Membrane Patches Research Articles

Double membrane formation in heterogeneous vesicles.

Lipids are capable of forming a variety of structures, including multi-lamellar vesicles. Layered lipid membranes are found in cell organelles, such as autophagosomes and mitochondria. Here, we present a mechanism for the formation of a double-walled vesicle (i.e., two lipid bilayers) from a unilamellar vesicle through the partitioning and phase separation of a small molecule. Using molecular dynamics simulations, we show that double membrane formation proceeds via a nucleation and growth process - i.e., after a critical concentration of the small molecules, a patch of double membrane nucleates and grows to cover the entire vesicle. We discuss the implications of this mechanism and theoretical approaches for understanding the evolution and formation of double membranes.

Membrane rolling induced by bacterial toxins

Membrane curvature effects are important in numerous cellular processes and many membrane interacting proteins induce spontaneous curvature upon membrane binding. Shiga and cholera toxins both belong to the AB5 family of toxins and consist of a toxic A subunit and a membrane-binding pentameric B subunit. Shiga and cholera toxins induce tubular membrane invaginations in cells and GUVs due to curvature effects and the toxins are known from MD simulations to induce curvature. Membrane invaginations have been linked to uptake of the toxins into cells. As a novel model system to experimentally characterize curvature-inducing proteins, we study the morphology induced in planar membrane patches. It was previously shown that annexins induce distinct morphologies in membrane patches including membrane rolling. In this study we show that the B subunits of Shiga and cholera toxins (STxB, CTxB) both induce roll-up of cell-sized membrane patches. Rolling starts from the free membrane edges of the patch and is completed within a few seconds. We characterize the branched roll morphology and find experimental estimates for the spontaneous curvature of the toxins based on the topography of rolls. The estimates are in agreement with previous MD simulations. We quantify the dynamics of rolling as induced by the toxins and demonstrate agreement with a theoretical model of the rolling dynamics. The model solves the equation of motion for a membrane roll and includes viscous drag and adhesion to the support. The results suggest that membrane rolling may be a general phenomenon displayed by many proteins that induce negative curvature in membranes with free edges.

A study of status of contralateral ear in unilateral chronic otitis media in 500 patients

<p class="abstract"><strong>Background:</strong> The aim of this study was to examine the contralateral ear clinically, audiologically and radiologically and compare the findings with squamous and mucosal type of chronic otitis media (COM).</p><p class="abstract"><strong>Methods:</strong> A cross sectional study conducted in patients attending ENT Department, Government Medical College, Amritsar between December 2016 to November 2018. The study was conducted in 500 patients of either sex aged between 8 to 60 years with unilateral COM without perforation or history of ear discharge in the contralateral ear. Otoscopy, pure tone audiometry and X-ray mastoid or high-resolution computed tomography temporal bone were done on the patients. The results were recorded and analysed with SPSS software. </p><p class="abstract"><strong>Results:</strong> 82% patients were found to have some abnormalities in the contralateral ear. On otoscopy abnormalities in contralateral ear includes retraction of tympanic membrane, thinning and tympanosclerotic patch, most common abnormality being retraction of tympanic membrane. Radiological analysis of contralateral mastoids showed 39% diploic and 22.8% sclerotic mastoids. Hearing status in the contralateral ear showed 45.6% patient with normal hearing, 48.6% with conductive hearing loss and 5.8% with mixed hearing loss. 40.4% contralateral ears had mild, 12% had moderate and 2% had severe hearing loss.</p><p class="abstract"><strong>Conclusions:</strong> Contralateral ear pathologies were more in those who had squamosal type of COM than who had mucosal type in the diseased ear. The contralateral ear shows unmistakable predilection towards developing COM in the future.</p><p class="abstract"> </p><p> </p>

Corneal Epithelial-Stromal Fibroblast Constructs to Study Cell-Cell Communication in Vitro.

