scholarly journals Insights into the Selectivity Mechanisms of Grapevine NIP Aquaporins

2020 ◽  
Vol 21 (18) ◽  
pp. 6697 ◽  
Author(s):  
Farzana Sabir ◽  
Antonella Di Pizio ◽  
Maria C. Loureiro-Dias ◽  
Angela Casini ◽  
Graça Soveral ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Substrate Selectivity ◽  
Channel Activity ◽  
Substrate Selection ◽  
Yeast Growth ◽  
Substrate Preferences ◽  
General Decline ◽  
Residue Substitution ◽  
Underlying Mechanisms ◽  
Aquaporin Family

Nodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the transport of physiologically relevant solutes. The present study intended to investigate how substrate selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within the pore and the possible underlying mechanisms. A mutational approach was used to interchange the ar/R residues between grapevine NIPs (VvTnNIP1;1 with VvTnNIP6;1, and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could alter the NIP channel activity, but it was not sufficient to interchange their substrate preferences. Further, homology modeling analyses evidenced that variations in the pore radius combined with the differences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance of H5 sequence for substrate selection. These results contribute to the knowledge of the overall determinants of substrate selectivity in NIPs.

scholarly journals Substrate Selection of Ascidian Larva: Wettability and Nano-Structures

2021 ◽  
Vol 9 (6) ◽  
pp. 634
Author(s):  
Euichi Hirose ◽  
Noburu Sensui
Keyword(s):  
The Body ◽  
Substrate Selection ◽  
Artificial Structures ◽  
Substrate Preferences ◽  
Nano Structures ◽  
Hydrophobic Materials ◽  
Faunal Communities ◽  
Adhesive Organs ◽  
Fouling Organisms ◽  
Selection Of

Ascidians are marine sessile chordates that comprise one of the major benthic animal groups in marine ecosystems. They sometimes cause biofouling problems on artificial structures underwater, and non-indigenous, invasive ascidian species can potentially and seriously alter native faunal communities. Ascidian larvae are usually tadpole-shaped, negatively phototactic, and adhere on substrates by secreting a glue from their adhesive organs. Although larvae often prefer hydrophobic surfaces, such as a silicone rubber, for settlement, hydrophobic materials are often used to reduce occurrence of fouling organisms on artificial structures. This inconsistency may indicate that an attractive surface for larvae is not always suitable for settlement. Micro-scale structures or roughness may enhance the settlement of ascidian larvae, but settlement is significantly reduced by a nano-scale nipple array (or moth-eye structure), suggesting functional properties of similar structures found on the body surfaces of various invertebrates. The substrate preferences of larvae should be one of the important bases in considering measures against biofouling, and this review also discusses the potential uses of materials to safely reduce the impacts of invasive species.

Experiments on Substrate Selection by Corophium Species: Films and Bacteria on Sand Particles

1964 ◽  
Vol 41 (3) ◽  
pp. 499-511
Author(s):  
P. S. MEADOWS
Keyword(s):  
Ionic Strength ◽  
Sea Water ◽  
Distilled Water ◽  
Substrate Selection ◽  
Corophium Volutator ◽  
Simple Method ◽  
Substrate Preferences ◽  
Solutions Of Electrolytes ◽  
Sand Particles ◽  
Micro Organisms

1. A simple method is described for determining the substrate preferences of Corophium volutator (Pallas) and Corophium arenarium Crawford. 2. If offered a choice of its own substrate with that of the other species each prefers its own. 3. Level of illumination and colour of substrate have little effect on choice. An animal's size and hence its age has little effect on its substrate preferences. 4. C. volutator prefers a substrate previously maintained under anaerobic conditions, C. arenarium vice versa. 5. Treatments which kill, inactivate, or remove micro-organisms render sands unattractive to Corophium. These include boiling, acid-cleaning, drying, and soaking in fixatives or distilled water. Attempts to make these sands attractive again failed. 6. Distilled water, and solutions of the non-electrolytes sucrose and glycerol at the same osmotic pressure as sea water, induce many bacteria to desorb from sand particles; smaller numbers are desorbed in the presence of solutions of electrolytes at the same ionic strength as sea water (NaCl, Na2SO4, KC1, MgSO4, MgCl2, CaCl2). Of all these, only distilled water and solutions of MgCl2 and CaCl2 reduce the attractive properties of sands. Hence the loss of bacteria from the surface of sand grains, though related to the ionic strength and composition of the medium, is not necessarily associated with a substrate becoming unattractive.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

