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.

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.

Potassium (K þ )-channel gating is choreographed by a complex interplay between external stimuli, K þ concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K þ , 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 þ 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 þ 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 þ -channel pore.

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.

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.

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 wellcharacterized 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. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

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.

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