SAP97 and Cortactin Remodeling in Arrhythmogenic Purkinje Cells

Because structural remodeling of several proteins, including ion channels, may underlie the abnormal action potentials of Purkinje cells (PCs) that survive in the 48 hr infarcted zone of the canine heart (IZPCs), we sought to determine the subcellular structure and function of the K_V1.5 (KCNA5) protein in single IZPCs. Clustering of the Kv1.5 subunit in axons is regulated by a synapse-associated protein, SAP97, and is linked to an actin-binding protein, cortactin, and an intercellular adhesion molecule, N-cadherin. To understand the functional remodeling of the Kv1.5 channel and its regulation in IZPCs, Kv1.5 currents in PCs were measured as the currents blocked by 10 µM RSD1379 using patch-clamp techniques. Immunocytochemistry and confocal imaging were used for both single and aggregated IZPCs vs normal PCs (NZPCs) to determine the relationship of Kv1.5 with SAP-97, cortactin and N-cadherin. In IZPCs, both the sarcolemma (SL) and intercalated disk (ID) Kv1.5 protein are abundant, and the amount of cytosolic Kv1.5 protein is greatly increased. SAP-97 is also increased at IDs and has notable cytosolic localization suggesting that SAP-97 may regulate the functional expression and stabilization of Kv1.5 channels in IZPCs. Cortactin, which is located with N-cadherin at IDs in NZPCs, remains at IDs but begins to dissociate from N-cadherin, often forming ring structures and colocalizing with Kv1.5 within IZPCs. At the same time, cortactin/Kv1.5 colocalization is increased at the ID, suggesting an ongoing active process of membrane trafficking of the channel protein. Finally, the Kv1.5 current, measured as the RSD1379-sensitive current, at +40 mV did not differ between NZPCs (0.81±0.24 pA/pF, n = 14) and IZPCs (0.83±0.21 pA/pF, n = 13, NS). In conclusion, the subcellular structural remodeling of Kv1.5, SAP97 and cortactin maintained and normalized the function of the Kv1.5 channel in Purkinje cells that survived myocardial infarction.     

Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway

Voltage-gated Na_v channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na_v1.5 is the predominant Na_v channel, and Na_v1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na_v1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na_v channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na_v1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na_v1.5 expression, abnormal Na_v1.5 membrane targeting, and reduced Na⁺ channel current density. We define the structural requirements on ankyrin-G for Na_v1.5 interactions and demonstrate that loss of Na_v1.5 targeting is caused by the loss of direct Na_v1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Na_v channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na_v channel organization in multiple excitable tissues.     

Being there: cellular targeting of voltage-gated sodium channels in the heart

Voltage-gated sodium (Na_v) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Na_v1.5, the principal Na_v channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Na_v1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Na_v1.5 and ankyrin-G is necessary for the expression of Na_v1.5 at the cardiomyocyte cell surface.     

The β1-Subunit of Nav1.5 Cardiac Sodium Channel Is Required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na_v1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na_v1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na_v1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β₁-subunit was essential for this dominant negative effect. Indeed, the absence of the β₁-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β₁-subunit was present. Our findings reveal a new role for β₁-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.     

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Na_v1.5 and Na_v1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Na_v1.5 or Na_v1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Na_v1.5 and Na_v1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Na_v1.5 or Na_v1.7. The recovery from inactivation of Na_v1.5 or Na_v1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Na_v1.5 than lidocaine, but not Na_v1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Na_v1.5, as compared to Na_v1.7; and mexiletine exhibits stronger use-dependent block of Na_v1.5. The differential gating properties of Na_v1.5 and Na_v1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.     

Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Nav1.5

Background

The cardiac sodium channel Na_v1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Na_v1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Na_v1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Na_v1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood.

Differential regulation of Na(v)beta subunits during myogenesis

Voltage-gated sodium channels (Na_v) consist of a pore-forming α subunit (Na_vα) associated with β regulatory subunits (Na_vβ). Adult skeletal myocytes primarily express Na_v1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na_v1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na_v1.5 currents than those recorded from myoblasts. Unlike myoblasts, I_Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na_vβ subunits modulate channel gating and pharmacology, the goal of the present work was to study Na_vβ subunits during myogenesis. All four Na_vβ (Na_vβ1-4) isoforms were present in L6E9 myocytes. While Na_vβ1-3 subunits were up-regulated by myogenesis, Na_vβ4 subunits were not. These results show that Na_vβ genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na⁺ channels during development and myotube formation.     

Benzoxazolinone aryl sulfonamides as potent,selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model

Studies on human genetics have suggested that inhibitors of the Na_v1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Na_v1.7 inhibitors with excellent selectivity against the Na_v1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Na_v1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.     

The discovery of benzenesulfonamide-based potent and selective inhibitors of voltage-gated sodium channel Nav1.7

The voltage gated sodium channel Na_v1.7 represents an interesting target for the treatment of pain. Human genetic studies have identified the crucial role of Na_v1.7 in pain signaling. Herein, we report the design and synthesis of a novel series of benzenesulfonamide-based Na_v1.7 inhibitors. Structural–activity relationship (SAR) studies were undertaken towards improving Na_v1.7 activity and minimizing CYP inhibition. These efforts resulted in the identification of compound 12k, a highly potent Na_v1.7 inhibitor with a thousand-fold selectivity over Na_v1.5 and negligible CYP inhibition.     

