Voltage-gated Potassium Channel Kv1 Research Articles

Inhibition of the Voltage-Gated Potassium Channel KV2.1 by RY785, a Kv2-Selective Small Molecule

Inhibition of the Voltage-Gated Potassium Channel KV2.1 by RY785, a Kv2-Selective Small Molecule

Functional Consequences of Incidentally Discovered KCNQ1 Variants Determined by Automated Electrophysiology

Mutations in KCNQ1, which encodes the voltage-gated potassium channel KV7.1, constitute a majority of cases with congenital long-QT syndrome (LQTS), and are also associated with short QT syndrome, familial atrial fibrillation, and sudden infant death syndrome. The increased availability and lower costs of genomic testing has led to the incidental discovery of KCNQ1 variants of unknown significance in persons without known heart rhythm disorders and in healthy individuals. We performed functional analyses of 37 heterozygous variants found incidentally in patients from Northwestern Memorial Hospital and the Geisinger Clinic who underwent whole genome or exome sequencing. Functional analysis was done using an established workflow that combines high efficiency electroporation of human KV7.1 into CHO cells stably expressing KCNE1, followed by automated planar patch-clamp recording. We analyzed variants alone first, and then co-expressed each variant with the WT channel to mimic patient heterozygous state. Twelve variants exhibited near normal peak current density (75-150% of WT), 16 exhibited mild loss-of-function (LOF; 25-75% of WT), while 9 had severe LOF (≤25% of WT). Co-expression of severe LOF variants with the WT channel rescued 1 variant, whereas 5 were partially rescued and 3 exhibited dominant-negative suppression of current that was consistent with autosomal dominant congenital LQTS. The rescued variant and 3 of the partially recued variants exhibited depolarizing shifts in activation (P≤0.01). Among mild LOF variants, 7 were rescued by WT co-expression, while the remainder continued to exhibit lower current density, but none of the mild LOF variants in this cohort exhibited dominant-negative behavior. Some variants rescued by co-expressing with the WT channel may represent recessive mutations. In summary, our results indicate that automated patch clamp recording can reveal important functional properties of incidental KCNQ1 variants that will help in classifying these variants.

Challenges Faced with Small Molecular Modulators of Potassium Current Channel Isoform Kv1.5.

The voltage-gated potassium channel Kv1.5, which mediates the cardiac ultra-rapid delayed-rectifier (IKur) current in human cells, has a crucial role in atrial fibrillation. Therefore, the design of selective Kv1.5 modulators is essential for the treatment of pathophysiological conditions involving Kv1.5 activity. This review summarizes the progress of molecular structures and the functionality of different types of Kv1.5 modulators, with a focus on clinical cardiovascular drugs and a number of active natural products, through a summarization of 96 compounds currently widely used. Furthermore, we also discuss the contributions of Kv1.5 and the regulation of the structure-activity relationship (SAR) of synthetic Kv1.5 inhibitors in human pathophysiology. SAR analysis is regarded as a useful strategy in structural elucidation, as it relates to the characteristics that improve compounds targeting Kv1.5. Herein, we present previous studies regarding the structural, pharmacological, and SAR information of the Kv1.5 modulator, through which we can assist in identifying and designing potent and specific Kv1.5 inhibitors in the treatment of diseases involving Kv1.5 activity.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

The Potassium Channel Kv1.5 Expression Alters During Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic, inflammatory, neurodegenerative disease with an autoimmune component. It was suggested that potassium channels, which are involved in crucial biological functions may have a role in different diseases, including MS and its animal model, experimental autoimmune encephalomyelitis (EAE). It was shown that voltage-gated potassium channels Kv1.5 are responsible for fine-tuning in the immune physiology and influence proliferation and differentiation in microglia and astrocytes. Here, we explored the cellular distribution of the Kv1.5 channel, together with its transcript and protein expression in the male rat spinal cord during different stages of EAE. Our results reveal a decrease of Kv1.5 transcript and protein level at the peak of disease, where massive infiltration of myeloid cells occurs,together with reactive astrogliosis and demyelination. Also, we revealed that the presence of this channel is not found in infiltrating macrophages/microglia during EAE. It is interesting to note that Kv1.5 channel is expressed only in resting microglia in the naïve animals. Predominant expression of Kv1.5 channel was found in the astrocytesin all experimental groups, while some vimentin+ cells, resembling macrophages, are devoid of Kv1.5 expression. Our results point to the possible link between Kv1.5 channel and the pathophysiological processes in EAE.

