block was repositioned before suture. No postoperative complications were reported. After 1 year, the patient presented asymptomatic, and a CBCT scan confirmed bone healing. The current 3D-printed guides may be planned and printed through a novel simplified workflow with a CBCT scan only, which allows its application in apical surgery for precise apex and surgical site location.

Voltage-gated proton channels in polyneopteran insects.

Voltage-gated proton channels (HV 1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about HV 1 channels in insects. A characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage-sensor motif are conserved structural elements in HV 1 channels. By analysing Transcriptome Shotgun Assembly (TSA) databases, we found 33 polyneopteran species meeting these structural requirements. Unexpectedly, an unusual natural variation Asp to glutamate (Glu) at SF was found in Phasmatodea and Mantophasmatodea. Additionally, we analysed the expression and function of HV 1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtHV 1 is strongly expressed in nervous tissue and shows pronounced inward proton conduction. This is the first study of a natural occurring Glu within the SF of a functional HV 1 and might be instrumental in uncovering the physiological function of HV 1 in insects.

Late discolouration of root-treated teeth and subsequent restorative retreatment: three case reports.

Root-filled teeth that receive ceramic veneers may experience subsequent coronal discolouration, which will likely negatively affect the quality of life of patients and their relationship with the clinician. This manuscript describes three cases of this undesirable aesthetic consequence in anterior teeth of young patients and the subsequent restorative reintervention, with satisfactory results. In all three reports, the root canal filling was performed using cements containing bismuth oxide that likely disassociated into reduced metallic crystals when in contact with LED light. The discolouration observed after veneer cementation was most likely caused by the accumulation of these bismuth metallic crystals. These cases highlight the importance of preventing this undesirable complication, which may also result in the potential subsequent further sacrifice of sound coronal dental structure, and the role of recall in aesthetically demanding clinical cases.

Remaining dentinal thickness after simulated post space preparation and the fit of prefabricated posts to root canal preparation shapes.

BACKGROUND: Post space preparation can increase the risk of experiencing perforation and root fracture. The authors assessed remaining dentin thickness after post space preparation and the fit of different prefabricated posts to root canal preparation systems in a theoretical model. METHODS: Ten datasets per type of tooth from cone-beam computed tomography were analyzed to determine the minimum root diameter from 5 through 13 mm from the apical foramen. The preparation shapes of 10 root canal preparation systems were calculated and compared with the root dimensions to determine the remaining dentin thickness. Eight post brands were compared with root dimensions to determine the areas where there was less than 1 mm dentin thickness. RESULTS: The average root canal preparation shape produced canal diameters of 0.57 mm at 5 mm from the canal terminus and 0.94 mm at 13 mm from the canal terminus. The mean post size tip diameter was 1.03 mm, which is larger than the dimensions of the average canal preparation. Post preparation would result in less than 1 mm of dentin thickness remaining in premolars, smaller roots of molars, and mandibular incisors. The area with less than 1 mm of dentin thickness was around the post tip. CONCLUSIONS: Endosequence Fiber Post (Brasseler USA), RelyX Fiber Post 3D (3M), and Rebilda (VOCO) were the better fitting posts with the root canal preparation shapes. PRACTICAL IMPLICATIONS: Many posts would result in less than 1 mm dentin thickness. Clinicians should use posts that fit their root canal preparations to minimize dentin removal.

Behaviour and animal welfare indicators of broiler chickens housed in an enriched environment.

The present study aimed to assess the influence of adding straw bales, step platforms, and laser projectors as environmental enrichment resources on the behaviour and welfare indicators of broiler chickens (Gallus gallus domesticus). A sample of 4,000 day-old male Cobb® 500 birds was used. The experimental treatments consisted of No Environmental Enrichment (NEE)-similar to a conventional environment; and Environmental Enrichment (EE)-environment enriched with straw bales, step platforms, and laser projectors, with four replicates per treatment of 500 animals. Behavioural characteristics (ethological observation through cameras, grab test, and modified touch test) and animal welfare indicators (pododermatitis and dorsal cranial myopathy) were assessed. The birds submitted to the EE treatment exhibited greater exploratory activity and expression of behaviours associated with comfort and welfare, whereas those in the NEE group were less active. Locomotion and play fighting behaviour decreased and behaviours associated with comfort increased as age advanced. The frequencies of interaction with laser spots and birds lying around straw bales were the highest in the 1st week (P<0.01). The behaviours of pecking at straw bales (P<0.0004), using the step platforms (P = 0.0001) and being on top of straw bales (P<0.0002) gradually increased. The chickens accessed the feeding troughs the most in the period of 0800 hours (P<0.0001) and expressed the highest frequencies of behaviours associated with comfort in the 1400 hours and 1700 hours periods. The birds in the EE group were calmer in face of human presence and touch and scored higher in animal welfare indicators. Adding straw bales, step platforms, and laser projectors increased locomotion, reduced expression of fear, and improved animal welfare indicators of broiler chickens.

Zinc accelerates respiratory burst termination in human PMN.

