Margaret Allen

Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7–12 but not 4‐week‐old mice. Inhibitors of PKA and of GluR2‐lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4‐week‐old WT mice. Inhibitors of GluR2‐lacking AMPA receptors also prevented LTP in 2 but not 3‐week‐old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2‐lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150‐anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8‐week‐old mice.

Properties of voltage-gated K+ currents expressed in Xenopus oocytes by mKv1.1, mKv1.2 and their heteromultimers as revealed by mutagenesis of the dendrotoxin-binding site in mKv1.1.

Two similar mouseShaker-like K+ channel genes, mKv1.1 and mKv1.2, have been shown to form heteromultimers in vivo. The predicted amino acid sequence of each channel is nearly identical in mice, rats and humans, suggesting that each has been highly conserved evolutionarily. Here we report the biophysical and pharmacological properties of each channel when expressed alone or when coexpressed inXenopus oocytes. The voltage sensitivities of activation were similar for both, but the voltages at which the K+ conductances were half-maximal (V1/2) were −37 mV and −27 mV for mKv1.1 and mKv1.2 respectively. Both displayed voltage-dependent, but incomplete, inactivation following a prepulse with mKv1.2 showing the greater degree of inactivation. For mKv1.1, the onset and recovery from inactivation could be described by single, slow time constants (2–4 s), whereas for mKv1.2 the onset and recovery from inactivation displayed a second, faster time constant (< 400 ms). Using a mutant mKv1.1 that is 100-fold less sensitive to dendrotoxin-I than mKv1.1, we demonstrate that this mutant mKv1.1 and wild-type mKv1.2 subunits can form heteromultimeric channels. With some exceptions, of unknown significance, the biophysical properties of the heteromultimeric channels formed by wild-type mKv1.1 and mKv1.2 subunits were intermediate between those of mKv1.1 and mKv1.2 homomultimers, but quantitatively more similar to the more abundant subunit.

Hypomyelination alters K+ channel expression in mouse mutants shiverer and Trembler

Voltage-gated K+ channels are localized to juxtaparanodal regions of myelinated axons. To begin to understand the role of normal compact myelin in this localization, we examined mKv1.1 and mKv1.2 expression in the dysmyelinating mouse mutants shiverer and Trembler. In neonatal wild-type and shiverer mice, the focal localization of both proteins in axon fiber tracts is similar, suggesting that cues other than mature myelin can direct initial K+ channel localization in shiverer mutants. In contrast, K+ channel localization is altered in hypomyelinated axonal fiber tracts of adult mutants, suggesting that abnormal myelination leads to channel redistribution. In shiverer adult, K+ channel expression is up-regulated in both axons and glia, as revealed by immunocytochemistry, RNase protection, and in situ hybridization studies. This up-regulation of K+ channels in hypomyelinated axon tracts may reflect a compensatory reorganization of ionic currents, allowing impulse conduction to occur in these dysmyelinating mouse mutants.

Mutations in AKAP5 Disrupt Dendritic Signaling Complexes and Lead to Electrophysiological and Behavioral Phenotypes in Mice

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIα or RIIβ as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and β-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.

AKAP150-anchored PKA activity is important for LTD during its induction phase

Protein kinase A (PKA) is thought to tonically maintain an enhanced level of postsynaptic AMPA receptor responses. Injection of PKA inhibitory peptides leads to a run‐down of AMPA receptor responses and prevents long‐term depression (LTD). This run‐down of AMPA receptor activity was proposed to occlude a further reduction that would otherwise constitute LTD. PKA is recruited to postsynaptic sites by the A kinase anchor protein AKAP150. We found that LTD was strongly impaired in acute hippocampal slices from 2‐week‐old mice in which the PKA binding site on AKAP150 had been genetically deleted (D36 mice). However, basal postsynaptic AMPA and NMDA receptor activity was indistinguishable between D36 and WT mice. During extracellular recordings of field EPSPs and during intracellular recording of EPSCs from hippocampal slices from WT mice, H‐89 and KT5720, two structurally different PKA inhibitors, inhibited LTD by more than 70% without affecting basal synaptic transmission or basal phosphorylation of serine 845 on GluR1. Collectively our data indicate that AKAP150‐anchored PKA activity is required to induce LTD and not merely to maintain a tonically heightened activity level of AMPA receptors as proposed earlier.

A prospective, randomized evaluation of effect of ventricular fibrillation duration on defibrillation thresholds in humans.