Paraneoplastic neuropathies. Antibodies spectrum

Small-cell lung cancer (SCLC) sometimes is associated with paraneoplastic neurological autoimmunity. Different antibodies have been used for diagnosis of this condition and for diagnosis and signs of therapy results. Therefore various types of antibodies play a pathogenic role in mechanisms of autoimmune processes in paraneoplastic neuropathies (PN). Under our observation were 59 patients with different type of PN, 37 from them suffered from SCLC with paraneoplastic neuropathies, and 11 from Gullian-Barre syndrome. Control group consists of 246 neurologically healthy donors. Cerebrospinal fluid (CSF) was obtained from urological patients during spinal anesthesia and consequent surgeries. We used ELISA and Western Blot analysis for detection of anti-myelin-associated glycoprotein antibodies (MAG), anti-ganglioside antibodies. which are principally associated with autoimmune peripheral neuropathies and anti-glutamic acid decarboxylase antibodies in serum and CSF of all patients and control group. Besides this AB against urinergic receptor channel P2X purinoceptor 7 (P2X7), the voltage-gated potassium channel KV1.3 and the voltage-gated sodium channel NaV1.7 as examples of targeting ion channels were studied. It was revealed that in 78 per cent of PN all AB were increased two and three-fold. Ion channels AB were especially raised. In conclusion, we can state that antibody response plays a key role in pathogenesis of all autoimmune neuropathies. Paraneoplastic pathology in this regard is essential. Future research has to be done consider ion channel involvement and autoimmune reactivity toward them.

Development of an Electrophysiological Assay for Kv7 Modulators on IonWorks Barracuda.

Relief from chronic pain continues to represent a large unmet need. The voltage-gated potassium channel Kv7.2/7.3, also known as KCNQ2/3, is a key contributor to the control of resting membrane potential and excitability in nociceptive neurons and represents a promising target for potential therapeutics. In this study, we present a medium throughput electrophysiological assay for the identification and characterization of modulators of Kv7.2/7.3 channels, using the IonWorks Barracuda™ automated voltage clamp platform. The assay combines a family of voltage steps used to construct conductance curves with a unique analysis method. Kv7.2/7.3 modulators shift the activation voltage and/or change the maximal conductance of the current, and both parameters have been used to quantify compound mediated effects. Both effects are expected to modulate neuronal excitability in vivo. The analysis method described assigns a single potency value that combines changes in activation voltage and maximal conductance and is expected to predict compound mediated changes in excitability.

Long-term obesogenic diet and targeted deletion of potassium channel Kv 1.3 have differing effects on voluntary exercise in mice.

Voluntary exercise is frequently employed as an intervention for obesity. The voltage‐gated potassium channel Kv1.3 is also receiving attention as a therapeutic target for obesity, in addition to potential therapeutic capabilities for neuroinflammatory diseases. To investigate the combinatorial effects of these two therapies, we have compared the metabolic status and voluntary exercise behavior of both wild‐type mice and a transgenic line of mice that are genetic knockouts for Kv1.3 when provided with a running wheel and maintained on diets of differing fat content and caloric density. We tracked the metabolic parameters and wheel running behavior while maintaining the mice on their assigned treatment for 6 months. Wild‐type mice maintained on the fatty diet gain a significant amount of bodyweight and adipose tissue and display significantly impaired glucose tolerance, though all these effects were partially reduced with provision of a running wheel. Similar to previous studies, the Kv1.3‐null mice were resistant to obesity, increased adiposity, and impaired glucose tolerance. Both wild‐type and Kv1.3‐null mice maintained on the fatty diet displayed increased wheel running activity compared to control‐fed mice, which was caused primarily by a significant increase in the amount of time spent running as opposed to an increase in running velocity. Interestingly, the patterns of running behavior differed between wild‐type and Kv1.3‐null mice. Kv1.3‐null mice spent significantly less time running during the light phase and displayed a decrease in running 1–2 h before the onset of the light phase, seemingly in anticipation of the dark‐to‐light phase transition. These studies indicate that voluntary exercise combats metabolic maladies and running behavior is modified by both consumption of an obesogenic diet and deletion of the Kv1.3 channel.

