Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

MICPA: a client-assisted channel assignment scheme for throughput enhancement in WLANs

The emergence of high-density wireless local area network (WLAN) deployments in recent years is a testament to the insatiable demands for wireless broadband services. The increased density of WLAN deployments brings with it the potential of increased capacity, extended coverage, and exciting new applications. However, the corresponding increase in contention and interference can significantly degrade throughputs, unless new challenges in channel assignment are effectively addressed. In this paper, a client-assisted channel assignment scheme that can provide enhanced throughput is proposed. A study on the impact of interference on throughput with multiple access points (APs)is first undertaken using a novel approach that determines the possibility of parallel transmissions. A metric with a good correlation to the throughput, i.e., the number of conflict pairs, is used in the client-assisted minimum conflict pairs (MICPA) scheme. In this scheme, measurements from clients are used to assist the AP in determining the channel with the minimum number of conflict pairs to maximize its expected throughput. Simulation results show that the client-assisted MICPA scheme can provide meaningful throughput improvements over other schemes that only utilize the AP’s measurements.

Use of synthetic CaV2.2 Ca2+ channel peptides to study presynaptic function

Small, synthetic peptides based on specific regions of voltage-gated Ca2+ channels (VGCCs) have been widely used to study Ca2+ channel function and have been instrumental in confirming the contribution of specific amino acid sequences to interactions with putative binding partners. In particular, peptides based on the Ca2+ channel Alpha Interaction Domain (AID) on the intracellular region connecting domains I and II (the I-II loop) and the SYNaptic PRotein INTerction (synprint) site on the II-III loop have been widely used. Emerging evidence suggests that such peptides may themselves possess inherent functionality, a property that may be exploitable for future drug design. Here, we review our recent work using synthetic Ca2+ channel peptides based on sequences within the CaV2.2 amino terminal and I-II loop, originally identified as molecular determinates for G protein modulation, and their effects on VGCC function. These CaV2.2 peptides act as inhibitory modules to decrease Ca2+ influx with direct effects on VGCC gating, ultimately leading to a reduction of synaptic transmission. CaV2.2 peptides also attenuate G protein modulation of VGCCs. Amino acid substitutions generate CaV2.2 peptides with increased or decreased inhibitory effects suggesting that synthetic peptides can be used to further probe VGCC function and, potentially, form the basis for novel therapeutic development.

A bank lending channel or a credit supply shock?

Shifts in credit supply could have a bearing on house prices e.g. through financial innovations and changes in regulation independently of the existence of a bank lending channel of monetary policy. This paper assesses the responses of US house prices to an exogenous credit supply shock and compares them with the effects from variations in credit supply associated with a bank lending channel. The contribution of the study is twofold. First, innovations in credit supply are identified using a mortgage mix variable, thereby accounting for the market-based financial intermediaries. As a robustness check a survey variable of bank lending standards for mortgage loans is also used. Second, the policy-induced credit supply effect on house prices is disentangled and compared with the effect from an exogenous credit supply shock. It is shown that in the first 3 years credit supply shocks affect house prices exogenously rather than through the bank lending channel. Monetary policy has still a large impact on house prices, even when the bank lending channel is ‘turned off’.

Compliant Multi-electrode Micro-channel Array for Recording Afferent Nerve Activity

We have fabricated a compliant neural interface to record afferent nerve activity. Stretchable gold electrodes were evaporated on a polydimethylsiloxane (PDMS) substrate and were encapsulated using photo-patternable PDMS. The built-in microstructure of the gold film on PDMS allows the electrodes to twist and flex repeatedly, without loss of electrical conductivity. PDMS microchannels (5mm long, 100μm wide, 100μm deep) were then plasma bonded irreversibly on top of the electrode array to define five parallel-conduit implants. The soft gold microelectrodes have a low impedance of ~200kΩ at the 1kHz frequency range. Teased nerves from the L6 dorsal root of an anaesthetized Sprague Dawley rat were threaded through the microchannels. Acute tripolar recordings of cutaneous activity are demonstrated, from multiple nerve rootlets simultaneously. Confinement of the axons within narrow microchannels allows for reliable recordings of low amplitude afferents. This electrode technology promises exciting applications in neuroprosthetic devices including bladder fullness monitors and peripheral nervous system implants.

