scholarly journals Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology

2016 ◽  
Vol 147 (6) ◽  
pp. 485-496 ◽  
Author(s):  
Maria Barthmes ◽  
Jun Liao ◽  
Youxing Jiang ◽  
Andrea Brüggemann ◽  
Christian Wahl-Schott
Keyword(s):  
Functional Data ◽  
Membrane Transporters ◽  
Transporter Proteins ◽  
Supported Membrane ◽  
Functional Studies ◽  
Sodium Calcium ◽  
Calcium Exchange ◽  
Electrophysiological Characterization ◽  
Insight Into

Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.

scholarly journals Unlocking the Reversal Potential of Solid Supported Membrane Electrophysiology to Determine Transport Stoichiometry

2020 ◽  
Author(s):  
Nathan E. Thomas ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Reversal Potential ◽  
New Technique ◽  
Supported Membrane ◽  
Functional Studies ◽  
A New Technique ◽  
And Function ◽  
Improved Methods ◽  
Insight Into ◽  
General Method

AbstractTransport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine transport stoichiometry are required to accurately characterize transporter activity. Reversal potential was previously shown to be a reliable, general method for determining the transport stoichiometry of ion-coupled transporters (Fitzgerald & Mindell, 2017). Here, we develop a new technique for measuring transport stoichiometry with greatly improved throughput using solid supported membrane electrophysiology (SSME). Using this technique, we are able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx. Our SSME method requires only small amounts of transporter and provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

scholarly journals Characterization of sodium-calcium exchange in rabbit renal arterioles

1996 ◽  
Vol 50 (6) ◽  
pp. 1856-1862 ◽  
Author(s):  
Beth C. Fowler ◽  
Pamela K. Carmines ◽  
Lawrence D. Nelson ◽  
P. Darwin Bell
Keyword(s):  
Sodium Calcium ◽  
Calcium Exchange ◽  
Sodium Calcium Exchange

scholarly journals Biosynthesis of UDP-xylose and UDP-arabinose in Sinorhizobium meliloti 1021: first characterization of a bacterial UDP-xylose synthase, and UDP-xylose 4-epimerase

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 260-269 ◽  
Author(s):  
Xiaogang Gu ◽  
Sung G. Lee ◽  
Maor Bar-Peled
Keyword(s):  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Glucuronic Acid ◽  
Functional Studies ◽  
H Nmr ◽  
New Information ◽  
Genes Encoding ◽  
Rhizobial Species ◽  
Insight Into

Sinorhizobium meliloti is a soil bacterium that fixes nitrogen after being established inside nodules that can form on the roots of several legumes, including Medicago truncatula. A mutation in an S. meliloti gene (lpsB) required for lipopolysaccharide synthesis has been reported to result in defective nodulation and an increase in the synthesis of a xylose-containing glycan. Glycans containing xylose as well as arabinose are also formed by other rhizobial species, but little is known about their structures and the biosynthetic pathways leading to their formation. To gain insight into the biosynthesis of these glycans and their biological roles, we report the identification of an operon in S. meliloti 1021 that contains two genes encoding activities not previously described in bacteria. One gene encodes a UDP-xylose synthase (Uxs) that converts UDP-glucuronic acid to UDP-xylose, and the second encodes a UDP-xylose 4-epimerase (Uxe) that interconverts UDP-xylose and UDP-arabinose. Similar genes were also identified in other rhizobial species, including Rhizobium leguminosarum, suggesting that they have important roles in the life cycle of this agronomically important class of bacteria. Functional studies established that recombinant SmUxs1 is likely to be active as a dimer and is inhibited by NADH and UDP-arabinose. SmUxe is inhibited by UDP-galactose, even though this nucleotide sugar is not a substrate for the 4-epimerase. Unambiguous evidence for the conversions of UDP-glucuronic acid to UDP-α-d-xylose and then to UDP-β-l-arabinose (UDP-arabinopyranose) was obtained using real-time 1H-NMR spectroscopy. Our results provide new information about the ability of rhizobia to form UDP-xylose and UDP-arabinose, which are then used for the synthesis of xylose- and arabinose-containing glycans.

scholarly journals Electrophysiological characterization of Af-Amt1 on a solid supported membrane

2012 ◽  
Vol 1817 ◽  
pp. S38-S39
Author(s):  
Tobias Wacker ◽  
Juan J. Garcia-Celma ◽  
Philipp Lewe ◽  
Susana L.A. Andrade
Keyword(s):  
Supported Membrane ◽  
Electrophysiological Characterization

Skeletal elements of crinoid echinoderms: High-resolution structural and microanalytical results

1983 ◽  
Vol 41 ◽  
pp. 194-195
Author(s):  
D. F. Blake ◽  
L. F. Allard ◽  
D. R. Peacor
Keyword(s):  
High Resolution ◽  
Marine Invertebrates ◽  
Sea Urchins ◽  
Structural Features ◽  
Sea Stars ◽  
High Resolution Tem ◽  
Relationship Of ◽  
The Relationship ◽  
Insight Into

Echinodermata is a phylum of marine invertebrates which has been extant since Cambrian time (c.a. 500 m.y. before the present). Modern examples of echinoderms include sea urchins, sea stars, and sea lilies (crinoids). The endoskeletons of echinoderms are composed of plates or ossicles (Fig. 1) which are with few exceptions, porous, single crystals of high-magnesian calcite. Despite their single crystal nature, fracture surfaces do not exhibit the near-perfect {10.4} cleavage characteristic of inorganic calcite. This paradoxical mix of biogenic and inorganic features has prompted much recent work on echinoderm skeletal crystallography. Furthermore, fossil echinoderm hard parts comprise a volumetrically significant portion of some marine limestones sequences. The ultrastructural and microchemical characterization of modern skeletal material should lend insight into: 1). The nature of the biogenic processes involved, for example, the relationship of Mg heterogeneity to morphological and structural features in modern echinoderm material, and 2). The nature of the diagenetic changes undergone by their ancient, fossilized counterparts. In this study, high resolution TEM (HRTEM), high voltage TEM (HVTEM), and STEM microanalysis are used to characterize tha ultrastructural and microchemical composition of skeletal elements of the modern crinoid Neocrinus blakei.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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