Function Of Channel Proteins Research Articles

Translational Utility of Organoid Models for Biomedical Research on Gastrointestinal Diseases.

Organoid models have recently been utilized to study 3D human-derived tissue systems to uncover tissuearchitecture and adultstem cellbiology. Patient-derived organoids unambiguously provide the most suitable in vitro system to study disease biology with the actual genetic background. With the advent of much improved and innovative approaches, patient-derived organoids can potentially be used in regenerative medicine. Various human tissues were explored to develop organoids due to their multifold advantage over the conventional in vitro cell line culture approach and in vivo models. Gastrointestinal (GI) tissues have been widely studied to establish organoids and organ-on-chip for screening drugs, nutraceuticals, and other small molecules having therapeutic potential. The function of channel proteins, transporters, and transmembrane proteins was also explained. The successful application of genome editing in organoids using the CRISPR-Cas approach has been reported recently. GI diseases such as Celiac disease (CeD), Inflammatory bowel disease (IBD), and common GI cancers have been investigated using several patient-derived organoid models. Recent advancements on organoid bio-banking and 3D bio-printing contributed significantly in personalized disease management and therapeutics. This article reviews the available literature on investigations and translational applications of patient-derived GI organoid models, notably on elucidating gut-microbial interaction and epigenetic modifications.

Next Generation Sequencing and Electromyography Reveal the Involvement of the P2RX6 Gene in Myopathy.

Ion channelopathies result from impaired ion channel protein function, due to mutations affecting ion transport across cell membranes. Over 40 diseases, including neuropathy, pain, migraine, epilepsy, and ataxia, are associated with ion channelopathies, impacting electrically excitable tissues and significantly affecting skeletal muscle. Gene mutations affecting transmembrane ionic flow are strongly linked to skeletal muscle disorders, particularly myopathies, disrupting muscle excitability and contraction. Electromyography (EMG) analysis performed on a patient who complained of weakness and fatigue revealed the presence of primary muscular damage, suggesting an early-stage myopathy. Whole exome sequencing (WES) did not detect potentially causative variants in known myopathy-associated genes but revealed a novel homozygous deletion of the P2RX6 gene likely disrupting protein function. The P2RX6 gene, predominantly expressed in skeletal muscle, is an ATP-gated ion channel receptor belonging to the purinergic receptors (P2RX) family. In addition, STRING pathways suggested a correlation with more proteins having a plausible role in myopathy. No previous studies have reported the implication of this gene in myopathy. Further studies are needed on patients with a defective ion channel pathway, and the use of in vitro functional assays in suppressing P2RX6 gene expression will be required to validate its functional role.

Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research.

Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

Evidence for significance of serine 1487 in β-adrenergic regulation of Cav1.2 channel protein function in genetically engineered mice

Stress responses are mediated through activation of the β-adrenergic signaling pathway. Chronic sympathetic stimulation can result in the development of hypertrophy, arrhythmia and sudden cardiac death but the molecular mechanisms are still not understood. We identified a single serine, following phosphorylation by cAMP-dependent protein kinase A, responsible for the increase in function of the human cardiac Cav1.2 channel protein in an artificial cell system. To confirm whether the serine is involved in in vivo channel stimulation by β-adrenergic signaling, we created CRISPR/Cas9 mutant mouse lines with glutamate and alanine-substitution of the serine1487 on Cav1.2 protein to mimic the phosphorylated and the non-phosphorylated protein. The average litter size (6.7 ± 0.7; N = 16 and 6.1 ± 0.4; N = 24 respectively) and survival rate (48.6% vs. 42.5%) were similar in the two lines, but homozygous mutation of Cav1.2 S1487E caused 100% mortality with a median survival of ~1 day. The relative size of the hearts of homozygous S1487A mice were smaller than S1487E (heart weight/tibial length: 7.0 ± 0.1; N = 3 vs 7.8 ± 0.3; N = 3). Administration of intraperitoneal injection of 20 mg/kg isoproterenol increased the heart rate up to 30% in both glutamate and alanine-substituted heterozygous mutants in a similar manner to the negatively genotyped littermates, but did not alter the heart rate of the homozygous S1487A mutants. Chronic isoproterenol treatment (20 mg/kg isoproterenol daily injection for 2 weeks) induced hypertrophy in the wild type and heterozygous S1497A mutant mice, but there were no significant changes in the posterior wall thickness of the heart of homozygous S1487A mutants.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration.

Genetic potassium channel-associated epilepsies: Clinical review of the Kv family.

