Voltage sensor ring in a native structure of a membrane-embedded potassium channel.

Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.

Cation dyshomeostasis and cardiomyocyte necrosis: the Fleckenstein hypothesis revisited.

An ongoing loss of cardiomyocytes to apoptotic and necrotic cell death pathways contributes to the progressive nature of heart failure. The pathophysiological origins of necrotic cell loss relate to the neurohormonal activation that accompanies acute and chronic stressor states and which includes effector hormones of the adrenergic nervous system. Fifty years ago, Albrecht Fleckenstein and coworkers hypothesized the hyperadrenergic state, which accompanies such stressors, causes cardiomyocyte necrosis based on catecholamine-initiated excessive intracellular Ca(2+) accumulation (EICA), and mitochondrial Ca(2+) overloading in particular, in which the ensuing dysfunction and structural degeneration of these organelles leads to necrosis. In recent years, two downstream factors have been identified which, together with EICA, constitute a signal-transducer-effector pathway: (i) mitochondria-based induction of oxidative stress, in which the rate of reactive oxygen metabolite generation exceeds their rate of detoxification by endogenous antioxidant defences; and (ii) the opening of the mitochondrial inner membrane permeability transition pore (mPTP) followed by organellar swelling and degeneration. The pathogenesis of stress-related cardiomyopathy syndromes is likely related to this pathway. Other factors which can account for cytotoxicity in stressor states include: hypokalaemia; ionized hypocalcaemia and hypomagnesaemia with resultant elevations in parathyroid hormone serving as a potent mediator of EICA; and hypozincaemia with hyposelenaemia, which compromise antioxidant defences. Herein, we revisit the Fleckenstein hypothesis of EICA in leading to cardiomyocyte necrosis and the central role played by mitochondria.

Structural basis for the prion-like MAVS filaments in antiviral innate immunity.

Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5ß-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.

A dyshomeostasis of electrolytes and trace elements in acute stressor states: impact on the heart.

Acute stressor states are associated with a homeostatic activation of the hypothalamic-pituitary-adrenal axis. A hyperadrenergic state follows and leads to a dyshomeostasis of several intra- and extracellular cations, including K, Mg, and Ca. Prolongation of myocardial repolarization and corrected QT interval (QTc) of the ECG are useful biomarkers of hypokalemia and/or hypomagnesemia and should be monitored to address the adequacy of cation replacement. A dyshomeostasis of several trace elements, including Zn and Se, are also found in critically-ill patients to compromise metalloenzyme-based antioxidant defenses. Collectively, dyshomeostasis of these electrolytes and trace elements have deleterious consequences on the myocardium: atrial and ventricular arrhythmias; induction of oxidative stress with reduced antioxidant defenses; and adverse myocardial remodeling, including cardiomyocytes lost to necrosis and replaced by fibrous tissue. To minimize such consequences during hyperadrenergic states, systematic surveillance of electrolytes and trace elements, together with QTc, are warranted. Plasma K and Mg should be maintained at > or =4.0 mEq/L and > or =2.0 mg/dL, respectively (the 4 and 2 rule).

Delay in the diagnosis of acute myocardial infarction: effect on quality of care and its assessment.

BACKGROUND: Delay in diagnosis of acute myocardial infarction (AMI) may affect quality of care and its assessment. OBJECTIVES: To examine over time the frequency of delay in AMI diagnosis and the effect of this delay on the quality of patient care and its assessment. METHODS: The authors examined the trend in coded admission diagnosis, age, comorbidities, procedures during hospitalization, and discharge status for 42,406 Connecticut Medicare cases with the principal discharge diagnosis of AMI for the time period 1992 through 2001. For 2,583 cases discharged in 1992 and 1993 and for 1,398 cases discharged in 1998 through 2001, the rates of administration of aspirin (ASA) and beta blocker (BB) on admission and discharge, by admission diagnosis, were ascertained. RESULTS: For patients discharged with the principal diagnosis of AMI over the decade examined, the proportion with this diagnosis on admission fell (59% to 40%, p < 0.001), the proportion with a non-acute coronary syndrome (ACS) admission diagnosis rose (18% to 26%, p < 0.001), and the population aged (proportion older than 85 years of age increased from 16% to 28%, p < 0.001). Patients with ACS as the admission diagnosis more frequently received cardiac catheterization (during 2000-2001, 39% versus 17%, p < 0.001), percutaneous coronary intervention (19% versus 4%, p < 0.001), and evidence-based therapy; during 1998-2001, opportunities to give ASA or BB on admission were fulfilled for 88% versus 73% (p < 0.001), and on discharge, for 87% versus 74% (p < 0.005). CONCLUSIONS: The diagnosis of AMI is delayed after admission for a significant proportion of cases who receive care that is measured to be of lower quality. There is a need to more effectively diagnose and treat these cases with delayed diagnosis and to develop new quality measures to address changes in the characteristics of patients who are hospitalized with AMI.