Creatine, guanidinoacetate and homoarginine in statin-induced myopathy.

Our study evaluated the effect of creatine and homoarginine in AGAT- and GAMT-deficient mice after simvastatin exposure. Balestrino and Adriano suggest that guanidinoacetate might explain the difference between AGAT- and GAMT-deficient mice in simvastatin-induced myopathy. We agree with Balestrino and Adriano that our data shows that (1) creatine possesses a protective potential to ameliorate statin-induced myopathy in humans and mice and (2) homoarginine did not reveal a beneficial effect in statin-induced myopathy. Third, we agree that guanidinoacetate can be phosphorylated and partially compensate for phosphocreatine. In our study, simvastatin-induced damage showed a trend to be less pronounced in GAMT-deficient mice compared with wildtype mice. Therefore, (phospo) guanidinoacetate cannot completely explain the milder phenotype of GAMT-deficient mice, but we agree that it might contribute to ameliorate statin-induced myopathy in GAMT-deficient mice compared with AGAT-deficient mice. Finally, we agree with Balestino and Adriano that AGAT metabolites should further be evaluated as potential treatments in statin-induced myopathy.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

Importance of lipid-pore loop interface for potassium channel structure and function.

Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.

Homoarginine supplementation improves blood glucose in diet-induced obese mice.

L-Homoarginine (hArg) is an endogenous amino acid which has emerged as a novel biomarker for stroke and cardiovascular disease. Low circulating hArg levels are associated with increased mortality and vascular events, whereas recent data have revealed positive correlations between circulating hArg and metabolic vascular risk factors like obesity or blood glucose levels. However, it is unclear whether hArg levels are causally linked to metabolic parameters. Therefore, the aim of our study was to investigate whether hArg directly influences body weight, blood glucose, glucose tolerance or insulin sensitivity. Here, we show that hArg supplementation (14 and 28 mg/mL orally per drinking water) ameliorates blood glucose levels in mice on high-fat diet (HFD) by a reduction of 7.3 ± 3.7 or 13.4 ± 3.8 %, respectively. Fasting insulin concentrations were slightly, yet significantly affected (63.8 ± 11.3 or 162.1 ± 39.5 % of control animals, respectively), whereas body weight and glucose tolerance were unaltered. The substantial augmentation of hArg plasma concentrations in supplemented animals (327.5 ± 40.4 or 627.5 ± 60.3 % of control animals, respectively) diminished profoundly after the animals became obese (129.9 ± 16.6 % in control animals after HFD vs. 140.1 ± 8.5 or 206.3 ± 13.6 %, respectively). This hArg-lowering effect may contribute to the discrepancy between the inverse correlation of plasma hArg levels with stroke and cardiovascular outcome, on the one hand, and the direct correlation with cardiovascular risk factors like obesity and blood glucose, on the other hand, that has been observed in human studies. Our results suggest that the glucose-lowering effects of hArg may reflect a compensatory mechanism of blood glucose reduction by hArg upregulation in obese individuals, without directly influencing body weight or glucose tolerance.

Erg potassium currents of neonatal mouse Purkinje cells exhibit fast gating kinetics and are inhibited by mGluR1 activation.

We investigated the subthreshold properties of an erg (ether-à-go-go-related gene) K(+) current in Purkinje cells of neonatal mice. Action potentials recorded from Purkinje cells in cerebellar slices exhibited a decreased threshold potential and increased frequency of spontaneous and repetitive activity following application of the specific erg channel blocker E-4031. Accommodation was absent before and after drug application. The erg current of these Purkinje cells activated at membrane potentials near -60 mV and exhibited fast gating kinetics. The functional importance of fast gating subthreshold erg channels in Purkinje cells was corroborated by comparing the results of action potential clamp experiments with erg1a, erg1b, erg2, and erg3 currents heterologously expressed in HEK cells. Computer simulations based on a NEURON model of Purkinje cells only reproduced the effects of the native erg current when an erg channel conductance like that of erg3 was included. Experiments with subunit-sensitive toxins (BeKm-1, APETx1) indicated that erg channels in Purkinje cells are presumably mediated by heteromeric erg1/erg3 or modified erg1 channels. Following mGluR1 activation, the native erg current was reduced by ∼70%, brought about by reduction of the maximal erg current and a shift of the activation curve to more positive potentials. The Purkinje cell erg current contributed to the sustained current component of the biphasic mGluR1 response. Activation of mGluR1 by the agonist 3,4-dihydroxyphenylglycol increased Purkinje cell excitability, similar to that induced by E-4031. The results indicated that erg currents can be modulated and may contribute to the mGluR1-induced plasticity changes in Purkinje cells.

Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity.

Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR.

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor interactome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications.

Guanidino compound ratios are associated with stroke etiology, internal carotid artery stenosis and CHA2DS2-VASc score in three cross-sectional studies.

INTRODUCTION: Guanidino compounds, including l-homoarginine (l-hArg), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA) and l-arginine (l-Arg) are associated with mortality, fatal strokes, stroke incidence, and atherosclerosis. OBJECTIVES: We aimed to study the association of guanidino compounds (l-hArg/ADMA and l-hArg/SDMA) with stroke etiology, internal carotid artery (ICA) stenosis and CHA2DS2-VASc score in patients with cerebrovascular disease. METHODS: We analyzed l-hArg, SDMA, ADMA, l-Arg, and compound molar ratios, i.e. l-hArg/ADMA and l-hArg/SDMA, in 272 patients with cerebrovascular disease in a cross-sectional discovery cohort and two cross-sectional validation cohorts of acute stroke patients from Germany (n = 137) and UK (n = 394). The guanidino compound levels were compared with clinical, imaging, and ultrasound parameters. RESULTS: Low l-hArg/ADMA and l-hArg/SDMA molar ratios predicted territorial infarcts (OR 1.74; 95% CI 1.34-2.26 and OR 1.64; 95% CI 1.26-2.15, respectively) and were associated with stroke subtypes due to large vessel disease or cardio-embolism (OR 1.52; 95% CI 1.12-2.06 and OR 2.01; 95% CI 1.35-3.00, respectively) in meta-analysis of the discovery and validation cohort data. In line with these results, a low l-hArg/ADMA and l-hArg/SDMA molar ratio was found in patients with ICA stenosis (OR 0.73; 95% CI 0.55-0.97 and OR 0.69; 95% CI 0.50-0.94, respectively) in the discovery and validation cohort. Furthermore, guanidino compound ratios (i.e. l-hArg/ADMA and l-hArg/SDMA) were strongly correlated with CHA2DS2-VASC score (p < .001) in all three cohorts. DISCUSSION: The results from these three cross-sectional studies reveal that guanidino compound ratios (i.e. l-hArg/ADMA and l-hArg/SDMA) can discriminate stroke etiologies, predict ICA stenosis and estimate risk prediction in patients with cerebrovascular disease.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 µM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin's action on acetylcholine release at mammalian neuromuscular junctions.

Solid-state NMR spectroscopy applied to a chimeric potassium channel in lipid bilayers.

We show that solid-state NMR can be used to investigate the structure and dynamics of a chimeric potassium channel, KcsA-Kv1.3, in lipid bilayers. Sequential resonance assignments were obtained using a combination of (15)N- (13)C and (13)C- (13)C correlation experiments conducted on fully labeled and reverse-labeled as well as C-terminally truncated samples. Comparison of our results with those from X-ray crystallography and solution-state NMR in micelles on the closely related KcsA K (+) channel provides insight into the mechanism of ion channel selectivity and underlines the important role of the lipid environment for membrane protein structure and function.

A Mouse Model of Creatine Transporter Deficiency Reveals Impaired Motor Function and Muscle Energy Metabolism.