scholarly journals AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria Paz Prada ◽  
Arsalan U. Syed ◽  
Gopireddy R. Reddy ◽  
Miguel Martín-Aragón Baudel ◽  
Víctor A. Flores-Tamez ◽  
...  
Keyword(s):  
Vascular Reactivity ◽  
Vascular Complications ◽  
Channel Activity ◽  
Camp Synthesis ◽  
Anchoring Protein ◽  
Extracellular Glucose ◽  
Elevated Glucose ◽  
Underlying Mechanisms ◽  
Purinergic Modulation ◽  
Human And Mouse

Abstract The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.

scholarly journals Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements.

1996 ◽  
Vol 16 (4) ◽  
pp. 1490-1499 ◽  
Author(s):  
S S Peng ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Functional Characterization ◽  
Structural Features ◽  
Mrna Degradation ◽  
Beta Globin ◽  
Globin Mrna ◽  
Transcription Inhibitors ◽  
Tightly Coupled ◽  
Underlying Mechanisms ◽  
Necessary And Sufficient ◽  
Domain I

AU-rich RNA-destabilizing elements (AREs) found in the 3' untranslated regions of many labile mRNAs encoding proto-oncoproteins and cytokines generally contain (i) one or more copies of the AUUUA pentanucleotide and (ii) a high content of uridylate and sometimes also adenylate residues. Recently, we have identified a potent ARE from the 3' untranslated region of c-jun proto-oncogene mRNA that does not contain the AUUUA motif. In an attempt to further our understanding of the general principles underlying mechanisms by which AREs direct rapid and selective mRNA degradation, in this study we have characterized the functionally important structural features and properties of this non-AUUUA ARE. Like AUUUA-containing AREs, this non-AUUUA ARE directs rapid shortening of the poly(A) tail as a necessary first step for mRNA degradation. It can be further dissected into three structurally and functionally distinct regions, designated domains I, II, and III. None of three domains alone is able to significantly destabilize the stable beta-globin mRNA. The two unlinked domains, I and III, together are necessary and sufficient for specifying the full destabilizing function of this non-AUUUA ARE. Domain II appears functionally dispensable but can partially substitute for domain I. Domain swaps made between the c-jun non-AUUUA and the c-fos AUUUA-containing AREs reveal that the two AREs, while sharing no sequence homology, appear to contain sequence domains that are structurally distinct but functionally overlapping and exchangeable. These data support the idea that the ultimate destabilizing function of an individual ARE is determined by its own unique combination of structurally distinct and functionally interdependent domains. Our polysome profile studies show tha the destabilizing function of the c-jun non-AUUUA ARE does not require any active transit by ribosomes of the mRNA bearing it, further corroborating that the destabilizing function of AREs is not tightly coupled to ongoing translation by ribosomes. Moreover, unlike AUUUA-containing AREs, the c-jun ARE is insensitive to blockage of its effects by addition of transcription inhibitors. Thus, our data provide further evidence for the existence of a novel class of ARE with unique properties.

ATP-sensitive K+ channels from aortic smooth muscle incorporated into planar lipid bilayers

1991 ◽  
Vol 261 (2) ◽  
pp. H604-H609 ◽  
Author(s):  
R. J. Kovacs ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Functional Characterization ◽  
Katp Channels ◽  
Membrane Preparation ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Aortic Smooth Muscle ◽  
Kca Channels ◽  
Significant Block

Glibenclamide binding sites were identified in a membrane preparation from canine aortic smooth muscle. The dissociation constant for [3H]glibenclamide binding was 10 +/- 2 nM, with a density of 420 +/- 108 fmol/mg protein. The properties of ATP-sensitive potassium (KATP) channels from the same membrane preparation incorporated into planar lipid bilayers were investigated. ATP was a potent inhibitor of the channels with half-maximal inhibition of channel activity by 41 microM ATP. Glibenclamide inhibited channel activity, and cromakalim activated the channel in the presence of ATP. Blockers of Ca(2+)-activated K+ (KCa) channels (charybdotoxin and tetraethylammonium ions) did not affect KATP channels in concentrations that caused significant block of KCa channels in bilayers. This membrane preparation should allow further biochemical and functional characterization of KATP channels and glibenclamide receptors in arterial smooth muscle.

