Paroxysmal depolarization shifts (PDS) have been described by epileptologists for the first time several decades ago, but controversy still exists to date regarding their role in epilepsy. In ...addition to the initial view of a lack of such a role, seemingly opposing hypotheses on epileptogenic and anti-ictogenic effects of PDS have emerged. Hence, PDS may provide novel targets for epilepsy therapy. Evidence for the roles of PDS has often been obtained from investigations of the multi-unit correlate of PDS, an electrographic spike termed "interictal" because of its occurrence during seizure-free periods of epilepsy patients. Meanwhile, interictal spikes have been found to be associated with neuronal diseases other than epilepsy, e.g., Alzheimer's disease, which may indicate a broader implication of PDS in neuropathologies. In this article, we give an introduction to PDS and review evidence that links PDS to pro- as well as anti-epileptic mechanisms, and to other types of neuronal dysfunction. The perturbation of neuronal membrane voltage and of intracellular Ca
that comes with PDS offers many conceivable pathomechanisms of neuronal dysfunction. Out of these, the operation of L-type voltage-gated calcium channels, which play a major role in coupling excitation to long-lasting neuronal changes, is addressed in detail.
This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis ...(AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis.
Key points
Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.
In this study, we identified the residues in five ...phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.
Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.
Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.
Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors.
The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.
Key points
Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.
In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.
Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.
Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.
Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors.
The release of transmitters at sympathoeffector junctions is not constant, but subject to modulation by a plethora of different mechanisms. In this respect, presynaptic receptors located on the ...sympathetic axon terminals are of utmost importance, because they are activated by exogenous agonists and by endogenous neurotransmitters. In the latter case, the transmitters that activate the presynaptic receptors of a nerve terminal may be released either from the very same nerve ending or from a different axon terminal, and the receptors involved are auto- and heteroreceptors, respectively. In terms of their structural and functional features, receptors of sympathetic axon terminals can be categorized as either ionotropic (transmitter-gated ion channels) or metabotropic (most commonly G protein-coupled) receptors. This review summarizes results on more than 30 different metabotropic and four different ionotropic receptors that have been found to control the amount of transmitter being released from sympathetic neurons. Each of these receptors may not only stimulate, facilitate, and reduce sympathetic transmitter release, respectively, but also interact with the functions of other receptors present on the same axonal varicosity. This provides a multitude of mechanisms that regulate the amount of sympathetic transmitter output. Accordingly, a sophisticated cross-talk within and between extra- and intracellular signals is integrated at axon terminals to adapt the strength of sympathoeffector transmission to a given situation. This will not only determine the function of the sympathetic nervous system in health and disease, but also therapeutic and untoward effects of drugs that bind to the presynaptic receptors in sympathetically innervated tissues.
Neuronal nitric oxide synthase (nNOS) is considered a regulator of Ca
v
1.2 L-type Ca
2+
channels and downstream Ca
2+
cycling in the heart. The commonest view is that nitric oxide (NO), generated by ...nNOS activity in cardiomyocytes, reduces the currents through Ca
v
1.2 channels. This gives rise to a diminished Ca
2+
release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca
2+
transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca
2+
cycling and contractility. Whole cell currents through L-type Ca
2+
channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Ca
v
1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Ca
v
1.2 channel function. We conclude that—against the currently prevailing view—basal Ca
v
1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca
2+
cycling and contractility occurs independently of direct regulation of Ca
v
1.2 channels by NO.
Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence ...suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug.
Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Na
1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Na
1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Na
1.5 inactivation parameters and inhibition of different VGSC isoforms.
Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Na
1.5), neuronal (Na
1.2), and skeletal muscle (Na
1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site.
Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.
ABSTRACT
Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. ...Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD.
Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal.
These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD.
BACKGROUND AND PURPOSE
Flupirtine is a non‐opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). ...Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.
EXPERIMENTAL APPROACH
Effects of flupirtine on native and recombinant voltage‐ and ligand‐gated ion channels were explored in patch‐clamp experiments using the following experimental systems: recombinant KIR3 and KV7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage‐gated Na+, Ca2+, inward rectifier K+, KV7 K+, and TRPV1 channels, as well as GABAA, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.
KEY RESULTS
Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage‐gated Na+ or Ca2+ channels, inward rectifier K+ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of KV7 K+ channels to more negative potentials and the gating of GABAA receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on KV7 channels and GABAA receptors was comparable, whereas in hippocampal neurons the effects on KV7 channels were more pronounced.
CONCLUSIONS AND IMPLICATIONS
These results indicate that flupirtine exerts its analgesic action by acting on both GABAA receptors and KV7 channels.
The muscular dystrophies caused by dystrophin deficiency, the so‐called dystrophinopathies, are associated with impaired cardiac contractility and arrhythmias, which considerably contribute to ...disease morbidity and mortality. Impaired Ca handling in ventricular cardiomyocytes has been identified as a causative factor for complications in the dystrophic heart, and restoration of normal Ca handling in myocytes has emerged as a promising new therapeutic strategy. In the present study, we explored the hypothesis that ivabradine, a drug clinically approved for the treatment of heart failure and stable angina pectoris, improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Therefore, ventricular cardiomyocytes were isolated from the hearts of adult dystrophin‐deficient DMDmdx rats, and the effects of acutely applied ivabradine on intracellular Ca transients were tested. In addition, the drug's acute impact on cardiac function in DMDmdx rats was assessed by transthoracic echocardiography. We found that administration of ivabradine to DMDmdx rats significantly improved cardiac function. Moreover, the amplitude of electrically induced intracellular Ca transients in ventricular cardiomyocytes isolated from DMDmdx rats was increased by the drug. We conclude that ivabradine enhances Ca release from the sarcoplasmic reticulum in dystrophic cardiomyocytes and thereby improves contractile performance in the dystrophic heart.
We have investigated the acute effects of the heart rate‐ lowering drug ivabradine on cardiac Ca handling and function in a rat model of Duchenne muscular dystrophy. Our findings suggest that the drug improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart.
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