scholarly journals Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier

1998 ◽  
Vol 332 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Steven VAYRO ◽  
Bryan LO ◽  
Mel SILVERMAN
Keyword(s):  
Glucose Transporter ◽  
Immunogold Labelling ◽  
Transport Activity ◽  
Wild Type ◽  
Functional Changes ◽  
Functional Studies ◽  
Kd Values ◽  
Function Relationship ◽  
Relationship Of ◽  
Glucose Carrier

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent α-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92±0.08 nmol/min per mg of protein and Km was 0.98±0.13 mM; for wild-type SGLT1, Vmax was 1.98±0.47 nmol/min per mg of protein and Km was 0.36±0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55±0.18 and 1.69±0.57 pmol/mg of protein respectively); Kd values were 0.46±0.15 and 0.51±0.11 µM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-0-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose

1993 ◽  
Vol 290 (2) ◽  
pp. 497-501 ◽  
Author(s):  
A Schürmann ◽  
K Keller ◽  
I Monden ◽  
F M Brown ◽  
S Wandel ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Binding Domains ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.

scholarly journals Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity

2021 ◽  
Author(s):  
Caroline Marie Teresa Neumann ◽  
Lena Lindtoft Rosenbæk ◽  
Rasmus Kock Flygaard ◽  
Michael Habeck ◽  
Jesper Lykkegaard Karlsen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Transmembrane Helix ◽  
Sodium Potassium ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
Regulation Of Transport ◽  
Function Relationship ◽  
Na Release ◽  
Relationship Of

The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl- and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 Å resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl-) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

scholarly journals Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics

2019 ◽  
Vol 152 (1) ◽  
Author(s):  
Toshiko Yamazawa ◽  
Haruo Ogawa ◽  
Takashi Murayama ◽  
Maki Yamaguchi ◽  
Hideto Oyamada ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Md Simulations ◽  
Hek293 Cells ◽  
Salt Bridges ◽  
Open Probability ◽  
Release Channel ◽  
Functional Studies ◽  
Function Relationship ◽  
Interdomain Interactions ◽  
Relationship Of

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation–contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure–function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

scholarly journals Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

2021 ◽  
Vol 22 (16) ◽  
pp. 9085
Author(s):  
Rafał Hołubowicz ◽  
Andrzej Ożyhar ◽  
Piotr Dobryszycki
Keyword(s):  
Wild Type ◽  
Positional Vertigo ◽  
Natural Variants ◽  
Self Association ◽  
Function Relationship ◽  
The Stability ◽  
And Function ◽  
Relationship Of ◽  
Wild Type Form ◽  
Paroxysmal Positional Vertigo

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

scholarly journals The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (22) ◽  
pp. 6968-6974 ◽  
Author(s):  
Alfredo Castañeda-García ◽  
Alexandro Rodríguez-Rojas ◽  
Javier R. Guelfo ◽  
Jesús Blázquez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Frequency ◽  
Wild Type ◽  
Apparent Lack ◽  
Fosfomycin Resistance ◽  
Function Relationship ◽  
Relationship Of ◽  
Specific Transport System ◽  
Insertion Mutants

ABSTRACT Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10−6. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10−11). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.

scholarly journals Allosteric Regulation of Catalytic Activity:Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

2001 ◽  
Vol 65 (3) ◽  
pp. 404-421 ◽  
Author(s):  
Kerstin Helmstaedt ◽  
Sven Krappmann ◽  
Gerhard H. Braus
Keyword(s):  
Conformational Changes ◽  
Classical Model ◽  
Allosteric Regulation ◽  
Chorismate Mutase ◽  
Aspartate Transcarbamoylase ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Function Relationship ◽  
Relationship Of ◽  
Entire Picture

SUMMARY Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail.

scholarly journals Structure-function relationship of serine protease-protein inhibitor interaction.

2001 ◽  
Vol 48 (2) ◽  
pp. 419-428 ◽  
Author(s):  
J Otlewski ◽  
M Jaskólski ◽  
O Buczek ◽  
T Cierpicki ◽  
H Czapińska ◽  
...  
Keyword(s):  
High Resolution ◽  
Structure Function ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Wild Type ◽  
Stability Parameters ◽  
X Ray ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Relationship Of

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

Deciphering the role of GLUT4 N-glycosylation in adipocyte and muscle cell models

2012 ◽  
Vol 445 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Nancy Zaarour ◽  
Marion Berenguer ◽  
Yannick Le Marchand-Brustel ◽  
Roland Govers
Keyword(s):  
Cell Surface ◽  
Basal Cell ◽  
Glucose Transporter ◽  
Insulin Response ◽  
Basal Cells ◽  
Cell Models ◽  
Transport Activity ◽  
Wild Type ◽  
Insulin Stimulation

GLUT4 (glucose transporter 4) is responsible for the insulin-induced uptake of glucose by muscle and fat cells. In non-stimulated (basal) cells, GLUT4 is retained intracellularly, whereas insulin stimulation leads to its translocation from storage compartments towards the cell surface. How GLUT4 is retained intracellularly is largely unknown. Previously, aberrant GLUT4 N-glycosylation has been linked to increased basal cell-surface levels, while N-glycosylation-deficient GLUT4 was found to be quickly degraded. As recycling and degradation of GLUT4 are positively correlated, we hypothesized that incorrect N-glycosylation of GLUT4 might reduce its intracellular retention, resulting in an increased cell-surface recycling, in increased basal cell-surface levels, and in enhanced GLUT4 degradation. In the present study, we have investigated N-glycosylation-deficient GLUT4 in detail in 3T3-L1 preadipocytes, 3T3-L1 adipocytes and L6 myoblasts. We have found no alterations in retention, insulin response, internalization or glucose transport activity. Degradation of the mutant molecule was increased, although once present at the cell surface, its degradation was identical with that of wild-type GLUT4. Our findings indicate that N-glycosylation is important for efficient trafficking of GLUT4 to its proper compartments, but once the transporter has arrived there, N-glycosylation plays no further major role in its intracellular trafficking, nor in its functional activity.

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