scholarly journals Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1)

2020 ◽  
Vol 295 (24) ◽  
pp. 8120-8134
Author(s):  
Nileeka Balasuriya ◽  
Norman E. Davey ◽  
Jared L. Johnson ◽  
Huadong Liu ◽  
Kyle K. Biggar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase B ◽  
Tumor Biology ◽  
Peptide Libraries ◽  
Substrate Selectivity ◽  
Activity Data ◽  
Cellular Processes ◽  
Substrate Preferences ◽  
Substrate Peptide ◽  
Kinase Substrates

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

scholarly journals BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

2014 ◽  
Vol 205 (2) ◽  
pp. 233-249 ◽  
Author(s):  
Pascal Béguin ◽  
Kazuaki Nagashima ◽  
Ramasubbu N. Mahalakshmi ◽  
Réjan Vigot ◽  
Atsuko Matsunaga ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Functional Characterization ◽  
Regulatory Protein ◽  
Binding Pocket ◽  
Neuroendocrine Cells ◽  
Cell Surface Expression ◽  
Channel Activity ◽  
Interaction Domain ◽  
Voltage Gated ◽  
Domain I

Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

scholarly journals It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin

2016 ◽  
Vol 311 (2) ◽  
pp. C166-C178 ◽  
Author(s):  
Martina P. Liebl ◽  
Thorsten Hoppe
Keyword(s):  
Binding Proteins ◽  
Covalent Attachment ◽  
Special Importance ◽  
Heat Shock Factors ◽  
Substrate Selection ◽  
E3 Ligases ◽  
Age Related ◽  
General Decline ◽  
Ubiquitin Binding ◽  
Proteolytic Pathways

Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases.

Abstract 17301: Functional Characterization of KCNK3 Mutants Associated With Pulmonary Arterial Hypertension Under Physiologically Relevant Heterozygous Conditions

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Michael S Bohnen ◽  
Danilo Roman-Campos ◽  
Cecile Terrenoire ◽  
Jack Jnani ◽  
Lei Chen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Resting Membrane Potential ◽  
Channel Activity ◽  
Loss Of Function ◽  
Pulmonary Arterial ◽  
Specific Impact

KCNK3 encodes a two-pore domain K+ channel, TASK-1, which is inhibited by extracellular acidity and hypoxia. Expressed in a variety of tissues, including human pulmonary artery smooth muscle cells (hPASMCs), the central nervous system, pancreas, and adrenal glands, TASK-1 contributes to the resting membrane potential of cells in which it is expressed. Recently, our group reported mutations in KCNK3 underlying idiopathic pulmonary arterial hypertension (PAH), resulting from loss of TASK-1 function, partially pharmacologically rescuable with ONO-RS-082. TASK-1 dimerizes in vivo, forming functional channels with another TASK-1 subunit or with the related TASK-3 channel. TASK-1 and TASK-3 often are expressed in the same cells, although it has been reported that TASK-1 alone is expressed in the lung. Our initial study examined mutant and wildtype (WT) homodimeric TASK-1 channels expressed heterologously in COS-7 cells. Here we further characterize PAH-linked TASK-1 mutations in physiologically relevant heterozygous conditions in COS-7 and hPASMC cell lines. We engineered heterodimeric channels consisting of one mutant and one WT subunit; compared this with co-expression of mutant and WT channels; and measured channel activity with whole cell patch clamp procedures. We found a mutation specific impact of heterozygosity on channel activity. One mutation, V221L, produces a shift in pH dependence accounting for loss of function at physiological pH 7.4, partially rescued by dimerization with a WT subunit, while another, G203D, produces near complete loss of function as a homo- or hetero-dimer. The presence of TASK-3 results in greater rescue of V221L TASK-1 activity at pH 7.4 than does WT TASK-1. Additionally, under current clamp we found that ONO-RS-082 hyperpolarizes the membrane potential in hPASMCs expressing WT or V221L TASK-1, reversible by selective block of TASK-1 with ML365. Together, our results suggest (a) TASK-1 mutant heterodimers exhibit loss of function with mutation specific severity; (b) TASK-3 may rescue mutant TASK-1 and underlie a tissue specific impact of the TASK-1 mutations observed clinically; and (c) PAH TASK-1 mutants can be pharmacologically modulated in hPASMCs and alter the critically important resting membrane potential.

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