Ubiquitin-proteasome system

The proteolytic active sites of the 26S proteasome are sequestered within the central chamber of its 20S catalytic core particle. Access to this chamber is through a narrow channel defined by the outer alpha subunits. Free proteasome 20S core particles are found in an autoinhibited state in which the N-termini of neighboring alpha subunits are anchored by an intricate lattice of interactions blocking access to the channel. Entry of substrates into proteasomes can be enhanced by attachment of activators or regulatory particles. An important part of this activation is channel gating; regulatory particles rearrange the blocking residues to form an open pore and promote substrate entry into the proteolytic chamber. Interestingly, some substrates can open the entrance themselves and thus facilitate their own destruction. In this review, we will discuss the mechanisms proposed for channel gating and the interactions required to maintain stable closed and open conformations.     

Dependence of 6β-acetoxy-7α-hydroxyroyleanone block of Kv1.2 channels on C-type inactivation

Voltage-gated K⁺ (Kv) channels exhibit slow or C-type inactivation during continuous depolarization. A selective pharmacological agent targeting C-type inactivation is hitherto lacking. Here, we report that 6β-acetoxy-7α-hydroxyroyleanone (AHR), a diterpenoid compound isolated from Taiwania cryptomerioides, can selectively modify C-type inactivation of Kv1.2 channels. Extracellular, but not intracellular, AHR (50 μM) dramatically accelerated the slow decay of Kv currents and left-shifted the steady-state inactivation curve. AHR blocked Kv currents with an IC₅₀ of 17.7 μM. AHR did not affect the kinetics and voltage-dependence of Kv1.2 channel activation. Channel block by AHR was independent of intracellular K⁺ concentration. In addition, effect of AHR was much attenuated in a Kv1.2 V370G mutant defective in C-type inactivation. Therefore, block of Kv1.2 channels by AHR did not appear to involve direct occlusion of the outer pore but depended on C-type inactivation. AHR could thus be a probe targeting Kv channel C-type inactivation gate.     

Interaction between bradykinin and voltage-sensitive sodium channels in myelinated nerve fibers

The effects of externally and internally applied bradykinin on the excitability of single myelinated nerve fibers were studied. External bradykinin (10 microM) slightly prolongs the action potential of a single myelinated nerve fiber; hence, when the fibers are stimulated by long-lasting pulses, this raises the frequency of repetitive firing in sensory fibers and evokes repetitive activity in motor fibers. Under voltage-clamp conditions, sodium channel inactivation is slowed, while sodium channel activation remains unaffected. Prolonged depolarization of the membrane leads to a maintained sodium current. The voltage dependence of the steady-state sodium current inactivation (h infinity) is shifted in the depolarized direction by approximately 10 mV. Internally applied bradykinin produces a frequency-dependent block of the sodium current. The phenomena described here imply that more than one site on the sodium channel is modified by bradykinin.     

Structure and assembly of the 20S proteasome

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain α-type subunits, the inner ones comprise β-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the α-type and β-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the β-type subunits are processed. The prosequence of the β-type subunits is essential for the assembly process and prevents protease activity of immature proteasomes.     

Trafficking and stability of voltage-gated calcium channels

Voltage-gated calcium channels are important mediators of calcium influx into electrically excitable cells. The amount of calcium entering through this family of channel proteins is not only determined by the functional properties of channels embedded in the plasma membrane but also by the numbers of channels that are expressed at the cell surface. The trafficking of channels is controlled by numerous processes, including co-assembly with ancillary calcium channel subunits, ubiquitin ligases, and interactions with other membrane proteins such as G protein coupled receptors. Here we provide an overview about the current state of knowledge of calcium channel trafficking to the cell membrane, and of the mechanisms regulating the stability and internalization of this important ion channel family.     

Interaction between bradykinin and voltage-sensitive sodium channels in myelinated nerve fibers

Summary The effects of externally and internally applied bradykinin on the excitability of single myelinated nerve fibers were studied. External bradykinin (10 M) slightly prolongs the action potential of a single myelinated nerve fiber; hence, when the fibers are stimulated by long-lasting pulses, this raises the frequency of repetitive firing in sensory fibers and evokes repetitive activity in motor fibers. Under voltage-clamp conditions, sodium channel inactivation is slowed, while sodium channel activation remains unaffected. Prolonged depolarization of th membrane leads to a maintained sodium current. The voltage dependence of the steady-state sodium current inactivation (h ) is shifted in the depolarized direction by 10 mV. Internally applied bradykinin produces a frequency-dependent block of the sodium current. The phenomena described here imply that more than one site on the sodium channel is modified by bradykinin.     