Voltage-gated sodium (NaV) channel blockade by plant cannabinoids does not confer anticonvulsant effects per se

Cannabidiol (CBD) is a non-psychoactive, well-tolerated, anticonvulsant plant cannabinoid, although its mechanism(s) of seizure suppression remains unknown. Here, we investigate the effect of CBD and the structurally similar cannabinoid, cannabigerol (CBG), on voltage-gated Na+ (NaV) channels, a common anti-epileptic drug target. CBG’s anticonvulsant potential was also assessed in vivo. CBD effects on NaV channels were investigated using patch-clamp recordings from rat CA1 hippocampal neurons in brain slices, human SH-SY5Y (neuroblastoma) cells and mouse cortical neurons in culture. CBG effects were also assessed in SH-SY5Y cells and mouse cortical neurons. CBD and CBG effects on veratridine-stimulated human recombinant NaV1.1, 1.2 or 1.5 channels were assessed using a membrane potential-sensitive fluorescent dye high-throughput assay. The effect of CBG on pentyleneterazole-induced (PTZ) seizures was assessed in rat. CBD (10M) blocked NaV currents in SH-SY5Y cells, mouse cortical neurons and recombinant cell lines, and affected spike parameters in rat CA1 neurons; CBD also significantly decreased membrane resistance. CBG blocked NaV to a similar degree to CBD in both SH-SY5Y and mouse recordings, but had no effect (50-200mg/kg) on PTZ-induced seizures in rat. CBD and CBG are NaV channel blockers at micromolar concentrations in human and murine neurons and recombinant cells. In contrast to previous reports investigating CBD, CBG had no effect upon PTZ-induced seizures in rat, indicating that NaV blockade per se does not correlate with anticonvulsant effects.

Inhibition of the cardiac Na+ channel Nav1.5 by carbon monoxide

Sub-lethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognised CO-sensitive intracellular signalling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of nitric oxide (NO) formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to dithiothreitol immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, L-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor L-NAME, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada-syndrome like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation and is dependent on channel redox state.

Calibration of structure in a distributed forecasting model for a semiarid flash flood: dynamic surface storage and channel roughness

Flash floods pose a significant danger for life and property. Unfortunately, in arid and semiarid environment the runoff generation shows a complex non-linear behavior with a strong spatial and temporal non-uniformity. As a result, the predictions made by physically-based simulations in semiarid areas are subject to great uncertainty, and a failure in the predictive behavior of existing models is common. Thus better descriptions of physical processes at the watershed scale need to be incorporated into the hydrological model structures. For example, terrain relief has been systematically considered static in flood modelling at the watershed scale. Here, we show that the integrated effect of small distributed relief variations originated through concurrent hydrological processes within a storm event was significant on the watershed scale hydrograph. We model these observations by introducing dynamic formulations of two relief-related parameters at diverse scales: maximum depression storage, and roughness coefficient in channels. In the final (a posteriori) model structure these parameters are allowed to be both time-constant or time-varying. The case under study is a convective storm in a semiarid Mediterranean watershed with ephemeral channels and high agricultural pressures (the Rambla del Albujón watershed; 556 km 2 ), which showed a complex multi-peak response. First, to obtain quasi-sensible simulations in the (a priori) model with time-constant relief-related parameters, a spatially distributed parameterization was strictly required. Second, a generalized likelihood uncertainty estimation (GLUE) inference applied to the improved model structure, and conditioned to observed nested hydrographs, showed that accounting for dynamic relief-related parameters led to improved simulations. The discussion is finally broadened by considering the use of the calibrated model both to analyze the sensitivity of the watershed to storm motion and to attempt the flood forecasting of a stratiform event with highly different behavior.