The respiratory burst of phagocytes is essential for human survival. Innate immune defence against pathogens relies strongly on reactive oxygen species (ROS) production by the NADPH oxidase (NOX2). ROS kill pathogens while the translocation of electrons across the plasma membrane via NOX2 depolarizes the cell. Simultaneously, protons are released into the cytosol. Here, we compare freshly isolated human polymorphonuclear leukocytes (PMN) to the granulocytes-like cell line PLB 985. We are recording ROS production while inhibiting the charge compensating and pH regulating voltage-gated proton channel (HV1). The data suggests that human PMN and the PLB 985 generate ROS via a general mechanism, consistent of NOX2 and HV1. Additionally, we advanced a mathematical model based on the biophysical properties of NOX2 and HV1. Our results strongly suggest the essential interconnection of HV1 and NOX2 during the respiratory burst of phagocytes. Zinc chelation during the time course of the experiments postulates that zinc leads to an irreversible termination of the respiratory burst over time. Flow cytometry shows cell death triggered by high zinc concentrations and PMA. Our data might help to elucidate the complex interaction of proteins during the respiratory burst and contribute to decipher its termination.

Announcing the call for the Issue Focus on the 2nd Costa Rican Biophysics Symposium-virtual meeting, March 2021.

This Commentary is a call for submissions for the upcoming Issue Focus that will highlight some of the scientific topics discussed during the 2nd Costa Rica Biophysics Symposium.

Guided endodontics: The impact of new technologies on complex case solution.

Discuss the impact of new diagnostic and planning technologies on the resolution of a clinical case of an upper central incisor with lateral perforation, root canal calcification and apical periodontitis. A 44-year-old woman sought treatment because of a colour change in an anterior tooth. The tooth had already been endodontically accessed, and she reported that two different clinicians had failed to locate the root canal. A Cone Beam Computed Tomography scan showed excessive wear and root perforation in the middle third, as well as pulp canal obliteration in the apical third. The perforation was treated using a biomaterial, and the root canal was located using guided endodontics. This treatment protocol was used to access, prepare, medicate with calcium hydroxide for 21 days and fill the root canal. Treatment results were satisfactory at 6-month follow-up.

Cone Beam Computed Tomography Assessment of the Volume of Dental Tissue Removed During Endodontic Access.

Introduction: The purpose of this study was to determine the volume of dental tissue removed during endodontic access preparation with cone-beam computed tomography (CBCT) comparing two different software. Methods and Materials: CBCT images of 20 teeth were obtained before and after endodontic access performed with spherical and conical diamond burs. The images were taken with i-CAT Precise system with 0.25 mm voxel size. Digital Imaging and Communications in Medicine (DICOM) images were loaded on two different software programs (Materialise® and InVersalius®), and a 3D reconstruction of the CBCT images was performed on both programs. The baseline volume (BV), and the final volume (FV) were obtained, and the lost tissue volume (LV) was calculated using the formula: LV=BV-FV. The t-test was used to compare initial and final volumes and also to compare the two programs, using a significance level of 5%. Results: The volumetric data calculated for the Materialise  and the InVesalius  programs were, respectively: BV-mean of 441.79±85.08 mm3 and 442.01±84.83 mm3; FV-mean of 426.75±83.88 mm3 and 426.94±83.75 mm3; LV-mean of 15.04±4.32 mm3 and 15.07±4.16 mm3. No statistically significant difference was found in the volumes calculated by either program for initial, final, or removed tissue (P>0.05). However, there was a significant difference between the BV and LV calculated on the same program (P<0.05). Conclusion: Our in vitro study showed that CBCT was able to determine the volume of dental tissue removed in the endodontic access preparation of extracted human teeth, regardless of the software program used.

Guided Endodontics: Volume of Dental Tissue Removed by Guided Access Cavity Preparation-An Ex Vivo Study.

INTRODUCTION: Guided endodontics emerged as an alternative method capable of providing greater precision and accuracy to endodontic treatments. The aim of the present study was to compare the volume of dental tissue removed after guided endodontic access (GEA) and conventional endodontic access (CEA) to mandibular incisors and upper molars. METHODS: Twenty extracted human mandibular incisors and upper molars were selected and submitted to cone-beam computed tomographic (CBCT) examination. They were divided into 2 groups, G1 (mandibular incisors) and G2 (maxillary molars), and subdivided into G1a (CEA), G1b (GEA), G2a (CEA), and G2b (GEA). The Digital Imaging and Communications in Medicine files obtained by examining the CBCT scans were transferred to InVesalius software (Renato Archer Information Technology Center, Campinas, SP, Brazil) to calculate the initial volume of each tooth. G1b and G2b teeth were scanned with a device to plan and print the guides. After gaining endodontic access, new CBCT examinations were performed to calculate the final volume of each sample unit. The Student t test for independent samples compared the volumes among the groups. RESULTS: G1 group had an average volume reduction of 31.667 mm3 (10.62%) using CEA and 26.523 mm3 (10.65%) using GEA with no significant difference among the groups (P = .960). There was an average volume reduction of 62.526 mm3 (5.86%) in the G2 group using CEA and 45.677 mm3 (4.11%) using GEA with a significant difference among the groups (P = .004). CONCLUSIONS: GEA preserved a greater volume of dental tissue in extracted upper human molars than CEA; however, there was no significant difference between CEA and GEA in the volume of dental tissue removed from mandibular incisors.

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

The HV 1 voltage-gated proton (HV 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of HV 1 is Zn2+ . Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV 1 from Nicoletia phytophila, NpHV 1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 µm. Zn2+ inhibition in NpHV 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpHV 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

Assessing Structural Determinants of Zn2+ Binding to Human HV1 via Multiple MD Simulations.

Voltage-gated proton channels (HV1) are essential for various physiological tasks but are strongly inhibited by Zn2+ cations. Some determinants of Zn2+ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn2+ binds to monomeric HV1 despite evidence that HV1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn2+ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 µs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human HV1. The dimer interface is more efficient for attracting and binding Zn2+ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.