Potassium currents in cardiac cells

The kinetic properties of the inwardly rectifying K current and the transient outward current in cardiac cells were investigated. In sheep Purkinje fibers superfused with Na-free K-free solution, time-dependent changes in the conductance of the inward rectifier are described. In patch clamp experiments the inward rectifier inactivates during hyperpolarization, as can be seen by a decrease in the open state probability. Using whole cell clamp on ventricular myocytes it is demonstrated that the inactivation during hyperpolarization is due to blocking of the channel by external Na, Mg and Ca. The channels responsible for the transient outward current in cow, sheep and rabbit Purkinje fibers are identified using single channel recording. It is demonstrated that in all three preparations the channels are K-selective. The channel in cow Purkinje cells has a large conductance and is regulated by voltage and internal Ca concentration. The channels identified in the sheep and rabbit cells have a much smaller conductance.     

GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/β and β-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or β-catenin knockdown with shRNAs decreased the levels of phosphorylated-β-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both “in vitro” and “in vivo”, therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and β-catenin at the AIS.     

Tauroursodeoxycholic acid prevents E22Q Alzheimer’s Aβ toxicity in human cerebral endothelial cells

The vasculotropic E22Q mutant of the amyloid-β (Aβ) peptide is associated with hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s) of toxicity and nature of the AβE22Q toxic assemblies are not completely understood. Comparative assessment of structural parameters and cell death mechanisms elicited in primary human cerebral endothelial cells by AβE22Q and wild-type Aβ revealed that only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet structure, achieving a modest degree of oligomerization with a secondary structure that remained a mixture of β and random conformations. The endogenous molecule tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered apoptosis but did not significantly change the secondary structures and fibrillogenic propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing new perspectives for modulation of amyloid toxicity. Received 20 November 2008; received after revision 12 December 2008; accepted 12 January 2009     

Molecular and cellular basis of small- and intermediate-conductance, calcium-activated potassium channel function in the brain

Small conductance calcium-activated potassium (SK or K_Ca2) channels link intracellular calcium transients to membrane potential changes. SK channel subtypes present different pharmacology and distribution in the nervous system. The selective blocker apamin, SK enhancers and mice lacking specific SK channel subunits have revealed multifaceted functions of these channels in neurons, glia and cerebral blood vessels. SK channels regulate neuronal firing by contributing to the afterhyperpolarization following action potentials and mediating I_AHP, and partake in a calcium-mediated feedback loop with NMDA receptors, controlling the threshold for induction of hippocampal long-term potentiation. The function of distinct SK channel subtypes in different neurons often results from their specific coupling to different calcium sources. The prominent role of SK channels in the modulation of excitability and synaptic function of limbic, dopaminergic and cerebellar neurons hints at their possible involvement in neuronal dysfunction, either as part of the causal mechanism or as potential therapeutic targets. Received 23 April 2008; received after revision 29 May 2008; accepted 4 June 2008     

Correlation between inhibition of stimulatory membrane repolarization and positive inotropic response caused by ouabain in rat heart]

During a stimulus train, the diastolic membrane potential of rat atria exhibits a depolarization phase followed by a slower repolarization phase which has been attributed to the activation of an electrogenic sodium pump (ATPase Na+, K+). This pump seems to be all the more active as stimulation frequency is higher. The parallel evolution of the sodium pump inhibition and a positive inotropic effect in response to ouabain perfusion, suggests that the enzymatic inhibition is directly involved in the development of the cardiotonic effect of digitalis.     