Next-generation sequencing has enhanced discovery of many disease-associated genes in previously unexplained epilepsies, mainly in developmental and epileptic encephalopathies and familial epilepsies. We now classify these disorders according to the underlying molecular pathways, which encompass a diverse array of cellular and sub-cellular compartments/signalling processes including voltage-gated ion-channel defects. With the aim to develop and increase the use of precision medicine therapies, understanding the pathogenic mechanisms and consequences of disease-causing variants has gained major relevance in clinical care. The super-family of voltage-gated potassium channels is the largest and most diverse family among the ion channels, encompassing approximately 80 genes. Key potassium channelopathies include those affecting the KV, KCa and Kir families, a significant proportion of which have been implicated in neurological disease. As for other ion channel disorders, different pathogenic variants within any individual voltage-gated potassium channel gene tend to affect channel protein function differently, causing heterogeneous clinical phenotypes. The focus of this review is to summarise recent clinical developments regarding the key voltage-gated potassium (KV) family-related epilepsies, which now encompasses approximately 12 established disease-associated genes, from the KCNA-, KCNB-, KCNC-, KCND-, KCNV-, KCNQ- and KCNH-subfamilies.

An Artificial Molecular Shuttle Operates in Lipid Bilayers for Ion Transport.

Inspired by natural biomolecular machines, synthetic molecular-level machines have been proven to perform well-defined mechanical tasks and measurable work. To mimic the function of channel proteins, we herein report the development of a synthetic molecular shuttle, [2]rotaxane 3, as a unimolecular vehicle that can be inserted into lipid bilayers to perform passive ion transport through its stochastic shuttling motion. The [2]rotaxane molecular shuttle is composed of an amphiphilic molecular thread with three binding stations, which is interlocked in a macrocycle wheel component that tethers a K+ carrier. The structural characteristics enable the rotaxane to transport ions across the lipid bilayers, similar to a cable car, transporting K+ with an EC50 value of 1.0 μM (3.0 mol % relative to lipid). We expect that this simple molecular machine will provide new opportunities for developing more effective and selective ion transporters.

Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca2+ channel (Cav1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel.

Numerical methods for a Poisson-Nernst-Planck-Fermi model of biological ion channels.

Numerical methods are proposed for an advanced Poisson-Nernst-Planck-Fermi (PNPF) model for studying ion transport through biological ion channels. PNPF contains many more correlations than most models and simulations of channels, because it includes water and calculates dielectric properties consistently as outputs. This model accounts for the steric effect of ions and water molecules with different sizes and interstitial voids, the correlation effect of crowded ions with different valences, and the screening effect of polarized water molecules in an inhomogeneous aqueous electrolyte. The steric energy is shown to be comparable to the electrical energy under physiological conditions, demonstrating the crucial role of the excluded volume of particles and the voids in the natural function of channel proteins. Water is shown to play a critical role in both correlation and steric effects in the model. We extend the classical Scharfetter-Gummel (SG) method for semiconductor devices to include the steric potential for ion channels, which is a fundamental physical property not present in semiconductors. Together with a simplified matched interface and boundary (SMIB) method for treating molecular surfaces and singular charges of channel proteins, the extended SG method is shown to exhibit important features in flow simulations such as optimal convergence, efficient nonlinear iterations, and physical conservation. The generalized SG stability condition shows why the standard discretization (without SG exponential fitting) of NP equations may fail and that divalent Ca(2+) may cause more unstable discrete Ca(2+) fluxes than that of monovalent Na(+). Two different methods-called the SMIB and multiscale methods-are proposed for two different types of channels, namely, the gramicidin A channel and an L-type calcium channel, depending on whether water is allowed to pass through the channel. Numerical methods are first validated with constructed models whose exact solutions are known. The experimental data of both channels are then used to verify and explain novel features of PNPF as compared with previous PNP models. The PNPF currents are in accord with the experimental I-V (V for applied voltages) data of the gramicidin A channel and I-C (C for bath concentrations) data of the calcium channel with 10(-8)-fold bath concentrations that pose severe challenges in theoretical simulations.

GW25-e0549 RNA interference targeting E637K mutation rescues hERG channel currents and restores its kinetic properties

The purpose of this study was to investigate the role of small interference RNA (siRNA) on expression of E637K-hERG (human ether-a-go-go-related gene) mutant and whether it can be used to rescue the mutant’s dominant-negative suppressive effects on hERG protein channel function. Western blot was

Calcium influx and male fertility in the context of the sperm proteome: an update.