Cell–cell communication plays a fundamental role in mediating corneal wound healing following injury or infection. Depending on the severity of the wound, regeneration of the cornea and the propensity for scar development are influenced by the acute resolution of the pro-fibrotic response mediated by closure of the wound via cellular and tissue contraction. Damage of the corneal epithelium, basement membrane, and anterior stroma following a superficial keratectomy is known to lead to significant provisional matrix deposition, including secretion of fibronectin and thrombospondin-1, as well as development of a corneal scar. In addition, corneal wounding has previously been shown to promote release of extracellular vesicles from the corneal epithelium, which, in addition to soluble factors, may play a role in promoting tissue regeneration. In this study, we report the development and characterization of a co-culture system of human corneal epithelial cells and corneal stromal fibroblasts cultured for 4 weeks to allow extracellular matrix deposition and tissue maturation. The secretion of provisional matrix components, as well as small and large extracellular vesicles, was apparent within the constructs, suggesting cell–cell communication between epithelial and stromal cell populations. Laminin-1β was highly expressed by the corneal epithelial layer with the presence of notable patches of basement membrane identified by transmission electron microscopy. Interestingly, we identified expression of collagen type III, fibronectin, and thrombospondin-1 along the epithelial–stromal interface similar to observations seen in vivo following a keratectomy, as well as expression of the myofibroblast marker, α-smooth muscle actin, within the stroma. Our results suggest that this corneal epithelial–stromal model may be useful in the study of the biochemical phenomena that occur during corneal wound healing.

Amniotic membrane for retinal detachment due to paravascular retinal breaks over patchy chorioretinal atrophy in pathologic myopia.

To describe a new surgical technique, using a human amniotic membrane patch, in two cases of retinal detachment with paravascular retinal breaks over patchy chorioretinal atrophy in pathologic myopia, already underwent pars plana vitrectomy with the internal limiting membrane peeling for myopic foveoschisis. Surgical technique description with surgical video. A 23-gauge pars plana vitrectomy was performed. A human amniotic membrane patch was implanted under the neuroretina to seal the posterior retinal break. Standard silicone oil tamponade was performed at the end of the surgery. The patients were positioned face down after the operation for the first week. Optical coherence tomography scans were used in the follow-ups. The 2 weeks postoperative optical coherence tomography showed a new tissue growth over the human amniotic membrane patch. The 3 months optical coherence tomography showed the new tissue entirely covering the human amniotic membrane plug. Visual acuity improved from 20/2000 (2 LogMAR) to 20/250 (1.1 LogMAR) 3 months after the operation in both patients. The silicone oil was extracted 2 months after surgery, and no recurrences were observed. The patient's visual acuity remained stable at 20/250 after the silicone oil extraction. In these complex cases, human amniotic membrane transplantation can be a valid option, when internal limiting membrane has already been peeled in previous surgeries, to seal the posterior retinal breaks over high myopic chorioretinal atrophy, with encouraging results.

Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.

Does the classical solid-shell element with the assumed natural strain method satisfy the three-dimensional patch test for arbitrary geometry?

In the present study, the ability of the classical solid-shell element to satisfy the membrane patch test is examined. Theoretical and numerical investigations showed that the classical solid-shell element fails to satisfy the membrane patch test when the elements' referential covariant base vectors in the thickness directions are coordinate-dependent. This deficiency has motivated the development of a new solid-shell formulation in the present study for modeling elastic thin structures and shell-like applications. Within the new solid-shell element, a modified Green-Lagrange strain tensor is constructed by adopting both the assumed natural inhomogeneous strain (ANIS) and the enhanced assumed strain (EAS) methods. The developed formulation is implemented in the commercial finite element software Abaqus for numerical simulations. An extensive numerical study is carried out for testing the accuracy of the developed finite element. To this end, the accuracy of the ANIS solid-shell element incorporating the proposed set of 21 EAS parameters is compared to the classical ANS solid-shell element with the same set of the EAS parameters. It is shown that the ANIS solid-shell element combined with the set of 21 EAS parameters is of great accuracy and excellent performance considering both the linear and the non-linear regimes.

Cytosolic GABA inhibits anion transport by wheat ALMT1.

Anion transport by aluminium-activated malate transporter (ALMT) proteins is negatively regulated by gamma-aminobutyric acid (GABA), which increases in concentration during stress. Here, the interaction between GABA and wheat (Triticum aestivum, Ta) TaALMT1 heterologously-expressed in Xenopus laevis oocytes was investigated. GABA inhibited anion transport by TaALMT1 in membrane patches from the cytosolic, not extracellular membrane face, via a reduction in open probability (NPopen ), not an inhibition of channel current magnitude. TaALMT1 currents in patches frequently exhibited rundown with complete removal of cytosolic factors, but were partially sustained by protein kinase C dependent phosphorylation. When applied to whole oocytes a GABA-analogue-BODIPY conjugate inhibited TaALMT1 anion currents from the cytoplasmic face only, whereas free GABA inhibited from both the inside and outside consistent with GABA traversing the TaALMT1 pore then acting from the inside. We propose GABA does not competitively inhibit ALMT conductance through the same pore but rather leads to an allosteric effect, reducing anion channel opening frequency. Across plants GABA is a conserved regulator of anion transport via ALMTs - a family with numerous physiological roles beyond Al3+ tolerance. Our data suggests that a GABA-ALMT interaction from the cytosolic face has the potential to form part of a novel plant signalling pathway.

