DOI: 10.5061/dryad.p2ngf1vzc
title: Data from: Redox state of NAD modulates the activation of Na-bicarbonate cotransporter NBCe1-B by IRBIT and L-IRBIT

description: 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.
description: 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
 (<em>I<sub>ND96</sub></em>) and CO2/HCO3-
 solutions (<em>I<sub>HCO3</sub></em>) represented
 the net HCO3- dependent current mediated by NBCe1
 (<em>I<sub>NBC</sub></em>). The slope-conductance
 of <em>I<sub>NBC</sub></em> 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.
description: # 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](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.