Voltage-Gated Potassium Channel Kv1.3 as a Target in Therapy of Cancer.

Voltage-gated potassium channel Kv1.3 is an integral membrane protein, which is selectively permeable for potassium ions and is activated upon a change of membrane potential. Channel activation enables transportation of potassium ions down their electrochemical gradient. Kv1.3 channel is expressed in many cell types, both normal and cancer. Activity of the channel plays an important role in cell proliferation and apoptosis. Inhibition of Kv1.3 channel may be beneficial in therapy of several diseases including some cancer disorders. This review focuses on Kv1.3 channel as a new potentially attractive molecular target in cancer therapy. In the first part, changes in the channel expression in selected cancer disorders are described. Then, the role of the channel activity in cancer cell proliferation and apoptosis is presented. Finally, it is shown that some low molecular weight organic inhibitors of the channel including selected biologically active plant-derived polycyclic compounds may selectively induce apoptosis of Kv1.3-expressing cancer cells while sparing normal cells and healthy organs. These compounds may be promising candidates for putative application in therapy of some cancer disorders, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), or B-type chronic lymphocytic leukemia (B-CLL).

Expression of the Voltage-Gated Potassium Channel Kv1.3 in Lesional Skin from Patients with Cutaneous T-Cell Lymphoma and Benign Dermatitis

Background: The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by effector memory T cells (T_EM) and plays an important role in their activation and proliferation. Mycosis fungoides (MF), the most common subtype of cutaneous T-cell lymphoma (CTCL), was recently proposed to be a malignancy of skin-resident T_EM. However, the expression of Kv1.3 in CTCL has not been investigated. Objectives: This study aims to examine the expression of Kv1.3 in situ and in vitro in CTCL. Methods: The expression of Kv1.3 was examined by immunohistochemistry in skin lesions from 38 patients with MF, 4 patients with Sézary syndrome (SS), and 27 patients with benign dermatosis. In 4 malignant T-cell lines of CTCL (Myla2059, PB2B, SeAx, and Mac2a) and a non-malignant T-cell line (MyLa1850), the expression of Kv1.3 was determined by flow cytometry. The proliferation of those cell lines treated with various concentrations of Kv1.3 inhibitor ShK was measured by ³H-thymdine incorporation. Results: Half of the MF patients (19/38) displayed partial Kv1.3 expression including 1 patient with moderate Kv1.3 positivity, while the other half (19/38) exhibited Kv1.3 negativity. An almost identical distribution was observed in patients with benign conditions, that is, 44.4% (12/27) were partially positive for Kv1.3 including 1 patient with moderate Kv1.3 positivity, while 55.6% (15/27) were Kv1.3 negative. In contrast, 3 in 4 SS patients displayed partial Kv1.3 positivity including 2 patients with weak staining and 1 with moderate staining, while 1 in 4 SS patients was Kv1.3 negative. In addition, all malignant T-cell lines, and a non-malignant T-cell line, displayed low Kv1.3 surface expression with a similar pattern. Whereas 2 cell lines (PB2B and Mac2a) were sensitive to Kv1.3 blockade, the other 2 (Myla2059 and SeAx) were completely resistant. Conclusions: We provide the first evidence of a heterogeneous Kv1.3 expression in situ in CTCL lesions.

Generation of an induced pluripotent stem cell (iPSC) line (HIHDNEi003-A) from a patient with developmental and epileptic encephalopathy carrying a KCNA2 (p.Thr374Ala) mutation.

De novo mutations in the KCNA2 gene, encoding the voltage-gated potassium channel KV1.2, have been identified to cause early-onset developmental and epileptic encephalopathies (DEE). KV1.2 channels conduct delayed-rectifier type K+ currents and play a crucial role in action potential repolarization. In this study we reprogrammed fibroblasts from a 6-months-old male patient with DEE carrying a de novo point mutation (c.1120A > G, p.Thr374Ala) in KCNA2 to induced pluripotent stem cells. Their pluripotency was verified by the capability to differentiate into all three germ layers and the expression of several pluripotency markers on RNA and protein levels.

Expression and function of Kv1.3 channel in malignant T cells in Sézary syndrome.

