Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

Neuropeptides commonly signal by metabotropic GPCRs. In some mollusks and cnidarians, RFamide neuropeptides mediate fast ionotropic signaling by peptide-gated ion channels that belong to the DEG/ENaC family. Here we describe a neuropeptide system with a dual mode of signaling by both a peptide-gated ion channel and a GPCR. We identified and characterized a peptide-gated channel in the marine annelid Platynereis dumerilii that is specifically activated by Wamide myoinhibitory peptides derived from the same proneuropeptide. The myoinhibitory peptide-gated ion channel (MGIC) belongs to the DEG/ENaC family and is paralogous to RFamide-gated ion channels. Platynereis myoinhibitory peptides also activate a previously described GPCR, MAG. We measured the potency of all Wamides on both MGIC and MAG and identified peptides that preferentially activate one or the other receptor. Analysis of a single-cell transcriptome resource indicates that MGIC and MAG signal in distinct target neurons. The identification of a Wamide-gated ion channel suggests that peptide-gated channels are more diverse and widespread in animals than previously appreciated. The possibility of neuropeptide signaling by both ionotropic and metabotropic receptors to different target cells in the same organism highlights an additional level of complexity in peptidergic signaling networks.-Schmidt, A., Bauknecht, P., Williams, E. A., Augustinowski, K., Gründer, S., Jékely, G. Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry.

Acid-sensing ion channels (ASICs) are widely expressed proton-gated Na(+) channels playing a role in tissue acidosis and pain. A trimeric composition of ASICs has been suggested by crystallization. Upon coexpression of ASIC1a and ASIC2a in Xenopus oocytes, we observed the formation of heteromers and their coexistence with homomers by electrophysiology, but could not determine whether heteromeric complexes have a fixed subunit stoichiometry or whether certain stoichiometries are preferred over others. We therefore imaged ASICs labeled with green and red fluorescent proteins on a single-molecule level, counted bleaching steps from GFP and colocalized them with red tandem tetrameric mCherry for many individual complexes. Combinatorial analysis suggests a model of random mixing of ASIC1a and ASIC2a subunits to yield both 2:1 and 1:2 ASIC1a:ASIC2a heteromers together with ASIC1a and ASIC2a homomers.

Functional and pharmacological characterization of two different ASIC1a/2a heteromers reveals their sensitivity to the spider toxin PcTx1.

Acid Sensing Ion Channels (ASICs) detect extracellular proton signals and are involved in synaptic transmission and pain sensation. ASIC subunits assemble into homo- and heteromeric channels composed of three subunits. Single molecule imaging revealed that heteromers composed of ASIC1a and ASIC2a, which are widely expressed in the central nervous system, have a flexible 2:1/1:2 stoichiometry. It was hitherto not possible, however, to functionally differentiate these two heteromers. To have a homogenous population of ASIC1a/2a heteromers with either 2:1 or 1:2 stoichiometry, we covalently linked subunits in the desired configuration and characterized their functional properties in Xenopus oocytes. We show that the two heteromers have slightly different proton affinity, with an additional ASIC1a subunit increasing apparent affinity. Moreover, we found that zinc, which potentiates ASIC2a-containing ASICs but not homomeric ASIC1a, potentiates both heteromers. Finally, we show that PcTx1, which binds at subunit-subunit interfaces of homomeric ASIC1a, inhibits both heteromers suggesting that ASIC2a can also contribute to a PcTx1 binding site. Using this functional fingerprint, we show that rat cortical neurons predominantly express the ASIC1a/2a heteromer with a 2:1 stoichiometry. Collectively, our results reveal the contribution of individual subunits to the functional properties of ASIC1a/2a heteromers.

Toxin binding reveals two open state structures for one acid-sensing ion channel.

Of the three principal conformations of acid-sensing ion channels (ASICs)--closed, open and desensitized--only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.