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of 55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Na_v1.5; human ether-à-go-go-related gene     

Structural modeling of human cardiac sodium channel pore domain

The pore domain of human voltage-dependent cardiac sodium channel Na_v1.5 (hNa_v1.5) is the crucial binding targets for anti-arrhythmics drugs and some local anesthetic drugs but its three-dimensional structure is still lacking. This has affected the detailed studies of the binding features and mechanism of these drugs. In this paper, we present a structural model for open-state pore domain of hNa_v1.5 built using single template ROSETTA-membrane homology modeling with the crystal structure of Na_vMs. The assembled structural models are evaluated by rosettaMP energy and locations of binding sites. The modeled structures of the pore domain of hNa_v1.5 in open state will be helpful to explore molecular mechanism of a state-dependent drug binding and help designing new drugs.     

Ionic exchanges in isolated and open-circuited toad skin

Summary Net influxes of Na and Cl and effluxes of K and H (J _Na,J _Cl,J _K andJ _H) and volume flowJ _v across isolated open-circuited toad skins were measured using rotating chambers and a small volume of external solution, the ion fluxes being determined by chemical analysis of the external solution, in the range of 0.2 to 5.0mm external Na concentration. In this concentration range, with skin potential varying with (Na) _e ,J _Na is a linear function of the Na electrochemical potential difference across the skin, as expected on irreversible thermodynamic grounds. TheL _Na coefficient calculated asJ _Na/ _Na is equal to 5.5×10^–12 mole² joule^–1 cm^–2 min^–1, which is similar to values obtained for the same species in the short-circuited state and in higher ranges of (Na) _e . A positive correlation is observed betweenJ _Na andJ _K whenJ _Na varied with (Na) _e and also whenJ _Na varies in randomly selected skins. Antidiuretic hormone stimulatesJ _Na,J _K andJ _v in the range of 0.2 to 5.0mm (Na) _e and lowers the Na concentration in the equivalent solution absorbed by the skin (calculated asJ _Na/J _v ). Substitution of external Cl by SO₄ has no effect onJ _Na,J _K andJ _H and also in the skin potential in the range of (Na) _e studied. Substitution of external Na by K abolishesJ _Cl and reverses the skin polarity, the external solution now being positive to the internal one. Na removal from the external solution also reducesJ _K almost to zero.J _H is significantly reduced in this condition; however, a basal secretion still persists with (Na) _e equal to zero. The results of these experiments can be tentatively interpreted in terms of electrical coupling between ion fluxes, since only the procedures that result in alterations of skin potential are followed by changes in the rates of ion transport. The existence of other coupling mechanisms cannot be ruled out.     

Recent progress in sodium channel modulators for pain

Voltage-gated sodium channels (Na_vs) are an important family of transmembrane ion channel proteins and Na_v drug discovery is an exciting field. Pharmaceutical investment in Na_vs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Na_v structural biology pave the way for a future of fruitful prospective Na_v drug discovery.     

Multiple machine learning methods aided virtual screening of NaV1.5 inhibitors

Na_v1.5 sodium channels contribute to the generation of the rapid upstroke of the myocardial action potential and thereby play a central role in the excitability of myocardial cells. At present, the patch clamp method is the gold standard for ion channel inhibitor screening. However, this method has disadvantages such as high technical difficulty, high cost and low speed. In this study, novel machine learning models to screen chemical blockers were developed to overcome the above shortage. The data from the ChEMBL Database were employed to establish the machine learning models. Firstly, six molecular fingerprints together with five machine learning algorithms were used to develop 30 classification models to predict effective inhibitors. A validation and a test set were used to evaluate the performance of the models. Subsequently, the privileged substructures tightly associated with the inhibition of the Na_v1.5 ion channel were extracted using the bioalerts Python package. In the validation set, the RF-Graph model performed best. Similarly, RF-Graph produced the best result in the test set in which the Prediction Accuracy (Q) was 0.9309 and Matthew's correlation coefficient was 0.8627, further indicating the model had high classification ability. The results of the privileged substructures indicated Sulfa structures and fragments with large Steric hindrance tend to block Na_v1.5. In the unsupervised learning task of identifying sulfa drugs, MACCS and Graph fingerprints had good results. In summary, effective machine learning models have been constructed which help to screen potential inhibitors of the Na_v1.5 ion channel and key privileged substructures with high affinity were also extracted.     