Creatine serves as fast energy buffer in organs of high-energy demand such as brain and skeletal muscle. L-Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase are responsible for endogenous creatine synthesis. Subsequent uptake into target organs like skeletal muscle, heart and brain is mediated by the creatine transporter (CT1, SLC6A8). Creatine deficiency syndromes are caused by defects of endogenous creatine synthesis or transport and are mainly characterized by intellectual disability, behavioral abnormalities, poorly developed muscle mass, and in some cases also muscle weakness. CT1-deficiency is estimated to be among the most common causes of X-linked intellectual disability and therefore the brain phenotype was the main focus of recent research. Unfortunately, very limited data concerning muscle creatine levels and functions are available from patients with CT1 deficiency. Furthermore, different CT1-deficient mouse models yielded conflicting results and detailed analyses of their muscular phenotype are lacking. Here, we report the generation of a novel CT1-deficient mouse model and characterized the effects of creatine depletion in skeletal muscle. HPLC-analysis showed strongly reduced total creatine levels in skeletal muscle and heart. MR-spectroscopy revealed an almost complete absence of phosphocreatine in skeletal muscle. Increased AGAT expression in skeletal muscle was not sufficient to compensate for insufficient creatine transport. CT1-deficient mice displayed profound impairment of skeletal muscle function and morphology (i.e., reduced strength, reduced endurance, and muscle atrophy). Furthermore, severely altered energy homeostasis was evident on magnetic resonance spectroscopy. Strongly reduced phosphocreatine resulted in decreased ATP/Pi levels despite an increased inorganic phosphate to ATP flux. Concerning glucose metabolism, we show increased glucose transporter type 4 expression in muscle and improved glucose clearance in CT1-deficient mice. These metabolic changes were associated with activation of AMP-activated protein kinase - a central regulator of energy homeostasis. In summary, creatine transporter deficiency resulted in a severe muscle weakness and atrophy despite different compensatory mechanisms.

Cognitive performance of 20 healthy humans supplemented with L-homoarginine for 4 weeks.

l-homoarginine (l-hArg) is an endogenous non-proteinogenic amino acid. Low l-hArg concentrations are associated with increased all-cause mortality, fatal strokes, and worse outcome after stroke. On the other hand, oral supplementation with l-hArg in mice improved neurological deficits and preserved cardiac function in experimental models of stroke and heart failure, respectively. Recently, oral supplementation with 125 mg daily l-hArg capsules in healthy volunteers demonstrated increased l-hArg plasma levels. Therefore, oral l-hArg supplementation could represent a potential treatment for patients with cerebrovascular disease. In addition to vascular physiology, animal studies have suggested that l-hArg might play a role in synapse function, neurotransmitter metabolism and cognitive training. However the direct influence of l-hArg on cognitive function has not been studied so far. In this study, cognitive performance in healthy humans was analyzed concerning memory, learning, and attention following supplementation with placebo or l-hArg for 4 weeks. Our results did not reveal any effects on cognition, neither impairment nor improvement, upon l-hArg supplementation. Therefore, potential l-hArg treatment is not expected to cause any acute neurocognitive or behavioral side effects.

Tetraphenylporphyrin derivative specifically blocks members of the voltage-gated potassium channel subfamily Kv1.

Tetraphenylporphyrin derivatives represent a promising class of high-affinity ligands for voltage-gated potassium (Kv) channels. Herein, we investigated the mode of Kv channel block of one tetraphenylporphyrin derivative, por3, using electrophysiological methods, structure-based mutagenesis, and solid-state NMR spectroscopy. The combined data showed that por3 specifically blocks Kv1.x channels. Unexpectedly, 2 different por3 binding modes lead to Kv1.x channel block exerted through multiple por3 binding sites: first, por3 interacts in a highly cooperative and specific manner with the voltage sensor domain stabilizing closed Kv1 channel state(s). Therefore, stronger depolarization is needed to activate Kv1.x channels in the presence of por3. Second, por3 bind to a single site at the external pore entrance to block the ion conduction pathway of activated Kv1.x channels. This block is voltage-independent. Por3 appears to have equal affinities for voltage-sensor and pore. However, at negative voltage and low por3 concentration, por3 gating modifier properties prevail due to the high cooperativity of binding. By contrast, at positive voltages, when Kv1.x channels are fully activated, por3 pore blocking properties predominate.

A structural link between inactivation and block of a K+ channel.

Gating the ion-permeation pathway in K(+) channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K(+) channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K(+) channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K(+) channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K(+) channel in a membrane setting.