scholarly journals An arginine residue in the outer segment of hASIC1a TM1 affects both proton affinity and channel desensitization

2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Zhuyuan Chen ◽  
Georg Kuenze ◽  
Jens Meiler ◽  
Cecilia M. Canessa
Keyword(s):  
Steady State ◽  
Proton Affinity ◽  
Outer Segment ◽  
Pain Perception ◽  
Transmembrane Domain ◽  
High Energy ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Acid Sensing Ion Channels ◽  
Channel Opening

Acid-sensing ion channels (ASICs) respond to changes in pH in the central and peripheral nervous systems and participate in synaptic plasticity and pain perception. Understanding the proton-mediated gating mechanism remains elusive despite the of their structures in various conformational states. We report here that R64, an arginine located in the outer segment of the first transmembrane domain of all three isoforms of mammalian ASICs, markedly impacts the apparent proton affinity of activation and the degree of desensitization from the open and preopen states. Rosetta calculations of free energy changes predict that substitutions of R64 in hASIC1a by aromatic residues destabilize the closed conformation while stabilizing the open conformation. Accordingly, F64 enhances the efficacy of proton-mediated gating of hASIC1a, which increases the apparent pH50 and facilitates channel opening when only one or two subunits are activated. F64 also lengthens the duration of opening events, thus keeping channels open for extended periods of time and diminishing low pH-induced desensitization. Our results indicate that activation of a proton sensor(s) with pH50 equal to or greater than pH 7.2–7.1 opens F64hASIC1a, whereas it induces steady-state desensitization in wildtype channels due to the high energy of activation imposed by R64, which prevents opening of the pore. Together, these findings suggest that activation of a high-affinity proton-sensor(s) and a common gating mechanism may mediate the processes of activation and steady-state desensitization of hASIC1a.

scholarly journals A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism

2021 ◽  
Vol 8 ◽  
Author(s):  
Alessandro Crnjar ◽  
Susanne M. Mesoy ◽  
Sarah C. R. Lummis ◽  
Carla Molteni
Keyword(s):  
Ligand Binding ◽  
Ion Channel ◽  
Drug Targets ◽  
Transmembrane Domain ◽  
Active Role ◽  
Extracellular Domain ◽  
Single Mutation ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Mutant Receptors

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

Reduced substrate oxidation in postischemic myocardium: 13C and 31P NMR analyses

1990 ◽  
Vol 258 (5) ◽  
pp. H1357-H1365 ◽  
Author(s):  
E. D. Lewandowski ◽  
D. L. Johnston
Keyword(s):  
Steady State ◽  
31P Nmr ◽  
Tca Cycle ◽  
High Energy ◽  
Substrate Oxidation ◽  
High Energy Phosphates ◽  
Atp Content ◽  
And Control ◽  
13C And 31P Nmr ◽  
Postischemic Myocardium

13C and 31P nuclear magnetic resonance (NMR) spectra were used to assess substrate oxidation and high-energy phosphates in postischemic (PI) isolated rabbit hearts. Phosphocreatine (PCr) increased in nonischemic controls on switching from glucose perfusion to either 2.5 mM [3-13C]pyruvate (120%, n = 7) or [2-13C]acetate (114%, n = 8, P less than 0.05). ATP content, oxygen consumption (MVO2), and hemodynamics (dP/dt) were not affected by substrate availability in control or PI hearts. dP/dt was 40-60% lower in PI hearts during reperfusion after 10 min ischemia. Hearts reperfused with either pyruvate (n = 11) or acetate (n = 8) regained preischemic PCr levels within 45 s. Steady-state ATP levels were 55-70% of preischemia with pyruvate and 52-60% with acetate. Percent maximum [4-13C]glutamate signal showed reduced conversion of pyruvate to glutamate via the tricarboxylic acid (TCA) cycle at 4-min reperfusion (PI = 24 +/- 4%, means +/- SE; Control = 48 +/- 4%). The increase in 13C signal from the C-4 position of glutamate was similar to control hearts within 10.5 min. The increase in [4-13C]glutamate signal from acetate was not different between PI and control hearts. The ratio of [2-13C]Glu:[4-13C]Glu, reflecting TCA cycle activity, was reduced in PI hearts with acetate for at least 10 min (Control = 0.76 +/- 0.03; PI = 0.51 +/- 0.09) until steady state was reached. Despite rapid recovery of oxidative phosphorylation, contractility remained impaired and substrate oxidation was significantly slowed in postischemic hearts.

scholarly journals A radical departure from the ‘steady-state’ concept in cosmology

1966 ◽  
Vol 290 (1421) ◽  
pp. 162-176 ◽  
Keyword(s):  
Steady State ◽  
Cosmic Rays ◽  
High Energy ◽  
Expansion Rate ◽  
Coupling Constant ◽  
De Sitter ◽  
Observable Universe ◽  
State Expansion ◽  
The Masses ◽  
Creation Of Matter