Contributions of cellular electrophysiology to the understanding of the electrocardiogram

Summary The understanding of cardiac action potential and membrane currents has broadened the theoretical foundation and enhanced the clinical usefulness of the electrocardiogram. An improved understanding of the morphology of the electrocardiographic waveform has resulted from: correlations between V_max of depolarization and QRS complex, plateau of the ventricular action potential and S-T segment, terminal repolarization and T-wave, from definitions of action potential differences responsible for the T-wave, and recordings of action potential alternans. Cellular electrophysiology has contributed to the understanding of certain mechanisms of cardiac standstill. Many disturbances of conduction and refractoriness associated with ventricular arrhythmias can be attributed to the following derangements at the cellular level: slowing of terminal repolarization, development of diastolic depolarization in fibers with stable resting membrane potential, afterdepolarizations, currents of injury resulting from non-uniform polarization, increased dispersion of action potential durations, and co-existence of slow conduction and short premature action potentials.     

An abundant, truncated human sulfonylurea receptor 1 splice variant has prodiabetic properties and impairs sulfonylurea action

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K_ATP) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K_ATP channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K_ATP-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.     

Understanding BACE1: essential protease for amyloid-β production in Alzheimer’s disease

The identification of the aspartic protease BACE1 (β-secretase) was a defining event in research aimed at understanding the molecular mechanisms that underlie Alzheimer’s disease (AD) pathogenesis. This is because BACE1 catalyses the rate limiting step in the production of amyloid-β (Aβ) the principal component of plaque pathology in AD, the excessive production of which is believed to be a primary cause of neurodegeneration, and cognitive dysfunction in AD. Subsequent discoveries showed that genetic deletion of BACE1 completely abolishes Aβ production and deposition in vivo, and that BACE1 activity is significantly increased in AD brain. In this review we present current knowledge on BACE1, discussing its structure, function and complex regulation with a view to understanding BACE1 function in the brain, and BACE1 as a target in blocking aberrant Aβ production in AD. Received 15 May 2008; received after revision 13 June 2008; accepted 18 June 2008     

Conotoxins of the O-superfamily affecting voltage-gated sodium channels

The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While ω-conotoxins and κ-conotoxins block Ca²⁺ and K⁺ channels, respectively, the closely related δ- and μO-conotoxins affect voltage-gated Na⁺ channels (Na_v channels). δ-conotoxins mainly remove the fast inactivation of Na_v channels and, thus, functionally resemble long-chain scorpion α-toxins. μO-conotoxins are functionally similar to μ-conotoxins, since they inhibit the ion flow through Na_v channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na_v channels. Received 27 December 2006; received after revision 30 January 2007; accepted 19 February 2007     

Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278)

To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y₁₉₁MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85β regulatory subunits and p110α, p110δ and p110β catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.     

Experimental and theoretical work on excitation and excitation-contraction coupling in the heart

A combination of experimental and theoretical work has been used to investigate the movements of calcium during cardiac excitation. In addition to calcium entry through several types of calcium channel, calcium efflux occurs to balance the entry during each cycle of activity. Measurements of net membrane calcium movements have been made with the right time resolution by Don Hilgemann in Los Angeles by investigating fast extracellular calcium transients. This work shows that, in mammalian cardiac cells, net calcium exit occurs quite early during repolarization and is nearly complete by the time the resting potential is re-established. These results correlate very well indeed with measurements made in the Oxford laboratory of calcium-activated inward current in single cardiac myocytes. Both approaches are consistent with the view that calcium efflux occurs largely through the sodium-calcium exchange process. Modelling of this process in equations developed recently with Dario DiFrancesco, Susan Noble and Don Hilgemann succeeds in reproducing both the ionic current changes and the fast extracellular calcium transients.     

Das Na-Transportsystem während der Erregungsprozesse am Ranvier-Knoten isolierter markhaltiger Nervenfasern

Summary (1) An attempt is made to interpret the action potential of medullated nerve fibres of the frog in terms of permeability changes to Na ions.(2) In keeping with a suggestion byHodgkin andHuxley, the following assumptions are made: (a) a carrier system is responsible for transporting Na ions across the nodal membrane; (b) in a resting fibre (high membrane potential) its transport capacity is small; (c) depolarization has a dual effect on the transport system, namely activation of carrier groups as a quick reaction and inactivation of the whole system as a slower reaction.(3) The following experimental observations are interpreted on the ground of these assumptions: threshold for depolarization, threshold for repolarization, absolute and relative refractory period, alteration of the action potential on application of a hypertonic (5%) NaCl solution.

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Eine vorläufige Mitteilung erfolgte auf der Tagung des Schweiz. Vereins für Physiologie usw. in Fribourg 1956. Helv. physiol. Acta14, C35–36 (1956).