The effect of ventricular fibrillation duration in humans on defibrillation efficacy as it pertains to the time of intervention of an automatic implantable defibrillator is unknown. If a difference in defibrillation efficacy exists in the early period after ventricular fibrillation onset, it may affect algorithms used by antiarrhythmic devices for arrhythmia detection and therapy. Therefore, a prospective, randomized evaluation was performed of the effect of ventricular fibrillation durations of 10 s and 20 s on defibrillation thresholds in 10 survivors of sudden cardiac arrest undergoing implantation of an automatic cardioverter defibrillator. The initial duration of ventricular fibrillation was chosen randomly. Subsequently, each patient served as his or her own control for the alternate duration of ventricular fibrillation to that chosen initially. The mean leading edge defibrillation threshold voltage was 411 +/- 114 V when ventricular fibrillation persisted for 10 s and 419 +/- 125 V when it persisted for 20 s (p = 0.73). The mean defibrillation threshold current was 11.4 +/- 2.8 A when ventricular fibrillation persisted for 10 s and 11.4 +/- 3.2 A when it persisted for 20 s (p = 0.97). The delivered energy defibrillation threshold was 11.5 +/- 5.9 J when ventricular fibrillation persisted for 10 s and 12.0 +/- 6.9 J when it persisted for 20 s (p = 0.67). These findings show that the defibrillation threshold does not change between 10 and 20 s of ventricular fibrillation in out-of-hospital survivors of cardiac arrest at the time of surgical implantation of an automatic defibrillator. The data may have influence on the programming of defibrillator detection algorithms.

Anterior sternal retraction for reoperative median sternotomy

The incidence of reoperative median sternotomy for repeat cardiac surgery is increasing. Reoperative median sternotomy is associated with a higher morbidity and mortality than first-time cardiac surgery. A portion of this morbidity and mortality may be due to direct injury to the heart and great vessels in the process of reopening the sternum. We report a new technique utilizing anterior sternal retraction that allows division of adhesions between the undersurface of the sternum and the heart and great vessels under direct vision. This technique enables the surgeon to minimize the risk of serious injury to these underlying structures during reoperative cardiac surgery.

Interactions of snake dendrotoxins with potassium channels

Publisher Summary Peptide toxins isolated from the venom of a number of Elapidae snake species have proven useful as high-affinity probes for the characterization of ligand- and voltage-gated ion channels. A good example of the utility of snake toxins has been the use of the dendrotoxins in the isolation and characterization of voltage-gated potassium channels. The dendrotoxins are seven peptide toxins isolated from the venom of three species of African mamba snakes. The published amino acid sequences of five dendrotoxins are discussed in this chapter. These dendrotoxins can be grouped into two subfamilies based on amino acid sequence homology. This chapter describes methods used to investigate the interaction of four of these dendrotoxins, α -DTX, γ -DTX, and δ -DTX from Dendroaspis angusticeps, and DTX-I from Dendroaspis polylepis, with mKv1.1, a mouse member of the Shaker subfamily of voltage-gated potassium channel α subunits. As will become apparent, each of the dendrotoxins can display unique properties as high-affinity ligands for this potassium channel subunit, even though two of them have a high degree of amino acid sequence homology (α-DTX and DTX-I).

Hemodynamic effects of oral amiodarone on left ventricular function before and after global ischemia.

We evaluated the hemodynamic effects of amiodarone on left ventricular (LV) function after global ischemia. Amiodarone was administered orally at 15 mg/kg/day for an average of 28 days to a group of 10 dogs. The concentration of amiodarone in serum and LV myocardium was 0.40 +/- 0.24 micrograms/ml and 44 +/- 27.0 micrograms/g, respectively. This experimental group and a control group of 10 dogs each underwent 120 min of aortic cross-clamping with cold crystalloid cardioplegia resulting in an average myocardial temperature of 15 degrees C. LV function measurements by pulse-transit sonomicrometry and Millar solid-state micromanometers were processed by a minicomputer. Compared to the nontreated control group, oral amiodarone for 28 days produced depression of LV contractility which was reflected by lower slope of the end-systolic pressure-volume relation (Evmax), percentage shortening of segment length, left ventricular pressure-segment length loop area, and slope of the end-systolic pressure-segment length relation (Esmax). After ischemia, percentage recovery of LV global function (Evmax and mean velocity of circumferential fiber shortening) and regional function (Esmax) was significantly better in the amiodarone group than in the control group. We conclude that oral amiodarone for 28 days results in a depression of LV contractility but the combination of amiodarone and ischemia does not act synergistically to further depress postischemic LV function.

Evaluating indicators of human well-being for ecosystem-based management

ABSTRACT Introduction: Interrelated social and ecological challenges demand an understanding of how environmental change and management decisions affect human well-being. This paper outlines a framework for measuring human well-being for ecosystem-based management (EBM). We present a prototype that can be adapted and developed for various scales and contexts. Scientists and managers use indicators to assess status and trends in integrated ecosystem assessments (IEAs). To improve the social science rigor and success of EBM, we developed a systematic and transparent approach for evaluating indicators of human well-being for an IEA. Methods: Our process is based on a comprehensive conceptualization of human well-being, a scalable analysis of management priorities, and a set of indicator screening criteria tailored to the needs of EBM. We tested our approach by evaluating more than 2000 existing social indicators related to ocean and coastal management of the US West Coast. We focused on two foundational attributes of human well-being: resource access and self-determination. Outcomes and Discussion: Our results suggest that existing indicators and data are limited in their ability to reflect linkages between environmental change and human well-being, and extremely limited in their ability to assess social equity and justice. We reveal a critical need for new social indicators tailored to answer environmental questions and new data that are disaggregated by social variables to measure equity. In both, we stress the importance of collaborating with the people whose well-being is to be assessed. Conclusion: Our framework is designed to encourage governments and communities to carefully assess the complex tradeoffs inherent in environmental decision-making.