Convolutional neural network approach for automatic tympanic membrane detection and classification

Otitis media (OM) is a term used to describe the inflammation of the middle ear. The clinical inspection of the tympanic membrane is conducted visually by experts. Visual inspection leads to limited variability among the observers and includes human-induced errors. In this study, we sought to solve these problems using a novel diagnostic model based on a faster regional convolutional neural network (Faster R-CNN) for tympanic membrane detection, and pre-trained CNNs for tympanic membrane classification. The experimental study was conducted on a new eardrum dataset. The Faster R-CNN was initially applied to the original images. The number of images in the dataset was subsequently increased using basic image augmentation techniques such as flip and rotation. We also evaluated the success of the model in the presence of various noise effects. The original and automatically extracted tympanic membrane patches were finally input separately to the CNNs. The AlexNet, VGGNets, GoogLeNet, and ResNets models were employed. This resulted in an average precision of 75.85% in the tympanic membrane detection. All CNNs in the classification produced satisfactory results, with the proposed approach achieving an accuracy of 90.48% with the VGG-16 model. This approach can potentially be used in future otological clinical decision support systems to increase the diagnostic accuracy of the physicians and reduce the overall rate of misdiagnosis. Future studies will focus on increasing the number of samples in the eardrum dataset to cover a full range of ontological conditions. This would enable us to realize a multi-class classification in OM diagnosis.

Cardiovascular disease's effects on red blood cells’ morphology and values of haemoglobin oxygen-binding capacity

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant-associated membrane patch (RAMP), once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of the spatiotemporal lipid collective assembly at the plasma membrane as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

Conjunctival Benign Fibrous Histiocytoma in a 57-year-old man

Purpose. To report a case of benign fibrous histiocytoma of the conjunctiva involving the cornea, an uncommon ocular surface tumor. Methods. A 57-year-old patient came in our service complaining of a progressively enlarging conjunctival mass temporally to the limbus and invading the adjacent cornea of the left eye. Results. The approach consisted in surgical excision followed by cryotherapy on the edges and on the base of the excision site and amniotic membrane patch reconstruction of the ocular surface defect. Pathologic examination and immunohistochemistry were performed in order to establish the diagnosis. No recurrences appeared in 8 months of follow up. Conclusions. Fibrous histiocytoma might be easily misdiagnosed as it is exceedingly rare. Complete resection with careful inspection of edges is advised. Cryotherapy at the base and borders of the resection site is recommended as both benign and malignant tumors can show recurrence. Amniotic membrane should always be regarded as an efficient option in reconstruction of broad surface defects after tumor resection. Abbreviations: FH = fibrous histiocytoma, CIN = corneal intraepithelial neoplasia, SSCA = squamous cell carcinoma, AM = amniotic membrane, MMC = topical mitomycin-C, 5-FU = 5-fluorouracil, BCVA = best corrected visual acuity.

Voltage-gated proton channels exist in the plasma membrane of human oocytes.

Do human oocytes express voltage-gated proton channels? Human oocytes exhibit voltage-gated proton currents. Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. N/A. Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. NIH R35GM126902 (TED), Bears Care (DM). No competing interests. N/A.

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

Cup-to-vesicle transition of a fluid membrane with spontaneous curvature.

The disk-to-vesicle transition of a fluid membrane with no spontaneous curvature is well described by the competition between edge line and curvature energies. However, the transition of asymmetric membranes with spontaneous curvatures is not yet understood. In this study, the shape of the fluid membrane patch with a constant spontaneous curvature and its closing transition to a vesicle is investigated using theory and meshless membrane simulations. It is confirmed that the (meta)stable and transient membranes are well approximated by spherical caps. The membrane Gaussian modulus can be estimated from the cup shape of membrane patches as well as from the transition probability, although the latter estimate gives slightly smaller negative values. Furthermore, the self-assembly dynamics of membranes are presented, in which smaller vesicles are formed at higher spontaneous curvatures, higher edge line tension, and lower density.

