Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.

Acid-sensing ion channels (ASICs) are known to be primarily activated by extracellular protons. Recently, we characterized a novel nonproton ligand (2-guanidine-4-methylquinazoline, GMQ), which activates the ASIC3 channel subtype at neutral pH. Using an interactive computational-experimental approach, here we extend our investigation to delineate the architecture of the GMQ-sensing domain in the ASIC3 channels. We first established a GMQ binding mode and revealed that residues Glu-423, Glu-79, Leu-77, Arg-376, Gln-271, and Gln-269 play key roles in forming the GMQ-sensing domain. We then verified the GMQ binding mode using ab initio calculation and mutagenesis and demonstrated the critical role of the above GMQ-binding residues in the interplay among GMQ, proton, and Ca 2؉ in regulating the function of ASIC3. Additionally, we showed that the same residues involved in coordinating GMQ responses are also critical for activation of the ASIC3 E79C mutant by thiol-reactive compound DTNB. Thus, a range of complementary techniques provide independent evidence for the structural details of the GMQ-sensing domain at atomic level, laying the foundation for further investigations of endogenous nonproton ligands and gating mechanisms of the ASIC3 channels. Acid-sensing ion channels (ASICs)4 are proton-gated cation channels (1-3), opening in response to extracellular pH reduction. To date, at least six ASIC subunits encoded by four distinctive genes (asic1, asic2, asic3, and asic4) have been identified (1, 2): 1a, 1b, 2a, 2b, 3, and 4. The first low pH crystal structure of chicken ASIC1 has been resolved (4, 5), revealing significant insights into many fundamental issues about these trimeric ion channels (6). Recently, we discovered a nonproton ligand, 2-guanidine-4-methylquinazoline (GMQ) (see Fig. 1A), which activates ASIC3 channels under neutral pH conditions (7). Remarkably, GMQ-and acid-induced (pH 5.0) currents exhibit distinct kinetics. The acid-induced currents desensitized rapidly, whereas the GMQ-evoked currents showed no desensitization when GMQ was continuously present. Using electrophysiological analysis of a series of ASIC3 channel mutants, we demonstrated that the carboxyl-carboxylate interaction pair, Glu-79 -Glu-423, is crucial for sustained activation of ASIC3 channels at neutral pH. Furthermore, using cysteine substitution mutants, we showed that covalent modification at E79C by DTNB, an Ellman's reagent used to modify and quantitatively detect sulfhydryl groups in proteins, was able to activate the channel, suggesting the importance of the Glu-79 residue in channel gating (7). Although the key role of Glu-79 and Glu-423 in GMQ-ASIC3 interactions has been elucidated, the makeup of this nonproton ligand-sensing domain remains largely unknown. In this study, taking advantage of computational approaches (8) and the available high resolution threedimensional structure of ASIC channels (4, 5), we aimed to delineate the architecture of the nonproton ligand-sensing domain as well as the GMQ binding mode in ASIC3 ...

The budding yeast Sir2 (silent information regulator 2) protein is the founding member of the sirtuin family of NAD-dependent histone/protein deacetylases. Its function in transcriptional silencing requires both the highly conserved catalytic domain and a poorly understood N-terminal regulatory domain (Sir2N). We determined the structure of Sir2 in complex with a fragment of Sir4, a component of the transcriptional silencing complex in Saccharomyces cerevisiae. The structure shows that Sir4 is anchored to Sir2N and contacts the interface between the Sir2N and the catalytic domains through a long loop. We discovered that the interaction between the Sir4 loop and the interdomain interface in Sir2 is critical for allosteric stimulation of the deacetylase activity of Sir2. These results bring to light the structure and function of the regulatory domain of Sir2, and the knowledge should be useful for understanding allosteric regulation of sirtuins in general.