Sensitivity of a hydraulic model to changes in channel erosion during extreme flooding

Recent research into flood modelling has primarily concentrated on the simulation of inundation flow without considering the influences of channel morphology. River channels are often represented by a simplified geometry that is implicitly assumed to remain unchanged during flood simulations. However, field evidence demonstrates that significant morphological changes can occur during floods to mobilise the boundary sediments. Despite this, the effect of channel morphology on model results has been largely unexplored. To address this issue, the impact of channel cross-section geometry and channel long-profile variability on flood dynamics is examined using an ensemble of a 1D-2D hydraulic model (LISFLOOD-FP) of the 1:2102 year recurrence interval floods in Cockermouth, UK, within an uncertainty framework. A series of hypothetical scenarios of channel morphology were constructed based on a simple velocity based model of critical entrainment. A Monte-Carlo simulation framework was used to quantify the effects of channel morphology together with variations in the channel and floodplain roughness coefficients, grain size characteristics, and critical shear stress on measures of flood inundation. The results showed that the bed elevation modifications generated by the simplistic equations reflected a good approximation of the observed patterns of spatial erosion despite its overestimation of erosion depths. The effect of uncertainty on channel long-profile variability only affected the local flood dynamics and did not significantly affect the friction sensitivity and flood inundation mapping. The results imply that hydraulic models generally do not need to account for within event morphodynamic changes of the type and magnitude modelled, as these have a negligible impact that is smaller than other uncertainties, e.g. boundary conditions. Instead morphodynamic change needs to happen over a series of events to become large enough to change the hydrodynamics of floods in supply limited gravel-bed rivers like the one used in this research.

Channel equalization for indoor lighting communications networks

We consider indoors communications networks using modulated LEDs to transmit the information packets. A generic indoor channel equalization formulation is proposed assuming the existence of both line of sight and diffuse emitters. The proposed approach is of relevance to emergent indoors distributed sensing modalities for which various lighting based network communications protocols are considered.

Oxygen-dependent hydroxylation by Factor Inhibiting HIF (FIH) regulates the TRPV3 ion channel

Factor Inhibiting HIF (FIH) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ankyrin repeat domain. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygendependent asparaginyl hydroxylation is likely to extend to other ion channels.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Efficient incorporation of channel cross-section geometry uncertainty into regional and global scale flood inundation models

This paper investigates the challenge of representing structural differences in river channel cross-section geometry for regional to global scale river hydraulic models and the effect this can have on simulations of wave dynamics. Classically, channel geometry is defined using data, yet at larger scales the necessary information and model structures do not exist to take this approach. We therefore propose a fundamentally different approach where the structural uncertainty in channel geometry is represented using a simple parameterization, which could then be estimated through calibration or data assimilation. This paper first outlines the development of a computationally efficient numerical scheme to represent generalised channel shapes using a single parameter, which is then validated using a simple straight channel test case and shown to predict wetted perimeter to within 2% for the channels tested. An application to the River Severn, UK is also presented, along with an analysis of model sensitivity to channel shape, depth and friction. The channel shape parameter was shown to improve model simulations of river level, particularly for more physically plausible channel roughness and depth parameter ranges. Calibrating channel Manning’s coefficient in a rectangular channel provided similar water level simulation accuracy in terms of Nash-Sutcliffe efficiency to a model where friction and shape or depth were calibrated. However, the calibrated Manning coefficient in the rectangular channel model was ~2/3 greater than the likely physically realistic value for this reach and this erroneously slowed wave propagation times through the reach by several hours. Therefore, for large scale models applied in data sparse areas, calibrating channel depth and/or shape may be preferable to assuming a rectangular geometry and calibrating friction alone.