Stearylamine Causes the Formation of Neutrophil Extracellular Traps Independently of Reactive Oxygen Species

Neutrophils can form extracellular traps that consist of chromatin and granule proteins and can, acting as a net, trap bacteria. The trap formation process called NETosis that has been thoroughly characterized in cells exposed to phorbol ester (PMA) takes 2–3 h and depends on reactive oxygen species produced by NADPH oxidase (this is called classical NETosis). The aim of the present work was to study the distinctive features of the NETosis process evoked by stearylamine (SA) dissolved in DMSO or incorporated into phosphatidylcholine (PC) liposomes and to compare it to NETosis evoked by PMA. Human neutrophils were incubated with 0.2 mg/mL SA (at a 2% DMSO content in the medium) or with cationic PC liposomes that contained SA (PC–SA liposomes; PC and SA concentrations 1.8 and 0.2 mg/mL, respectively). Confocal fluorescence microscopy of fixed neutrophil preparations showed that SA (dissolved in DMSO or incorporated into liposomes) caused the formation of neutrophil extracellular traps. NETosis kinetics were analyzed in the real-time mode in live cells exposed to fluorescently labeled PC–SA liposomes. PC–SA liposomes added to the neutrophils were shown to adsorb to isolated patches of the plasma membrane at first and to occupy the entire membrane surface as the incubation time was prolonged. This was accompanied by chromatin decondensation and fusion of the nuclear material and the cytoplasm, that is, the stages observed for phorbol ester-induced NETosis. However, SA-evoked NETosis was much faster (30–90 min) than PMA-evoked NETosis. It is necessary to emphasize that SA did not induce an oxidative burst (in contrast to PMA), as revealed by analysis of luminol-dependent chemiluminescence. Trap formation by neutrophils exposed to PC–SA liposomes was not affected by apocynin and DPI (NADPH oxidase inhibitors) or catalase, and this also shows that SA stimulates ROS-independent formation of neutrophil extracellular traps.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, β-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.

Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development.

Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1's presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.

Molecular Lipid Films on Microengineering Materials.

In this study, we have systematically investigated the formation of molecular phospholipid films on a variety of solid substrates fabricated from typical surface engineering materials and the fluidic properties of the lipid membranes formed on these substrates. The surface materials comprise of borosilicate glass, mica, SiO2, Al (native oxide), Al2O3, TiO2, ITO, SiC, Au, Teflon AF, SU-8, and graphene. We deposited the lipid films from small unilamellar vesicles (SUVs) by means of an open-space microfluidic device, observed the formation and development of the films by laser scanning confocal microscopy, and evaluated the mode and degree of coverage, fluidity, and integrity. In addition to previously established mechanisms of lipid membrane-surface interaction upon bulk addition of SUVs on solid supports, we observed nontrivial lipid adhesion phenomena, including reverse rolling of spreading bilayers, spontaneous nucleation and growth of multilamellar vesicles, and the formation of intact circular patches of double lipid bilayer membranes. Our findings allow for accurate prediction of membrane-surface interactions in microfabricated devices and experimental environments where model membranes are used as functional biomimetic coatings.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (IK1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the “epestfind” webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical IK1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of IK1 channels.

Temperature effect on memristive ion channels.

Neuron shows distinct dependence of electrical activities on membrane patch temperature, and the mode transition of electrical activity is induced by the patch temperature through modulating the opening and closing rates of ion channels. In this paper, inspired by the physical effect of memristor, the potassium and sodium ion channels embedded in the membrane patch are updated by using memristor-based voltage gate variables, and an external stimulus is applied to detect the variety of mode selection in electrical activities under different patch temperatures. It is found that each ion channel can be regarded as a physical memristor, and the shape of pinched hysteresis loop of memristor is dependent on both input voltage and patch temperature. The pinched hysteresis loops of two ion-channel memristors are dramatically enlarged by increasing patch temperature, and the hysteresis lobe areas are monotonously reduced with the increasing of excitation frequency if the frequency of external stimulus exceeds certain threshold. However, for the memristive potassium channel, the AREA1 corresponding to the threshold frequency is increased with the increasing of patch temperature. The amplitude of conductance for two ion-channel memristors depends on the variation of patch temperature. The results of this paper might provide insights to modulate the neural activities in appropriate temperature condition completely, and involvement of external stimulus enhance the effect of patch temperature.