Data from: Redox state of NAD modulates the activation of Na-bicarbonate cotransporter NBCe1-B by IRBIT and L-IRBIT
Data files
Mar 22, 2024 version files 155.89 KB
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Figure_1.xlsx
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Figure_2.xlsx
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Figure_S2.xlsx
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README.md
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Abstract
NAD is known as a coenzyme for many enzymes catalyzing redox reactions or as a substrate for enzymes for second messenger cADP-ribose generation or protein deacetylation. IRBIT and L-IRBIT are structurally conserved with dehydrogenases but lacking catalytic activity. By protein interaction, the IRBITs instead modulate many proteins of fundamental biological importance. Among these is the Na+-HCO3− cotransporter NBCe1-B, which plays a central role in intracellular pH (pHi) regulation and epithelial electrolyte transport. Here, we demonstrate that NAD modulates NBCe1-B activation by serving as a cofactor of the IRBITs. Blocking NAD salvage pathway decreases NBCe1-B activation by the IRBITs. NAD+ administration enhances, whereas NADH decreases NBCe1-B activity. Our study reveals a new role of NAD and greatly expands our appreciation of NAD biology. Because NAD redox state fluctuates greatly with metabolic status, our work provides insight into how, via IRBITs, energy metabolism could affect pHi regulation and many other IRBIT-dependent processes.
README: Data from: Redox state of NAD modulates the activation of Na-bicarbonate cotransporter NBCe1-B by IRBIT and L-IRBIT
https://doi.org/10.5061/dryad.p2ngf1vzc
Description of the data and file structure
The study examined the effect of nicotinamide adenine dinucleotide on the activation of Na+ /HCO3– cotransporter NBCe1-B by IRBIT or L-IRBIT.
The data spreadsheet contains data for the bar graphs presented in the manuscript, including:
(1) the data about NBCe1 activity, indexed by the slope conductance (GNBC, in μS), in Figures 1D, 1E, 1F, 2A, 2B, 2C, 3A, 3B, 4F, 5B, 5E, S2C, S4A, S4B, S4C, S4D, S5A, and S5B. Conductance was calculated based on the NBCe1-mediated currents recorded by voltage clamp with Xenopus laevis oocytes.
(2) the data of the cellular levels (in pmol/cell) of NAD+ and NADH in Figures 1G, 1H, 1I, and S6A. Figures 1J and S6B represent the ratio of NAD+/NADH. Levels of NAD+ and NADH in oocytes were determined by using WST-8 colorimetric assay.
(3) the data of relative expression levels of NBCe1 and IRBIT in Figures S3A, S3B, S3C, and S3D. The expression of NBCe1 or IRBIT was assayed by western blotting and quantified by densitometry analysis with Image J.
Methods
NBCe1 activity was determined by using two-electrode voltage clamp with an OC-725C oocyte clamp (Warner Instruments, Hamden, Ct, USA) controlled by pCLAMP10.2 (Molecular Devices, San Jose, CA, USA). The oocyte was first perfused with ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH7.50) and then with CO2/HCO3- solution (in mM: 63 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, followed by titrarion to pH 7.50, and the addition of 33 NaHCO2, then bubbled with 5% CO2 balanced with 95% N2). Voltage clamp recordings were acquired in both ND96 and CO2/HCO3- solutions. The difference between the current in ND96 (IND96) and CO2/HCO3- solutions (IHCO3) represented the net HCO3- dependent current mediated by NBCe1 (INBC). The slope-conductance of INBC between ±40mV was used as an index for NBCe1 activity.
The expression level data of NBCe1 and IRBIT was determined by western blotting assay and quantified by densitometric assay with ImageJ.
The cellular level of NAD+ and NADH was determined by using a commercially available WST-8 colorimetric assay kits.