Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

Abstract

Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cysloop family of ligand-gated ion channels, such as the heteromeric GABA-A abg receptors. The actions of these guanidine compounds on the homomeric GABA-A r1 receptor remains unclear, especially in light of how many GABA-A abg receptor modulators have different effects in the GABA-A r1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A r1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A r1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A r1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A r1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

5-(N, N-Hexamethylene) amiloride is a GABA-Aρ1 receptor positive allosteric modulator

“…The positive allosteric modulatory activity of HMA in the mutant receptor was unexpected, as the same mutation abolished amiloride activity in a previous study. 14 We speculate that the isoleucine to asparagine mutation facilitated HMA interaction, possibly through accommodating the HMA azapine ring within the site. Additionally, the I15'N mutation may alter the activity of the GABA-A r1 receptor and modify the amiloride/amiloride-derivative site, where the site is a distinct site separate from the channel's second transmembrane domain 15' site.…”

“…Previously, we reported that the GABA-A r1 (I15'N) mutant receptor was insensitive to amiloride. 14 This site within the interface of 2 neighboring GABA-A r1 receptor subunits is an appealing candidate for an amiloride derivative site of action. To further probe this site, we assessed the mutant receptor for HMA, phenamil, and benzamil activity.…”

“…The HMA azapine ring may account for the apparent reduction in potency compared to amiloride studied previously. 14 The increase in size and retention of the exposed guanidine group in HMA provided additional information regarding the scale of a potential amiloride/amiloride-derivative binding site within the hGABA-A r1 receptor.…”

“…The ability for guanidine compounds such as HMA, along with the previously studied amiloride, 14 suggest that there may be other compounds that may prove to be efficacious at the GABA-A r1 receptor. Our work has shown that guanidine compounds that have a planar geometry (amiloride and HMA) could be candidates for screening against the GABA-A r1 receptor and the rest of the GABA-A receptor family.…”

“…These solutions were described previously. 14 Test solution pH was checked and if deviated from pH 7.4, was re-established following dilution of the test compounds. An array of capillary tubes was used to expose patched cells to the test solutions.…”