Blecker, L. M. | Search bioRxiv

bioRxiv2026

The proximal N-terminus of IRAG is required for potentiation of HCN4 channels

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 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, 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, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

biophysics↗

bioRxiv2021

The diazepam binding inhibitors modulation of the GABA-A receptor is subunit-dependent

First synthesized in the 1950s, benzodiazepines are widely prescribed drugs that exert their anxiolytic, sedative and anticonvulsant actions by binding to GABA-A receptors, the main inhibitory ligand-gated ion channel in the brain. Scientists have long theorized that there exists an endogenous benzodiazepine, or endozepine, in the brain. While there is indirect evidence suggesting a peptide, the diazepam binding inhibitor, is capable of modulating the GABA-A receptor, direct evidence of the modulatory effects of the diazepam binding inhibitor is limited. Here we take a reductionist approach to understand how purified diazepam binding inhibitor interacts with and affects GABA-A receptor activity. We used two-electrode voltage clamp electrophysiology to study how the effects of diazepam binding inhibitor vary with GABA-A receptor subunit composition, and found that GABA-evoked currents from 3-containing GABA-A receptors are weakly inhibited by the diazepam binding inhibitor, while currents from 5-containing receptors are positively modulated. We also used in silico protein-protein docking to visualize potential diazepam binding inhibitor/GABA-A receptor interactions that revealed diazepam binding inhibitor bound at the benzodiazepine /{gamma} binding site interface, which provides a structural framework for understanding diazepam binding inhibitor effects on GABA-A receptors. Our results provide novel insights into mechanisms underlying how the diazepam binding inhibitor modulates GABA-mediated inhibition in the brain.

neuroscience↗