Abstract
Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. In this study, we reported that AMI paradoxically opened homomeric ASIC3 and heteromeric ASIC3 plus ASIC1b channels at neutral pH and synergistically enhanced channel activation induced by mild acidosis (pH 7.2 to 6.8). The characteristic profile of AMI stimulation of ASIC3 channels was reminiscent of the channel activation by the newly identified nonproton ligand, 2-guanidine-4-methylquinazoline. Using site-directed mutagenesis, we showed that ASIC3 activation by AMI, but not its inhibitory effect, was dependent on the integrity of the nonproton ligand sensing domain in ASIC3 channels. Moreover, the structure-activity relationship study demonstrated the differential requirement of the 5-amino group in AMI for the stimulation or inhibition effect, strengthening the different interactions within ASIC3 channels that confer the paradoxical actions of AMI. Furthermore, using covalent modification analyses, we provided strong evidence supporting the nonproton ligand sensing domain is required for the stimulation of ASIC3 channels by AMI. Finally, we showed that AMI causes pain-related behaviors in an ASIC3-dependent manner. These data reinforce the idea that ASICs can sense nonproton ligands in addition to protons. The results also indicate caution in the use of AMI for studying ASIC physiology and in the development of AMI-derived ASIC inhibitors for treating pain syndromes.
Highlights
Acid-sensing ion channels (ASICs) are activated by extracellular protons and are inhibited by amiloride
We demonstrate that AMI activates and sensitizes ASIC3 channels, AMI has been widely used as an inhibitor for many ion channels and transporters, such as Naϩ/Hϩ exchanger [32], low threshold calcium channel [33], glycine receptors [34], and the epithelial sodium channel/degenerin family [23], including ASIC channels [2]
Using mutagenesis and covalent modification analysis, we have revealed that the nonproton ligand sensing domain lined by residues around Glu-423 and Glu-79 [26] is required for the stimulatory action of AMI
Summary
Acid-sensing ion channels (ASICs) are activated by extracellular protons and are inhibited by amiloride. Acid-sensing ion channels (ASICs), which belong to the epithelial sodium channel/degenerin family, are activated by extracellular protons and are inhibited by amiloride (AMI), an important pharmacological tool for studying all known members of epithelial sodium channel/degenerin. AMI is a common blocker for all members of the epithelial sodium channel/degenerin family [23] It inhibits acid-induced currents from most known ASIC channels [1,2,3], and the AMI blockade is often used to demonstrate the physiological function of ASICs. A premise for its use in the study of ASIC physiology is that AMI does not directly or indirectly affect ASIC functions other than inhibiting channel activity; some studies have suggested that AMI may paradoxically stimulate ASICs. In addition to its blocking activity, AMI was found to stimulate the “Deg” mutation (at Gly-430) but not the wild-type (WT) ASIC2a channel [24] through exposing extracellular membrane-proximal residues in a similar fashion as that by protons [25]. Using site-directed mutagenesis, electrophysiological recording, and covalent modification, we demonstrate that the nonproton ligand sensing domain is required for stimulation of ASIC function by AMI
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