Freshly ejaculated spermatozoa are incapable or poorly capable of fertilizing an oocyte. The fertilization aptness of spermatozoa depends on the appropriate and time-dependent acquisition of hyperactivation, chemotaxis, capacitation, and the acrosome reaction, where calcium (Ca2+) is extensively involved in almost every step. A literature review showed that several ion channel proteins are likely responsible for regulation of the Ca2+ uptake in spermatozoa. Therefore, manipulation of the functions of channel proteins is closely related to Ca2+ influx, ultimately affecting male fertility. Recently, it has been shown that, together with different physiological stimuli, protein-protein interaction also modifies the Ca2+ influx mechanism in spermatozoa. Modern proteomic analyses have identified several sperm proteins, and, therefore, these findings might provide further insight into understanding the Ca2+ influx, protein functions, and regulation of fertility. The objective of this review was to synthesize the published findings on the Ca2+ influx mechanism in mammalian spermatozoa and its implications for the regulation of male fertility in the context of sperm proteins. Finally, Pathway Studio (9.0) was used to catalog the sperm proteins that regulate the Ca2+ influx signaling by using the information available from the PubMed database following a MedScan Reader (5.0) search.

Defective channels lead to an impaired skin barrier.

Channels are integral membrane proteins that form a pore, allowing the passive movement of ions or molecules across a membrane (along a gradient), either between compartments within a cell, between intracellular and extracellular environments or between adjacent cells. The ability of cells to communicate with one another and with their environment is a crucial part of the normal physiology of a tissue that allows it to carry out its function. Cell communication is particularly important during keratinocyte differentiation and formation of the skin barrier. Keratinocytes in the skin epidermis undergo a programme of apoptosis-driven terminal differentiation, whereby proliferating keratinocytes in the basal (deepest) layer of the epidermis stop proliferating, exit the basal layer and move up through the spinous and granular layers of the epidermis to form the stratum corneum, the external barrier. Genes encoding different families of channel proteins have been found to harbour mutations linked to a variety of rare inherited monogenic skin diseases. In this Commentary, we discuss how human genetic findings in aquaporin (AQP) and transient receptor potential (TRP) channels reveal different mechanisms by which these channel proteins function to ensure the proper formation and maintenance of the skin barrier.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101

Comparison of diverse orthologs is a powerful tool to study the structure and function of channel proteins. We investigated the response of human, killifish, pig, and shark cystic fibrosis transmembrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTR(inh)-172, glibenclamide, and GlyH-101. In three systems, including organ perfusion of the shark rectal gland, primary cultures of shark rectal gland tubules, and expression studies of each ortholog in cRNA microinjected Xenopus laevis oocytes, we observed fundamental differences in the sensitivity to inhibition by these channel blockers. In organ perfusion studies, shark CFTR was insensitive to inhibition by CFTR(inh)-172. This insensitivity was also seen in short-circuit current experiments with cultured rectal gland tubular epithelial cells (maximum inhibition 4 ± 1.3%). In oocyte expression studies, shark CFTR was again insensitive to CFTR(inh)-172 (maximum inhibition 10.3 ± 2.5% at 25 μM), pig CFTR was insensitive to glibenclamide (maximum inhibition 18.4 ± 4.4% at 250 μM), and all orthologs were sensitive to GlyH-101. The amino acid residues considered responsible by previous site-directed mutagenesis for binding of the three inhibitors are conserved in the four CFTR isoforms studied. These experiments demonstrate a profound difference in the sensitivity of different orthologs of CFTR proteins to inhibition by CFTR blockers that cannot be explained by mutagenesis of single amino acids. We believe that the potency of the inhibitors CFTR(inh)-172, glibenclamide, and GlyH-101 on the CFTR chloride channel protein is likely dictated by the local environment and the three-dimensional structure of additional residues that form the vestibules, the chloride pore, and regulatory regions of the channel.

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.