Discovery of novel 4-phenyl-2-(pyrrolidinyl)nicotinamide derivatives as potent Nav1.1 activators

The voltage-gated sodium channel, Na_v1.1, is predominantly expressed in parvalbumin-positive fast spiking interneurons and has been genetically linked to Dravet syndrome. Starting from a high throughput screening hit isoxazole derivative 5, modifications of 5 via combinations of IonWorks and Q-patch assays successfully identified the nicotinamide derivative 4. Its increasing decay time constant (tau) of Na_v1.1 currents at 0.03?μM along with significant selectivity against Na_v1.2, Na_v1.5, and Na_v1.6 and acceptable brain exposure in mice was observed. Compound 4 is a promising Na_v1.1 activator that can be used to analyze pathophysiological functions of the Na_v1.1 channel towards treating various central nervous system diseases.     

Bone marrow mesenchymal stem cells protected post‐infarcted myocardium against arrhythmias via reversing potassium channels remodelling

Bone marrow mesenchymal stem cells (BMSCs) emerge as a promising approach for treating heart diseases. However, the effects of BMSCs‐based therapy on cardiac electrophysiology disorders after myocardial infarction were largely unclear. This study was aimed to investigate whether BMSCs transplantation prevents cardiac arrhythmias and reverses potassium channels remodelling in post‐infarcted hearts. Myocardial infarction was established in male SD rats, and BMSCs were then intramyocardially transplanted into the infarcted hearts after 3 days. Cardiac electrophysiological properties in the border zone were evaluated by western blotting and whole‐cell patch clamp technique after 2 weeks. We found that BMSCs transplantation ameliorated the increased heart weight index and the impaired LV function. The survival of infarcted rats was also improved after BMSCs transplantation. Importantly, electrical stimulation‐induced arrhythmias were less observed in BMSCs‐transplanted infarcted rats compared with rats without BMSCs treatment. Furthermore, BMSCs transplantation effectively inhibited the prolongation of action potential duration and the reduction of transient and sustained outward potassium currents in ventricular myocytes in post‐infarcted rats. Consistently, BMSCs‐transplanted infarcted hearts exhibited the increased expression of K_V4.2, K_V4.3, K_V1.5 and K_V2.1 proteins when compared to infarcted hearts. Moreover, intracellular free calcium level, calcineurin and nuclear NFATc3 protein expression were shown to be increased in infarcted hearts, which was inhibited by BMSCs transplantation. Collectively, BMSCs transplantation prevented ventricular arrhythmias by reversing cardiac potassium channels remodelling in post‐infarcted hearts.     

Investigation of the selectivity of one type of small-molecule inhibitor for three Nav channel isoforms based on the method of computer simulation

Voltage-gated sodium (Na_v) channels play a pivotal role for the changes in membrane potential and belong to large membrane proteins that compose four voltage sensor domains (VSD1–4). In this study, we describe the binding mode and selectivity of one of the aryl sulfonamide sodium channel inhibitors, PF-04856264, for the VSD4s in Na_v1.4, Na_v1.5 and Na_v1.7, respectively, through molecular dynamics simulation and enhanced post-dynamics analyses. Our results show that there are three binding site regions (BSR1–3) in the combination of the ligand and receptors, of which BSR1 and BSR3 contribute to the selectivity and affinity of the ligand to the receptor. What’s more, the 39th residue (Y39 in VSD4^hNav1.4/ VSD4^hNav1.7 and A39 in VSD4^hNav1.5) and N42 in BSR1, the 84th residue (L84 in VSD4^hNav1.4, T84 in VSD4^hNav1.5, and M84 in VSD4^hNav1.7) in BSR2 and the conserved positive charged residues in BSR3 have major contributions to the interaction between the ligand and receptor. Further analysis reveals that if the 39th residue has a benzene ring structure, the connection of BSR1 and the ligand would be much stronger through π-stacking interaction. On the other hand, the strength and number of the hydrogen bonds formed by the ligand and the conserved arginines on S4 determine the contribution of BSR3 to the total free binding energy. We anticipate this study pave the way for the design of more effective and safe treatment for pain that selectively target Na_v1.7.     

An analysis of the direct effect of chloridazepoxide on mammalian cardiac tissues and crayfish and squid giant axons: possible basis of antiarrhythmic activity.

Chlordiazepoxide has been found to be antiarrhythmic $\text{in vivo}$. The purpose of the present investigation is to identify the mechanism(s) of such antiarrhythmic activity. In canine heart, chlordiazepoxide effectively depressed the enhanced repetitive discharges in subendocardial Purkinje fibers surviving acute myocardial infarction. Chlordiazepoxide altered the action potential characteristics of Purkinje fiber by shortening the APD₅₀, APD₁₀₀ and effective refractory period with little effect on the resting membrane potential. The maximal rate of upstroke (dv/dt) was significantly reduced only at 1 × 10^?4M and above in Purkinje fibers and the membrane response curve was consistently shifted to the right by chlordiazepoxide. The ventricular muscle was little affected by chlordiazepoxide except for the shortened APD₅₀ and reduced dv/dt. Chlordiazepoxide exerted nerve blocking potency comparable to lidocaine in the crayfish giant axon. Voltage-clamp experiments in squid axon showed that chlordiazepoxide suppressed both components of membrane current, the transient inward sodium current being diminished far greater than the steady-state potassium current. These results demonstrate a direct action on cardiac and axonal membranes which may be partially responsible for the antiarrhythmic activity of this agent.