Dataset Information

Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a).

ABSTRACT: Acid-Sensing Ion Channels (ASICs) are gated by extracellular protons and play important roles in physiological and pathological states, such as pain and stroke. ASIC1a and ASIC2a, two of the most highly expressed subunits in the brain, form functional homo- and hetero-meric (ASIC1a/2a) channels. The function of ASIC1a has been widely studied using psalmotoxin (PcTx1), a venom-derived peptide, as an ASIC1a-selective antagonist. Here, using whole-cell patch clamp, we show that PcTx1 has dual actions at ASIC1a/2a. It can either inhibit or potentiate the heteromeric channel, depending on the conditioning and stimulating pHs. Potent inhibition occurs only at conditioning pHs that begin to desensitize the channel (IC50?=?2.9?nM at pH7.0, a threshold pH for desensitization of ASIC1a/2a). By contrast, potent potentiation can occur at the physiological pH in both CHO cells (EC50?=?56.1?nM) and cortical neurons (threshold concentration?

SUBMITTER: Liu Y

PROVIDER: S-EPMC5940917 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a).

Liu Yi Y Hagan Rebecca R Schoellerman Jeffrey J

Scientific reports 20180508 1

Acid-Sensing Ion Channels (ASICs) are gated by extracellular protons and play important roles in physiological and pathological states, such as pain and stroke. ASIC1a and ASIC2a, two of the most highly expressed subunits in the brain, form functional homo- and hetero-meric (ASIC1a/2a) channels. The function of ASIC1a has been widely studied using psalmotoxin (PcTx1), a venom-derived peptide, as an ASIC1a-selective antagonist. Here, using whole-cell patch clamp, we show that PcTx1 has dual actio ...[more]

PMID: 29739981

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Project description:Trimeric acid-sensing ion channels (ASICs) contribute to neuronal signaling by converting extracellular acidification into excitatory sodium currents. Previous work with homomeric ASIC1a implicates conserved leucine (L7') and consecutive glycine-alanine-serine (GAS belt) residues near the middle, and conserved negatively charged (E18') residues at the bottom of the pore in ion permeation and/or selectivity. However, a conserved mechanism of ion selectivity throughout the ASIC family has not been established. We therefore explored the molecular determinants of ion selectivity in heteromeric ASIC1a/ASIC2a and homomeric ASIC2a channels using site-directed mutagenesis, electrophysiology, and molecular dynamics free energy simulations. Similar to ASIC1a, E18' residues create an energetic preference for sodium ions at the lower end of the pore in ASIC2a-containing channels. However, and in contrast to ASIC1a homomers, ion permeation through ASIC2a-containing channels is not determined by L7' side chains in the upper part of the channel. This may be, in part, due to ASIC2a-specific negatively charged residues (E59 and E62) that lower the energy of ions in the upper pore, thus making the GAS belt more important for selectivity. This is confirmed by experiments showing that the L7'A mutation has no effect in ASIC2a, in contrast to ASIC1a, where it eliminated selectivity. ASIC2a triple mutants eliminating both L7' and upper charges did not lead to large changes in selectivity, suggesting a different role for L7' in ASIC2a compared with ASIC1a channels. In contrast, we observed measurable changes in ion selectivity in ASIC2a-containing channels with GAS belt mutations. Our results suggest that ion conduction and selectivity in the upper part of the ASIC pore may differ between subtypes, whereas the essential role of E18' in ion selectivity is conserved. Furthermore, we demonstrate that heteromeric channels containing mutations in only one of two ASIC subtypes provide a means of functionally testing mutations that render homomeric channels nonfunctional.

Project description:ATP-sensitive potassium (K(ATP)) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. K(ATP) channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. K(ATP) channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional K(ATP) channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of K(ATP) channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K(+) currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional K(ATP) channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.

Dataset Information

Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a).

Publications

Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a).

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