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

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.

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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

Biochemical Journal ◽

10.1042/bj3320119 ◽

1998 ◽

Vol 332 (1) ◽

pp. 119-125 ◽

Cited By ~ 19

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201812235 ◽

2019 ◽

Vol 152 (1) ◽

Cited By ~ 4

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.

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ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV

Clinical Science ◽

10.1042/cs20200899 ◽

2020 ◽

Vol 134 (21) ◽

pp. 2851-2871 ◽

Cited By ~ 1

Author(s):

Lizelle Lubbe ◽

Gyles E. Cozier ◽

Delia Oosthuizen ◽

K. Ravi Acharya ◽

Edward D. Sturrock

Keyword(s):

Blood Pressure ◽

Blood Pressure Regulation ◽

Pressure Regulation ◽

X Ray Crystallography ◽

Physiological Processes ◽

Cryo Electron Microscopy ◽

Potential Development ◽

Function Relationship ◽

Relationship Of ◽

Functional Receptor

Abstract Angiotensin converting enzyme (ACE) is well-known for its role in blood pressure regulation via the renin–angiotensin aldosterone system (RAAS) but also functions in fertility, immunity, haematopoiesis and diseases such as obesity, fibrosis and Alzheimer’s dementia. Like ACE, the human homologue ACE2 is also involved in blood pressure regulation and cleaves a range of substrates involved in different physiological processes. Importantly, it is the functional receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 responsible for the 2020, coronavirus infectious disease 2019 (COVID-19) pandemic. Understanding the interaction between SARS-CoV-2 and ACE2 is crucial for the design of therapies to combat this disease. This review provides a comparative analysis of methodologies and findings to describe how structural biology techniques like X-ray crystallography and cryo-electron microscopy have enabled remarkable discoveries into the structure–function relationship of ACE and ACE2. This, in turn, has enabled the development of ACE inhibitors for the treatment of cardiovascular disease and candidate therapies for the treatment of COVID-19. However, despite these advances the function of ACE homologues in non-human organisms is not yet fully understood. ACE homologues have been discovered in the tissues, body fluids and venom of species from diverse lineages and are known to have important functions in fertility, envenoming and insect–host defence mechanisms. We, therefore, further highlight the need for structural insight into insect and venom ACE homologues for the potential development of novel anti-venoms and insecticides.

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Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-057 ◽

1983 ◽

Vol 61 (6) ◽

pp. 421-427 ◽

Cited By ~ 65

Author(s):

James R. Lepock ◽

Kwan-Hon Cheng ◽

Hisham Al-Qysi ◽

Jack Kruuv

Keyword(s):

Mammalian Cells ◽

Plasma Membranes ◽

Chinese Hamster ◽

Cell Killing ◽

Water Soluble ◽

Growth Data ◽

Protein Fluorescence ◽

Polar Groups ◽

Spin Resonance ◽

Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

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Structure–property–function relationship of fluorescent conjugated microporous polymers

Materials Chemistry Frontiers ◽

10.1039/d0qm00769b ◽

2021 ◽

Author(s):

M. G. Monika Bai ◽

H. Vignesh Babu ◽

V. Lakshmi ◽

M. Rajeswara Rao

Keyword(s):

Long Range ◽

Structure Property ◽

Porous Organic Polymers ◽

Aggregation Induced Emission ◽

Microporous Polymers ◽

Exciton Migration ◽

Wide Range ◽

Function Relationship ◽

Relationship Of ◽

Conjugated Microporous Polymers

Fluorescent porous organic polymers are a unique class of materials owing to their strong aggregation induced emission, long range exciton migration and permanent porosity, thus envisioned to possess a wide range of applications (sensing, OLEDs).

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Structure-Function Relationship of Human Parathyroid Hormone on Vitamin D Receptor Regulation in Osteoblast-Like Cells (ROS 17/2.8)

Vitamin D ◽

10.1515/9783110882513-085 ◽

1994 ◽

pp. 245-246

Keyword(s):

Vitamin D ◽

Parathyroid Hormone ◽

Structure Function ◽

Vitamin D Receptor ◽

Receptor Regulation ◽

Human Parathyroid Hormone ◽

Structure Function Relationship ◽

Function Relationship ◽

Relationship Of

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Morphology-Function Relationship of Thermoelectric Nanocomposite Films from PEDOT:PSS with Silicon Nanoparticles

Advanced Electronic Materials ◽

10.1002/aelm.201700181 ◽

2017 ◽

Vol 3 (8) ◽

pp. 1700181 ◽

Cited By ~ 27

Author(s):

Nitin Saxena ◽

Mihael Čorić ◽

Anton Greppmair ◽

Jan Wernecke ◽

Mika Pflüger ◽

...

Keyword(s):

Silicon Nanoparticles ◽

Nanocomposite Films ◽

Function Relationship ◽

Relationship Of

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High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis

Nature Communications ◽

10.1038/s41467-020-20490-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qiu Sun ◽

Alan Perez-Rathke ◽

Daniel M. Czajkowsky ◽

Zhifeng Shao ◽

Jie Liang

Keyword(s):

High Resolution ◽

Single Cell ◽

3D Models ◽

Computational Method ◽

Midblastula Transition ◽

Genome Wide ◽

Large Ensembles ◽

Chromatin Folding ◽

Function Relationship ◽

Relationship Of

AbstractSingle-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5–6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

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