Data from: The proximal N-terminus of IRAG is required for potentiation of HCN4 channels
Data files
Sep 09, 2026 version files 12.73 KB
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Dataforbleckerfinalboltzmann.csv
7.45 KB
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Dataforbleckerfinalkinetics.csv
1.88 KB
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README.md
3.40 KB
Abstract
The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 as well as a unique set of residues in the cAMP transduction center of HCN4, which is comprised of the N-terminal HCN domain (HCND), the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology on mutated IRAG and HCN4 channels, we determined the structural domains required for the regulation of HCN4 by IRAG. The data here represents the analysis of current vs voltage measurements where currents are elicited from HEK cells expressing the indicated versions of HCN4 and IRAG. We elicited currents by stepping from a holding potential of 0 mV down to successively more hyperpolarized potentials between -50 mV and -170 mV in 3 s voltage steps, and then stepping to -50 mV to elicit deactivating tail currents. Channel activation was determined from peak tail current amplitudes at –50 mV. Normalized tail current-voltage relationships were fit by single Boltzmann equations to yield values for the midpoint activation voltage (V1/2) and slope factor which are provided in this data set. In addition, time to half activation was measured which is the time point when the current has reached half of its peak value. Finally, the tau of channel deactivation was determined but fitting a single exponential to the decay of the tail currents as detailed in the manuscript. These data revealed that, similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also like LRMP, two point mutations in the C-linker region, near where the C-linker of one subunits contacts its neighboring subunit, were able to eliminate the effect of IRAG. Taken together these findings suggest a model whereby IRAG and LRMP make complex interactions with a number of intracellular domains of HCN4. For both proteins, regulation of HCN4 requires a portion of the N-terminus and at least some portions of the structural domains required to convert the binding of cAMP to the cyclic nucleotide binding domain (CNBD) to a change in channel gating.
Dataset DOI: 10.5061/dryad.2280gb67t
Description of the data and file structure
Patch clamp experiments were performed in either transiently transfected HEK293 cells, an HCN4 stable line in HEK293 cells (Zong et al., 2012), or a stable line in HEK293 cells of double mutant HCN4 P545A/T547F (HCN4 PT/AF).Cells were plated on sterile ECL-coated glass coverslips 24–48 hours prior to experiments. Cells on coverslip shards were transferred to the recording chamber and perfused (~0.5–1 mL/min) with extracellular solution containing (in mM): 30 KCl, 115 NaCl, 1 MgCl2, 1.8 CaCl2, 5.5 glucose, and 5 HEPES. Transiently transfected cells were identified by green fluorescence. Patch pipettes were pulled from borosilicate glass to a resistance of 1.0–3.0 MOhm when filled with intracellular solution containing (in mM): 130 K-Aspartate, 10 NaCl, 1 EGTA, 0.5 MgCl2, 5 HEPES, and 2 Mg-ATP. One mM cAMP was added to the intracellular solution as indicated.
All data shown here are the fits to individual current recordings. Channel activation was determined from peak tail current amplitudes at –50 mV following 3 s hyperpolarizing pulses to membrane potentials between –50 mV and –170 mV from a holding potential of 0 mV. Normalized tail current-voltage relationships were fit by single Boltzmann equations to yield values for the midpoint activation voltage (V1/2) and slope factor. For the first file the fits are to a single Boltzmann equation with two free parameters (V1/2 and slope). These two values are reported for each individual recording and the averages and SEM's are shown as well. Data comparisons are made in groups of four as detailed in the manuscript. In short, each condition has a control, +cAMP, +IRAG, and a +IRAG+cAMP condition. For the recordings with mutated IRAG protein, the +IRAG and +IRAG+cAMP conditions are compated to the WT HCN/WT IRAG data highlighted in orange. For all others, the relavent group of four is shaded the same color.
For the second file, there are two pieces of data reported. First is the time to half activation which represents the time point when the current has reached half of the peak value. Second is the tau of deactivation which is determined by a single exponential fit of the tail currents as detailed in the manuscript. There are only two different sets of comparisons for these data and they are grouped via the different shading on the table.
For all datasets, often the same number of recording were not made for each condition. For the empty cells, we have replaced the empty cell with NaN (not a number) which indicates no data for this cell. We elected to use NaN because it does not interfere with downstream analysis using a number of softwares (e.g., Matlab).
Files and variables
File: Dataforbleckerfinalboltzmann.csv
File: Dataforbleckerfinalkinetics.csv
Description: Values from the fit of GV curves from "The proximal N-terminus of IRAG is required for potentiation of HCN4 channels".
Variables
- V1/2 (sometimes Vhalf); in mV (millivolts)
- slope factor; unit-less
- tau of deactivation - in ms (milliseconds)
- time to half activation - in ms (milliseconds)
Code/software
Excel