Proteomic profile of osteoclast membrane proteins: Identification of Na+/H+ exchanger domain containing 2 and its role in osteoclast fusion

Osteoclast formation and bone resorption are multiple processes that involve the participation of specialized membrane structures and their associated proteins. In this study, we used an MS to analyze the profile of proteins associated with osteoclast membranes and focused on the function of channel proteins in osteoclast differentiation and function. We filtered out with a SEQUEST score greater than 10 and a peptide hit number of more than 2, resulting in the identification of 499 proteins that were commonly found in both macrophages and osteoclasts, 96 proteins selectively found in osteoclasts, and 179 proteins selectively found in macrophages. The proteins that were selectively found in osteoclasts were classified based on their localizations: plasma membrane (17%), ER/Golgi and lysosome/endosome (15%), mitochondrion (18%), nucleus (13%), cytosol (19%), and unknown (18%). Proteins associated with osteoclast function such as v-ATPase, IGF2R, TRAP, and cathepsin K were found in osteoclasts as previously shown. We found several ion channel proteins such as Ank and Nhedc2 and signaling molecules such as Dock5 and RAB-10 in osteoclasts. Inhibition of the Na(+)/H(+) exchanger family by amiloride suppressed RANKL-induced osteoclast fusion and bone resorption. In addition, shRNA for Nhedc2 inhibited osteoclast differentiation. Our results provide a proteomic profile of osteoclast membrane proteins and identify Nhedc2, which is probably associated with proton transport in osteoclasts, as a regulator of osteoclast function.

Normal-Mode Refinement of Anisotropic Thermal Parameters for Potassium Channel KcsA at 3.2 Å Crystallographic Resolution

We report a normal-mode method for anisotropic refinement of membrane-protein structures, based on a hypothesis that the global near-native-state disordering of membrane proteins in crystals follows low-frequency normal modes. Thus, a small set of modes is sufficient to represent the anisotropic thermal motions in X-ray crystallographic refinement. By applying the method to potassium channel KcsA at 3.2 A, we obtained a structural model with an improved fit with the diffraction data. Moreover, the improved electron density maps allowed for large structural adjustments for 12 residues in each subunit, including the rebuilding of 3 missing side chains. Overall, the anisotropic KcsA structure at 3.2 A was systematically closer to a 2.0 A KcsA structure, especially in the selectivity filter. Furthermore, the anisotropic thermal ellipsoids from the refinement revealed functionally relevant structural flexibility. We expect this method to be a valuable tool for structural refinement of many membrane proteins with moderate-resolution diffraction data.

Aquaporin expression in developing human teeth and selected orofacial tissues.

The aquaporin (AQP) family of membrane channel proteins function as selective pores through which water, glycerol, and other small solutes cross the cell plasma membrane. To date, 11 members of this transporter family, designated AQP0-10, have been cloned and characterized in humans. The AQPs are differentially expressed in temporospatial patterns, where different AQPs demonstrate distinct tissue distributions that may reflect differing cell membrane transport functions. The purpose of this study was to evaluate AQP expression in the developing human teeth by RT-PCR and Western blot analysis. To access the generality of AQP expression, selected other orofacial tissues were studied by RT-PCR. The presence of all eleven human AQPs was screened in each tissue by RT-PCR. Positive amplification products were verified by direct DNA sequencing. AQPs 1, 3, 4, 5, 6, and 10 were identified by RT-PCR in developing teeth, and AQP1, 3, 5, and 6 were confirmed by Western blot analysis. AQP 4 was not detected by Western blot analysis, and we were unable to test for the recently identified AQP10 due to unavailability of antibodies. AQPs detected in other orofacial tissues by RT-PCR included gingiva (AQP3, 7, 10); Meckel's cartilage (AQP1, 3, 4, 5, 6); submandibular gland (AQP1, 3, 4, 5, 6, 7); masseter muscle (AQP1, 3, 4, 7, 8, 9,10); and infrahyoid muscle (AQP1, 3, 4,10). These results demonstrate that multiple aquaporins are expressed in developing teeth and in selected orofacial tissues.

Crystal structure of the tetramerization domain of the Shaker potassium channel.

Voltage-dependent, ion-selective channels such as Na+, Ca2+ and K+ channel proteins function as tetrameric assemblies of identical or similar subunits. The clustering of four subunits is thought to create an aqueous pore centred at the four-fold symmetry axis. The highly conserved, amino-terminal cytoplasmic domain (approximately 130 amino acids) immediately preceding the first putative transmembrane helix S1 is designated T1. It is known to confer specificity for tetramer formation, so the heteromeric assembly of K+-channel subunits is an important mechanism for the observed channel diversity. We have determined the crystal structure of the T1 domain of a Shaker potassium channel at 1.55 A resolution. The structure reveals that four identical subunits are arranged in a four-fold symmetry surrounding a centrally located pore about 20 A in length. Subfamily-specific assembly is provided primarily by polar interactions encoded in a conserved set of amino acids at its tetramerization interface. Most highly conserved amino acids in the T1 domain of all known potassium channels are found in the core of the protein, indicating a common structural framework for the tetramer assembly.