scholarly journals Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding

2015 ◽  
Vol 290 (45) ◽  
pp. 27113-27123 ◽  
Author(s):  
Marcus Fischer ◽  
Adam P. Hopkins ◽  
Emmanuele Severi ◽  
Judith Hawkhead ◽  
Daniel Bawdon ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Selectivity Filter ◽  
High Affinity

scholarly journals Structural Similarities between Glutamate Receptor Channels and K+ Channels Examined by Scanning Mutagenesis

2001 ◽  
Vol 117 (4) ◽  
pp. 345-360 ◽  
Author(s):  
Victor A. Panchenko ◽  
Carla R. Glasser ◽  
Mark L. Mayer
Keyword(s):  
Dissociation Constant ◽  
Glutamate Receptors ◽  
Selectivity Filter ◽  
Periodic Pattern ◽  
K Channels ◽  
High Affinity ◽  
Aromatic Cage ◽  
Voltage Dependent ◽  
Complete Disruption ◽  
A Site

The pores of glutamate receptors and K+ channels share sequence homology, suggesting a conserved secondary structure. Scanning mutagenesis with substitution of alanine and tryptophan in GluR6 channels was performed based on the structure of KcsA. Our assay used disruption of voltage-dependent polyamine block to test for changes in the packing of pore-forming regions. Alanine scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A periodic pattern from F575 to M589 aligned with the pore helix in KcsA, whereas a cluster of sensitive positions around Q590, a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D567 to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 37 positions and a pM dissociation constant for Q590W. Molecular modeling with KcsA coordinates showed that GluR6 pore helix mutants disrupting polyamine block pack against M1 and M2, and are not exposed in the ion channel pore. In the selectivity filter, tryptophan creates an aromatic cage consistent with the pM dissociation constant for Q590W. A scan with glutamate substitution was used to map the cytoplasmic entrance to the pore based on charge neutralization experiments, which established that E594 was uniquely required for high affinity polyamine block. In E594Q mutants, introduction of glutamate at positions S593–L600 restored polyamine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that the pore region of glutamate receptors contains a helix and pore loop analogous to that found in K+ channels. At the cytoplasmic entrance of the channel, a negatively charged amino acid, located in an extended loop with solvent-exposed side chains, is required for high affinity polyamine block and probably attracts cations via a through space electrostatic mechanism.

scholarly journals Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Robert Warneke ◽  
Anja Poehlein ◽  
Janina Stautz ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Potassium Channel ◽  
Living Cell ◽  
Potassium Uptake ◽  
Selectivity Filter ◽  
Uptake System ◽  
Content Type ◽  
Apparent Affinity ◽  
High Affinity ◽  
External Potassium

ABSTRACT Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD. IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

scholarly journals Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter

2004 ◽  
Vol 101 (50) ◽  
pp. 17549-17554 ◽  
Author(s):  
S. E. Unkles ◽  
D. A. Rouch ◽  
Y. Wang ◽  
M. Y. Siddiqi ◽  
A. D. M. Glass ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Nitrate Transporter ◽  
High Affinity ◽  
Arginine Residues

Cofactor and Substrate Binding to Vanadium Chloroperoxidase Determined by UV−VIS Spectroscopy and Evidence for High Affinity for Pervanadate†

Biochemistry ◽  
2000 ◽  
Vol 39 (5) ◽  
pp. 1133-1141 ◽  
Author(s):  
Rokus Renirie ◽  
Wieger Hemrika ◽  
Sander R. Piersma ◽  
Ron Wever
Keyword(s):  
Substrate Binding ◽  
High Affinity ◽  
Vis Spectroscopy

scholarly journals To be, or not to be two sites: that is the question about LeuT substrate binding

2011 ◽  
Vol 138 (4) ◽  
pp. 467-471 ◽  
Author(s):  
Nicolas Reyes ◽  
Sotiria Tavoulari
Keyword(s):  
Binding Sites ◽  
Journal Club ◽  
Substrate Binding ◽  
Structural Data ◽  
Transport Proteins ◽  
Model System ◽  
Transport Mechanisms ◽  
High Affinity ◽  
The Central Nervous System ◽  
Substrate Binding Sites

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

scholarly journals Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

FEBS Letters ◽  
2006 ◽  
Vol 580 (21) ◽  
pp. 5049-5053 ◽  
Author(s):  
Lili Kandra ◽  
Maher Abou Hachem ◽  
Gyöngyi Gyémánt ◽  
Birte Kramhøft ◽  
Birte Svensson
Keyword(s):  
Substrate Binding ◽  
High Affinity ◽  
Binding Cleft

scholarly journals Identification of a Determinant of High Affinity Calcium Binding in the Selectivity Filter of a Mammalian Calcium Channel

2014 ◽  
Vol 106 (2) ◽  
pp. 331a
Author(s):  
Felix Findeisen ◽  
David Shaya ◽  
Fayal Abderemane-Ali ◽  
Cristina Arrigoni ◽  
Gildas Loussouarn ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Calcium Binding ◽  
Selectivity Filter ◽  
High Affinity
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