The voltage-gated potassium channel Kv1.3 (KCNA3) is expressed by a subset of chronically activated memory T cells and plays an important role in their activation and proliferation. Here, we show that primary malignant T cells isolated from patients with Sézary syndrome (SS) express Kv1.3 and are sensitive to potent Kv1.3 inhibitors ShK and Vm24, but not sensitive to a less potent inhibitor [N17A/F32T]-AnTx. Kv1.3 blockade inhibits CD3/CD28-induced proliferation and IL-9 expression by SS cells in a concentration-dependent manner. In parallel, CD3/CD28-mediated CD25 induction is inhibited, whereas Kv1.3 blockade has no effect on apoptosis or cell death as judged by Annexin V and PI staining. In conclusion, we provide the first evidence that malignant T cells in SS express functional Kv1.3 channels and that Kv1.3 blockade inhibits activation-induced proliferation as well as cytokine and cytokine receptor expression in malignant T cells, suggesting that Kv1.3 is a potential target for therapy in SS.

Deep Sequencing of Somatosensory Neurons Reveals Molecular Determinants of Intrinsic Physiological Properties

Dorsal root ganglion (DRG) sensory neuron subtypes defined by their invivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.

Impact of anti-CASPR2 autoantibodies from patients with autoimmune encephalitis on CASPR2/TAG-1 interaction and Kv1 expression

Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

Selective Axonal Expression of the Kv1 Channel Complex in Pre-myelinated GABAergic Hippocampal Neurons.

In myelinated fibers, the voltage-gated sodium channels Nav1 are concentrated at the nodal gap to ensure the saltatory propagation of action potentials. The voltage-gated potassium channels Kv1 are segregated at the juxtaparanodes under the compact myelin sheath and may stabilize axonal conduction. It has been recently reported that hippocampal GABAergic neurons display high density of Nav1 channels remarkably in clusters along the axon before myelination (Freeman et al., 2015). In inhibitory neurons, the Nav1 channels are trapped by the ankyrinG scaffold at the axon initial segment (AIS) as observed in pyramidal and granule neurons, but are also forming “pre-nodes,” which may accelerate conduction velocity in pre-myelinated axons. However, the distribution of the Kv1 channels along the pre-myelinated inhibitory axons is still unknown. In the present study, we show that two subtypes of hippocampal GABAergic neurons, namely the somatostatin and parvalbumin positive cells, display a selective high expression of Kv1 channels at the AIS and all along the unmyelinated axons. These inhibitory axons are also highly enriched in molecules belonging to the juxtaparanodal Kv1 complex, including the cell adhesion molecules (CAMs) TAG-1, Caspr2, and ADAM22 and the scaffolding protein 4.1B. Here, taking advantage of hippocampal cultures from 4.1B and TAG-1 knock-out mice, we observed that 4.1B is required for the proper positioning of Caspr2 and TAG-1 along the distal axon, and that TAG-1 deficiency induces alterations in the axonal distribution of Caspr2. However, the axonal expression of Kv1 channels and clustering of ankyrinG were not modified. In conclusion, this study allowed the analysis of the hierarchy between channels, CAMs and scaffolding proteins for their expression along hippocampal inhibitory axons before myelination. The early steps of channel compartmentalization preceding myelination may be crucial for stabilizing nerve impulses switching from a continuous to saltatory conduction during network development.

In vitro and in vivo characterization of a synthetic scorpion toxin AmmTx3, a potent inhibitor of cardiac voltage-gated potassium channel Kv4.2

Voltage-gated potassium channel Kv4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus , is a blocker of Kv4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac Kv4.2 channels remains unclear. The aim of this study was to: – produce a synthetic AmmTx3 (AmmTx3-NCL); – evaluate in vitro and in vivo, effects of this toxin on Kv4.2 channels compared to native toxin. We did chemical synthesis of AmmTx3 using the native chemical ligation strategy (NCL). We characterized its biological activity compared to native AmmTx3 using an automated patch-clamp system (Syncropatch 384 PE, Nanion) on HEK cells co-expressing Kv4.2 α subunit and two auxiliaries subunits (KChIP and DPP6). Effects of AmmTx3 and AmmTx3-NCL on cardiac cellular electrophysiology were studied by patch clamp on adult rat cardiomyocytes and hiPSC-CMs. Integrated cardiac effects of AmmTx3 were studied on ECG of wild-type mice. In this study, we report the total chemical synthesis of AmmTx3 and 3D structure was also determined. As expected, biological activity of folded AmmTx3-NCL is coherent to native toxin with a similar time course of Kv4.2 current inhibition without any change of activation kinetics. Characterisation of AmmTx3 effects on isolated cardiomyocytes, hiPSC-CMs and in vivo by ECG is ongoing. AmmTx3 toxin can be chemically synthesized and used as a Kv4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.