The results in this paper are based on an entirely different choice of the undetermined coupling constant f which appears in the theory of creation of matter. Previously f was chosen to make the steady-state expansion rate coincident with the observed expansion rate. Now that we take a much larger value for f , the corresponding steady-state expansion rate is much greater than the observed value. We interpret this difference as showing that we live in a wide, possibly temporary, fluctuation from the steady-state situation. The expansion rate in such a fluctuation follows the Einstein-de Sitter relations. The natural scale set by the new steady-state corresponds to the masses of clusters of galaxies, we obtain 10 13 M0 instead of 10 23 M@ for the ‘observable universe’. It is suggested that elliptical galaxies were formed early in the development of a fluctuation. Our discussion of high energy phenomena leads to im m ediate explanations of the energy spectrum of cosmic rays, of the presence of e + in cosmic rays and of the rate of energy production associated with radio sources.

scholarly journals Electrostatics, proton sensor, and networks governing the gating transition in GLIC, a proton-gated pentameric ion channel

2018 ◽  
Vol 115 (52) ◽  
pp. E12172-E12181 ◽  
Author(s):  
Haidai Hu ◽  
Kenichi Ataka ◽  
Anaïs Menny ◽  
Zaineb Fourati ◽  
Ludovic Sauguet ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Ion Channel ◽  
Transmembrane Domain ◽  
Hydrogen Bond Network ◽  
Transmembrane Region ◽  
Allosteric Site ◽  
Closed Channel ◽  
Open Conformation ◽  
Poisson Boltzmann ◽  
Bond Network

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure–function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson–Boltzmann/Debye–Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain–transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC’s gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

scholarly journals Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

2021 ◽  
Author(s):  
Toby S Turney ◽  
Vivian Li ◽  
Stephen G Brohawn
Keyword(s):  
Conformational Changes ◽  
Pancreatic Beta Cells ◽  
Low Ph ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Lipid Nanodiscs ◽  
Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

Transient changes in muscle high-energy phosphates during moderate exercise

1993 ◽  
Vol 75 (2) ◽  
pp. 648-656 ◽  
Author(s):  
G. D. Marsh ◽  
D. H. Paterson ◽  
J. J. Potwarka ◽  
R. T. Thompson
Keyword(s):  
Oxygen Consumption ◽  
Steady State ◽  
Recovery Period ◽  
High Energy ◽  
Second Order ◽  
Moderate Intensity ◽  
High Energy Phosphates ◽  
Order Model ◽  
Time Constants ◽  
First Order

The purpose of this study was to use 31P-nuclear magnetic resonance spectroscopy to examine changes in wrist flexor muscle metabolism during the transitions from rest to steady-state exercise (on-transient) and back to rest (off-transient). Five healthy young males (mean age 25 +/- 2 yr) performed a series of square-wave exercise tests, each consisting of 5 min of moderate-intensity work followed by a 5-min recovery period. The subjects repeated this protocol six times, and each individual's results were pooled before analysis. ATP and intracellular pH did not change significantly during exercise or recovery. Phosphocreatine (PCr) declined progressively at the onset of exercise, reaching a plateau after approximately 2 min. A reciprocal increase in Pi occurred during the onset of exercise. During the recovery period PCr was resynthesized, whereas Pi returned to resting levels. The data were plotted as a function of time and fit with both first- and second-order exponential growth or decay models; however, the second-order model did not significantly improve the fit of the data. Time constants for the first-order model of the on- and off-transient responses for both PCr and Pi were approximately 30 s. These values are nearly identical to the time constants for oxygen consumption during submaximal exercise that have been reported previously by several authors. The results of this study show that the metabolism of muscle PCr during steady-state exercise and recovery can be accurately described by a monoexponential model and, further, suggest that a first-order proportionality exists between metabolic substrate utilization and oxygen consumption.

scholarly journals SLC38A8 mutations result in arrested retinal development with loss of cone photoreceptor specialization

2020 ◽  
Vol 29 (18) ◽  
pp. 2989-3002 ◽  
Author(s):  
Helen J Kuht ◽  
Jinu Han ◽  
Gail D E Maconachie ◽  
Sung Eun Park ◽  
Seung-Tae Lee ◽  
...  
Keyword(s):  
Outer Segment ◽  
Transmembrane Domain ◽  
Retinal Development ◽  
Anterior Segment ◽  
Cone Photoreceptor ◽  
Photoreceptor Outer Segment ◽  
Multi Centre Study ◽  
Photoreceptor Layer ◽  
Foveal Hypoplasia

Abstract Foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis is an autosomal recessive disorder arising from SLC38A8 mutations. SLC38A8 is a putative glutamine transporter with strong expression within the photoreceptor layer in the retina. Previous studies have been limited due to lack of quantitative data on retinal development and nystagmus characteristics. In this multi-centre study, a custom-targeted next generation sequencing (NGS) gene panel was used to identify SLC38A8 mutations from a cohort of 511 nystagmus patients. We report 16 novel SLC38A8 mutations. The sixth transmembrane domain is most frequently disrupted by missense SLC38A8 mutations. Ninety percent of our cases were initially misdiagnosed as PAX6-related phenotype or ocular albinism prior to NGS. We characterized the retinal development in vivo in patients with SLC38A8 mutations using high-resolution optical coherence tomography. All patients had severe grades of arrested retinal development with lack of a foveal pit and no cone photoreceptor outer segment lengthening. Loss of foveal specialization features such as outer segment lengthening implies reduced foveal cone density, which contributes to reduced visual acuity. Unlike other disorders (such as albinism or PAX6 mutations) which exhibit a spectrum of foveal hypoplasia, SLC38A8 mutations have arrest of retinal development at an earlier stage resulting in a more under-developed retina and severe phenotype.

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