Carboxy-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking [Molecular Bases of Disease]

October 9th, 2015 by Stolting, G., Bungert–Plumke, S., Franzen, A., Fahlke, C.

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia and alkalosis. In recent years, several Bartter syndromeassociated mutations have been described that result in truncations of the intracellular carboxy-terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at W610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClCKb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyterminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltagedependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyterminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxy-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyterminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.
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Cardiac myosin binding protein C and Troponin-I phosphorylation independently modulate myofilament length dependent activation [Signal Transduction]

October 9th, 2015 by

β-adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA, and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA or DDD permeabilized myocytes (n=12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca2+] relationships were determined at two sarcomere lengths (SL=1.9 um and SL=2.3 um). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca2+] required to achieve 50% force activation at the two SL. We found that PKA mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67% and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL, while accelerating cross-bridge detachment and relaxation at short SL.
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GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase KRAS4B, Exposing the Effector Binding Site [Molecular Biophysics]

October 9th, 2015 by

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, while the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate HVR's disassociation in a nucleotide-independent manner, thereby upregulate oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way - abolishing GTP hydrolysis and facilitating effector binding.
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Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic {beta}-cells [Signal Transduction]

October 9th, 2015 by Wu, Y., Shyng, S.-L., Chen, P.-C.

In pancreatic β-cells, Kv2.1 channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β -cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase AMPK. Activation of AMPK mimics, whereas inhibition of AMPK occludes the effect of leptin. Inhibition of CaMKKβ;, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase PKA is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, while stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization; and pharmacologically-induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (KATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface KATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. The study supports a model in which leptin exerts concerted trafficking regulation of KATP and Kv2.1 channels to coordinately inhibit insulin secretion.

Coordination of Protein Phosphorylation and Dephosphorylation in Synaptic Plasticity [Signal Transduction]

October 9th, 2015 by Woolfrey, K. M., Dell'Acqua, M. L.

A central theme in nervous system function is equilibrium: synaptic strengths wax and wane, neuronal firing rates adjust up and down, and neural circuits balance excitation with inhibition. This push/pull regulatory theme carries through to the molecular level at excitatory synapses, where protein function is controlled through phosphorylation and dephoshorylation by kinases and phosphatases. However, these opposing enzymatic activities are only part of the equation, as scaffolding interactions and assembly of multi-protein complexes are further required for efficient, localized synaptic signaling. This review will focus on coordination of postsynaptic serine/threonine kinase and phosphatase signaling by scaffold proteins during synaptic plasticity.

A Disintegrin and Metalloprotease 10 is Indispensable for Maintenance of the Muscle Satellite Cell Pool [Cell Biology]

October 9th, 2015 by

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10, a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared to wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.

Dynamic Regulation of NMDA and AMPA Receptors by Posttranslational Modifications [Neurobiology]

October 9th, 2015 by Lussier, M. P., Sanz-Clemente, A., Roche, K. W.

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination and palmitoylation control the stability, trafficking and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site [Protein Structure and Folding]

October 9th, 2015 by Bachmann, A.-L., Mootz, H. D.

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal up-hill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N-terminus. The IntN fragment could be shortened from 37 aa to 28 aa and exchanged with the 25 aa IntN fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the IntC fragment of the GOS-TerL intein.
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Thematic Mini-Review Series: Molecular Mechanisms of Synaptic Plasticity [Signal Transduction]

October 9th, 2015 by Colbran, R. J.

The human brain contains ~86 billion neurons, which are precisely organized in specific brain regions and nuclei. High fidelity synaptic communication between subsets of neurons in specific circuits is required for most human behaviors, and is often disrupted in neuropsychiatric disorders. The presynaptic axon terminals of one neuron release neurotransmitters that activate receptors on multiple postsynaptic neuron targets to induce electrical and chemical responses. Typically, postsynaptic neurons integrate signals from multiple presynaptic neurons at thousands of synaptic inputs in order to control downstream communication to the next neuron in the circuit. Importantly, the strength (or efficiency) of signal transmission at each synapse can be modulated on time scales ranging up to the lifetime of the organism. This "synaptic plasticity" leads to changes in overall neuronal circuit activity, resulting in behavioral modifications. This series of mini-reviews will focus on recent advances in our understanding of the molecular and cellular mechanisms that control synaptic plasticity.

Interaction of the Jhd2 H3K4 demethylase with chromatin is controlled by histone H2A surfaces and restricted by H2B ubiquitination. [Genomics and Proteomics]

October 8th, 2015 by

Histone H3 lysine 4 (K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex, and removed by Jhd2, a JMJC-domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di- and tri-) of H3K4 methylation. Jhd2 also contains a PHD finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (F26 and E57) serve as a binding site for Jhd2 in vitro, and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA-seq, we have identified the functional target genes for Jhd2 and the H2A F26 and E57 residues. We demonstrate that H2A F26 and E57 residues control Jhd2's chromatin association and H3K4 demethylase functions during positive or negative regulation of transcription at target genes. Importantly, we show that H2BK123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.
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