Kathrin Sauter

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.

L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

Contribution of N- and C-terminal channel domains to Kv channel interacting proteins in a mammalian cell line

Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].

Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

A homozygous SCN5A mutation in a severe, recessive type of cardiac conduction disease

Cardiac sodium channels are key players in the generation and propagation of action potentials in the human heart. Heterozygous mutations in the SCN5A gene have been found to be associated with long QT syndrome, Brugada syndrome, and sinus node dysfunction (SND). Recently, overlapping arrhythmia phenotypes have been reported as well. Here we describe a novel recessive SCN5A mutation in a family originating from the German minority in White Russia. Four affected children with a history of early cardiac arrhythmia encompassing SND, conduction disease, and severe ventricular arrhythmias, are homozygous carriers of a novel SCN5A missense mutation (p.I230T) in the channel protein. Interestingly, the heterozygous mutation carriers had neither significant ECG abnormalities nor a history of cardiac events. Heterologous expression of SCN5A(I230T) channels revealed normal protein transport but altered biophysical sodium channel properties. Voltage range of both activation and inactivation were shifted in a way that resulted in decreased sodium current and loss of channel function. In conclusion, we describe a rare clinical condition with a novel SCN5A mutation causing a new type of complex cardiac arrhythmia. Unlike most previously reported sodium channelopathies, this overlap syndrome displays recessive inheritance characteristics and does not seem to follow simple Mendelian rules.© 2010 Wiley‐Liss, Inc.

Differential regulation of AMPK activation in leptin- and creatine-deficient mice.

AMP‐activated protein kinase (AMPK) is a key sensor and regulator of energy homeostasis. Previously, we demonstrated that intracellular energy depletion by L‐arginine:glycine amidinotransferase (AGAT) deficiency resulted in AMPK activation and protected from metabolic syndrome. In the present study, we show tissue‐specific leptin dependence of AMPK activation by energy depletion. We investigated leptin‐dependent AMPK regulation in AGAT‐ and leptin‐deficient (d/d ob/ob) mice. Like ob/ob mice, but unlike d/d mice, d/d ob/ob mice were obese and glucose intolerant. Therefore, leptin is a prerequisite for resistance to metabolic syndrome in AGAT‐deficient mice. Quantitative Western blots revealed a 4‐fold increase in AMPK activation in skeletal muscle of d/d ob/ob mice (P<0.001). However, AMPK activation was absent in white adipose tissue (WAT) and liver. Compared with blood glucose levels in ob/ob mice, fasting levels were still reduced and therefore did not show leptin dependence (wild‐type, 79.4±3.9 mg/dl; d/d, 68.4±3.2 mg/dl; P<0.05). In ob/ob mice and wild‐type mice, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR), in combination with leptin, augmented glucose tolerance compared with AICAR alone, whereas no improvement was found under conditions of high‐fatdiet feeding. These findings reveal a previously unknown synergistic AMPK activation by leptin and intracellular energy depletion, suggesting that AMPK activation can be therapeutically effective in metabolic syndrome only if leptin sensitivity is preserved.—Stockebrand, M., Sauter, K., Neu, A., Isbrandt, D., Choe, C., Differential regulation of AMPK activation in leptin‐ and creatine‐deficient mice. FASEBJ. 27, 4147‐4156 (2013). www.fasebj.org

Biophysical Properties of Kv3.1 Channels in SH-SY5Y Human Neuroblastoma Cells

Biophysical properties of delayed rectifier K channels in the human neuroblastoma SH-SY5Y were established using patch clamp recordings. The whole cell K+ conductance activated at membrane potentials positive to -20 mV. The midpoint of current activation was 9.6 +/- 5.1 mV, the equivalent charge was 3.7 +/-.6. Whole-cell currents inactivated slightly with time constants of 700 ms and 5 s. The K+ currents were sensitive to micromolar concentrations of TEA and 4-aminopyridine. RT-PCR experiments amplified a cDNA fragment specific for human Kv3.1 channels. Activation gating parameters in outside-out patches were shifted by approximately 14 mV in the hyperpolarizing direction.

Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT−/− mice

Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking l-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT−/− (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

Ret and Substrate-Derived TGF-β Maverick Regulate Space-Filling Dendrite Growth in Drosophila Sensory Neurons

Dendrite morphogenesis is a highly regulated process that gives rise to stereotyped receptive fields, which are required for proper neuronal connectivity and function. Specific classes of neurons, including Drosophila class IV dendritic arborization (C4da) neurons, also feature complete space-filling growth of dendrites. In this system, we have identified the substrate-derived TGF-β ligand maverick (mav) as a developmental signal promoting space-filling growth through the neuronal Ret receptor. Both are necessary for radial spreading of C4da neuron dendrites, and Ret is required for neuronal uptake of Mav. Moreover, local changes in Mav levels result in directed dendritic growth toward regions with higher ligand availability. Our results suggest that Mav acts as a substrate-derived secreted signal promoting dendrite growth within not-yet-covered areas of the receptive field to ensure space-filling dendritic growth.

Contribution of N- and C-terminal channel domains to Kv channel interacting proteins in a mammalian cell line: Kv4/KChIP interaction

Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.