Physiologically relevant acid-sensing ion channel (ASIC) 2a/3 heteromers have a 1:2 stoichiometry.

Acid-sensing ion channels (ASICs) sense extracellular protons and are involved in synaptic transmission and pain sensation. ASIC1a and ASIC3 are the ASIC subunits with the highest proton sensitivity. ASIC2a in contrast has low proton sensitivity but increases the variability of ASICs by forming heteromers with ASIC1a or ASIC3. ASICs are trimers and for the ASIC1a/2a heteromer it has been shown that subunits randomly assemble with a flexible 1:2/2:1 stoichiometry. Both heteromers have almost identical proton sensitivity intermediate between ASIC1a and ASIC2a. Here, we investigated the stoichiometry of the ASIC2a/3 heteromer. Using electrophysiology, we extensively characterized, first, cells expressing ASIC2a and ASIC3 at different ratios, second, concatemeric channels with a fixed subunit stoichiometry, and, third, channels containing loss-of-functions mutations in specific subunits. Our results conclusively show that only ASIC2a/3 heteromers with a 1:2 stoichiometry had a proton-sensitivity intermediate between ASIC2a and ASIC3. In contrast, the proton sensitivity of ASIC2a/3 heteromers with a 2:1 stoichiometry was strongly acid-shifted by more than one pH unit, which suggests that they are not physiologically relevant. Together, our results reveal that the proton sensitivity of the two ASIC2a/3 heteromers is clearly different and that ASIC3 and ASIC1a make remarkably different contributions to heteromers with ASIC2a.

The Conorfamide RPRFa Stabilizes the Open Conformation of Acid-Sensing Ion Channel 3 via the Nonproton Ligand-Sensing Domain.

Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na+ channel with important roles in pain. ASIC3 quickly desensitizes in less than a second, limiting its capacity to sense sustained acidosis during pain. RFamide neuropeptides are modulators of ASIC3 that slow its desensitization and induce a variable sustained current. The molecular mechanism of slowed desensitization and the RFamide binding site on ASIC3 are unknown. RPRFamide, a RFamide from the venom of a cone snail, has a comparatively high affinity for ASIC3 and strongly slows its desensitization. Here we show that covalent binding of a UV-sensitive RPRFamide variant to ASIC3 prevents desensitization, suggesting that RPRFamide has to unbind from ASIC3 before it can desensitize. Moreover, we show by in silico docking to a homology model of ASIC3 that a cavity in the lower palm domain, which is also known as the nonproton ligand-sensing domain, is a potential binding site of RPRFamide. Finally, using extensive mutagenesis of residues lining the nonproton ligand-sensing domain, we confirm that this domain is essential for RPRFamide modulation of ASIC3. As comparative analysis of ASIC crystal structures in the open and in the desensitized conformation suggests that the lower palm domain contracts during desensitization, our results collectively suggest that RPRFamide, and probably also other RFamide neuropeptides, bind to the nonproton ligand-sensing domain to stabilize the open conformation of ASIC3.

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.

The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.