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.
Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Niko
N
Joeres
Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Katrin
K
Augustinowski
Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Andreas
A
Neuhof
Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Marc
M
Assmann
Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany.
Stefan
S
Gründer
D062646:Acid Sensing Ion Channel Blockers / Q000494:pharmacology*; D062565:Acid Sensing Ion Channels / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002540:Cerebral Cortex / Q000166:cytology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013111:Spider Venoms / Q000494:pharmacology*; D014981:Xenopus
D062646:Acid Sensing Ion Channel Blockers; D062565:Acid Sensing Ion Channels; C568268:Asic1 protein, rat; C568425:Asic2 protein, rat; C413209:PcTX1 protein, Psalmopoeus cambridgei; D010455:Peptides; D021122:Protein Subunits; D013111:Spider Venoms