Epilepsy Gene Therapy Using an Engineered Potassium Channel.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

Antibodies and venom peptides: new modalities for ion channels.

Ion channels play fundamental roles in both excitable and non-excitable tissues and therefore constitute attractive drug targets for myriad neurological, cardiovascular and metabolic diseases as well as for cancer and immunomodulation. However, achieving selectivity for specific ion channel subtypes with small-molecule drugs has been challenging, and there currently is a growing trend to target ion channels with biologics. One approach is to improve the pharmacokinetics of existing or novel venom-derived peptides. In parallel, after initial studies with polyclonal antibodies demonstrated the technical feasibility of inhibiting channel function with antibodies, multiple preclinical programmes are now using the full spectrum of available technologies to generate conventional monoclonal and engineered antibodies or nanobodies against extracellular loops of ion channels. After a summary of the current state of ion channel drug discovery, this Review discusses recent developments using the purinergic receptor channel P2X purinoceptor 7 (P2X7), the voltage-gated potassium channel KV1.3 and the voltage-gated sodium channel NaV1.7 as examples of targeting ion channels with biologics.

Probing Kv1.3 Interactome with Proximity-Dependent Biotinylation

Protein-protein interactions (PPIs) are central to the proper functioning of the most basic molecular mechanisms underlying cellular life and are often perturbed in disease states. Traditional techniques contributed to the improvement of knowledge in this field but they demonstrated significant limitations. Recently, a new method called BioID (proximity dependent biotin identification) based on proximity-dependent labeling of proteins has been developed (Ruox et al., 2012 doi.org/10.1083/jcb.201112098). The technique exploits a promiscuous biotinylating enzyme, BirA, that can be fused to the protein of interest. In this work, we applied the BioID protocol to study the PPIs of the voltage gated potassium channel Kv1.3, that displays subcellular localization to various organelles in addition to being present in the plasmamembrane. Proteins identified by BioID are candidate interactors for the channel and represent the starting point to improve our knowledge about the role this protein plays in human diseases and to possibly to identify new pathways in which Kv1.3 is involved at the cellular level.

PKC Activation Decreases Kv1.5 Protein Expression through Accelerating Endocytic Channel Degradation

The voltage-gated potassium channel Kv1.5 is primarily expressed in the atria and conducts the ultra-rapidly delayed rectifier K+ current (IKur), which is important for atrial repolarization and closely related to atrial fibrillation. Elevated stress is associated with several cardiovascular outcomes, including atrial fibrillation in which Kv1.5 is thought to play a role. Since protein kinase C (PKC) activation is associated with stress, we examined the effects of PKC activation on Kv1.5 channels. Previous studies have shown that PKC regulates Kv1.5 channel when co-expressed with Kvβ1.2 or Kvβ1.3. In the present study, using Western blot analysis and whole-cell patch clamp recording in human embryonic kidney cells stably expressing Kv1.5 alone, we found that PKC activation by phorbol 12-myristate 13-acetate (PMA) at 10 nM nearly abolished Kv1.5 channel expression and current within 3 hours. Inhibition of PKC by Bisindolylmaleimide I completely prevented PMA-induced Kv1.5 channel reduction. Co-immunoprecipitation experiments showed that PMA treatment enhanced ubiquitination of Kv1.5 channels; proteasome inhibitors MG-132 or lactacystin partially prevented the PMA-induced Kv1.5 channel reduction. We also found that transfection of dominant-negative Rab5 partly impeded PMA-induced Kv1.5 channel reduction. Furthermore, truncation of the N-terminus of Kv1.5 up to residue 209 (ΔN209) abolished PMA-mediated reduction in expression and current of the channel. Intriguingly, deletion of a distinct motif in the N-terminus for non-receptor tyrosine kinase Src also abolished PMA-mediated reduction in Kv1.5 expression and current. We propose that interplay between PKC and Src mediates degradation of mature Kv1.5 channels partially through ubiquitination and Rab5-mediated endocytosis. These findings extend our understanding of the regulation of Kv1.5 channel by PKC activation. (Supported by the Canadian Institutes of Health Research).