Search Results (15)

Fluorescent labeling of membrane proteins is essential for exploring their functions, signaling pathways, interaction partners, and structural dynamics. Organic fluorophores are commonly used for this purpose due to their favorable photophysical properties and photostability. However, a persistent challenge is the inaccessibility of the surface-exposed cysteine residues required for site-specific labeling, as these residues often become sequestered within detergent micelles during protein extraction. To address this limitation, we developed an approach based on polymer-encapsulated nanodiscs that preserves the protein’s native-like lipid-bilayer environment while ensuring the accessibility of surface-exposed cysteine residues. In this method, His-tagged proteins embedded in native nanodiscs are retained on a nickel affinity column, allowing for simultaneous purification and labeling by adding fluorescent dyes. This versatile technique was demonstrated with two challenging-to-label membrane proteins, the potassium channel KvAP and the urea channel HpUreI, for which detergent-based labeling had failed. This opens new possibilities for studying a wide range of fluorescently labeled membrane proteins in near-native states, advancing applications in biophysics, structural biology, and drug discovery. Full article

Multidrug-resistant tuberculosis (MDR-TB) patients are treated with a standardised, short World Health Organization (WHO) regimen which includes clofazimine (CFZ) and bedaquiline (BDQ) antibiotics. These two antibiotics lead to the development of QT prolongation in patients, inhibiting potassium (K⁺) uptake by targeting the voltage-gated K⁺ (Kv)11.1 (hERG) channel of the cardiomyocytes (CMs). However, the involvement of these antibiotics to regulate other K⁺ transporters of the CMs, as potential mechanisms of QT prolongation, has not been explored. This study determined the effects of CFZ and BDQ on sodium, potassium–adenosine triphosphatase (Na⁺,K⁺-ATPase) activity of CMs using rat cardiomyocytes (RCMs). These cells were treated with varying concentrations of CFZ and BDQ individually and in combination (1.25–5 mg/L). Thereafter, Na⁺,K⁺-ATPase activity was determined, followed by intracellular adenosine triphosphate (ATP) quantification and cellular viability determination. Furthermore, molecular docking of antibiotics with Na⁺,K⁺-ATPase was determined. Both antibiotics demonstrated dose–response inhibition of Na⁺,K⁺-ATPase activity of the RCMs. The greatest inhibition was demonstrated by combinations of CFZ and BDQ, followed by BDQ alone and, lastly, CFZ. Neither antibiotic, either individually or in combination, demonstrated cytotoxicity. Molecular docking revealed an interaction of both antibiotics with Na⁺,K⁺-ATPase, with BDQ showing higher protein-binding affinity than CFZ. The inhibitory effects of CFZ and BDQ, individually and in combination, on the activity of Na⁺,K⁺-ATPase pump of the RCMs highlight the existence of additional mechanisms of QT prolongation by these antibiotics. Full article

Pacemaking activity in substantia nigra dopaminergic neurons is generated by the coordinated activity of a variety of distinct somatodendritic voltage- and calcium-gated ion channels. We investigated whether these functional interactions could arise from a common localization in macromolecular complexes where physical proximity would allow for efficient interaction and co-regulations. For that purpose, we immunopurified six ion channel proteins involved in substantia nigra neuron autonomous firing to identify their molecular interactions. The ion channels chosen as bait were Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 channel proteins, and the methods chosen to determine interactions were co-immunoprecipitation analyzed through immunoblot and mass spectrometry as well as proximity ligation assay. A macromolecular complex composed of Cav1.3, HCN, and SK3 channels was unraveled. In addition, novel potential interactions between SK3 channels and sclerosis tuberous complex (Tsc) proteins, inhibitors of mTOR, and between HCN4 channels and the pro-degenerative protein Sarm1 were uncovered. In order to demonstrate the presence of these molecular interactions in situ, we used proximity ligation assay (PLA) imaging on midbrain slices containing the substantia nigra, and we could ascertain the presence of these protein complexes specifically in substantia nigra dopaminergic neurons. Based on the complementary functional role of the ion channels in the macromolecular complex identified, these results suggest that such tight interactions could partly underly the robustness of pacemaking in dopaminergic neurons. Full article

K_V channel-interacting proteins (KChIP1-4) belong to a family of Ca²⁺-binding EF-hand proteins that are able to bind to the N-terminus of the K_V4 channel α-subunits. KChIPs are predominantly expressed in the brain and heart, where they contribute to the maintenance of the excitability of neurons and cardiomyocytes by modulating the fast inactivating-K_V4 currents. As the auxiliary subunit, KChIPs are critically involved in regulating the surface protein expression and gating properties of K_V4 channels. Mechanistically, KChIP1, KChIP2, and KChIP3 promote the translocation of K_V4 channels to the cell membrane, accelerate voltage-dependent activation, and slow the recovery rate of inactivation, which increases K_V4 currents. By contrast, KChIP4 suppresses K_V4 trafficking and eliminates the fast inactivation of K_V4 currents. In the heart, I_Ks, I_Ca,L, and I_Na can also be regulated by KChIPs. I_Ca,L and I_Na are positively regulated by KChIP2, whereas I_Ks is negatively regulated by KChIP2. Interestingly, KChIP3 is also known as downstream regulatory element antagonist modulator (DREAM) because it can bind directly to the downstream regulatory element (DRE) on the promoters of target genes that are implicated in the regulation of pain, memory, endocrine, immune, and inflammatory reactions. In addition, all the KChIPs can act as transcription factors to repress the expression of genes involved in circadian regulation. Altered expression of KChIPs has been implicated in the pathogenesis of several neurological and cardiovascular diseases. For example, KChIP2 is decreased in failing hearts, while loss of KChIP2 leads to increased susceptibility to arrhythmias. KChIP3 is increased in Alzheimer’s disease and amyotrophic lateral sclerosis, but decreased in epilepsy and Huntington’s disease. In the present review, we summarize the progress of recent studies regarding the structural properties, physiological functions, and pathological roles of KChIPs in both health and disease. We also summarize the small-molecule compounds that regulate the function of KChIPs. This review will provide an overview and update of the regulatory mechanism of the KChIP family and the progress of targeted drug research as a reference for researchers in related fields. Full article

Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients. Full article

The prevention of metastasis is a central goal of cancer therapy. Caveolin-1 (Cav-1) is a structural membrane and scaffolding protein shown to be a key regulator of late-stage breast cancer metastasis. However, therapeutic strategies targeting Cav-1 are still lacking. Here, we demonstrate that the pharmacological activation of potassium channel Kv11.1, which is uniquely expressed in MDA-MB-231 triple negative breast cancer cells (TNBCs) but not in normal MCF-10A cells, induces the dephosphorylation of Cav-1 Tyr-14 by promoting the Ca²⁺-dependent stimulation of protein tyrosine phosphatase 1B (PTP1B). Consequently, the dephosphorylation of Cav-1 resulted in its disassociation from β-catenin, which enabled the accumulation of β-catenin at cell borders, where it facilitated the formation of cell–cell adhesion complexes via interactions with R-cadherin and desmosomal proteins. Kv11.1 activation-dependent Cav-1 dephosphorylation induced with NS1643 also reduced cell migration and invasion, consistent with its ability to regulate focal adhesion dynamics. Thus, this study sheds light on a novel pharmacological mechanism of promoting Cav-1 dephosphorylation, which may prove to be effective at reducing metastasis and promoting contact inhibition. Full article

Excitable cells of the nervous and cardiovascular systems depend on an assortment of plasmalemmal potassium channels to control diverse cellular functions. Voltage-gated potassium (Kv) channels are central to the feedback control of membrane excitability in these processes due to their activation by depolarized membrane potentials permitting K⁺ efflux. Accordingly, Kv currents are differentially controlled not only by numerous cellular signaling paradigms that influence channel abundance and shape voltage sensitivity, but also by heteromeric configurations of channel complexes. In this context, we discuss the current knowledge related to how intracellular Kvβ proteins interacting with pore complexes of Shaker-related Kv1 channels may establish a modifiable link between excitability and metabolic state. Past studies in heterologous systems have indicated roles for Kvβ proteins in regulating channel stability, trafficking, subcellular targeting, and gating. More recent works identifying potential in vivo physiologic roles are considered in light of these earlier studies and key gaps in knowledge to be addressed by future research are described. Full article

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility. Full article

The K_V10.1 voltage-gated potassium channel is highly expressed in 70% of tumors, and thus represents a promising target for anticancer drug discovery. However, only a few ligands are known to inhibit K_V10.1, and almost all also inhibit the very similar cardiac hERG channel, which can lead to undesirable side-effects. In the absence of the structure of the K_V10.1–inhibitor complex, there remains the need for new strategies to identify selective K_V10.1 inhibitors and to understand the binding modes of the known K_V10.1 inhibitors. To investigate these binding modes in the central cavity of K_V10.1, a unique approach was used that allows derivation and analysis of ligand–protein interactions from molecular dynamics trajectories through pharmacophore modeling. The final molecular dynamics-derived structure-based pharmacophore model for the simulated K_V10.1–ligand complexes describes the necessary pharmacophore features for K_V10.1 inhibition and is highly similar to the previously reported ligand-based hERG pharmacophore model used to explain the nonselectivity of K_V10.1 pore blockers. Moreover, analysis of the molecular dynamics trajectories revealed disruption of the π–π network of aromatic residues F359, Y464, and F468 of K_V10.1, which has been reported to be important for binding of various ligands for both K_V10.1 and hERG channels. These data indicate that targeting the K_V10.1 channel pore is also likely to result in undesired hERG inhibition, and other potential binding sites should be explored to develop true K_V10.1-selective inhibitors as new anticancer agents. Full article

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression. Full article

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels. Full article

The K⁺ channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels. Full article

Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression—downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies. Full article

Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM. Full article

Human ether a-go-go related gene (hERG) or KV11.1 potassium channels mediate the rapid delayed rectifier current (I_Kr) in cardiac myocytes. Drug-induced inhibition of hERG channels has been implicated in the development of acquired long QT syndrome type (aLQTS) and fatal arrhythmias. Several marketed drugs have been withdrawn for this reason. Therefore, there is considerable interest in developing better tests for predicting drugs which can block the hERG channel. The drug-binding pocket in hERG channels, which lies below the selectivity filter, normally contains K⁺ ions and water molecules. In this study, we test the hypothesis that these water molecules impact drug binding to hERG. We developed 3D QSAR models based on alignment independent descriptors (GRIND) using docked ligands in open and closed conformations of hERG in the presence (solvated) and absence (non-solvated) of water molecules. The ligand–protein interaction fingerprints (PLIF) scheme was used to summarize and compare the interactions. All models delineated similar 3D hERG binding features, however, small deviations of about ~0.4 Å were observed between important hotspots of molecular interaction fields (MIFs) between solvated and non-solvated hERG models. These small changes in conformations do not affect the performance and predictive power of the model to any significant extent. The model that exhibits the best statistical values was attained with a cryo_EM structure of the hERG channel in open state without water. This model also showed the best R² of 0.58 and 0.51 for the internal and external validation test sets respectively. Our results suggest that the inclusion of water molecules during the docking process has little effect on conformations and this conformational change does not impact the predictive ability of the 3D QSAR models. Full article