Desensitization Of Acid-sensing Ion Channels Research Articles

Protein semisynthesis underscores the role of a conserved lysine in activation and desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are trimeric ion channels that open a cation-conducting pore in response to proton binding. Excessive ASIC activation during prolonged acidosis in conditions such as inflammation and ischemia is linked to pain and stroke. A conserved lysine in the extracellular domain (Lys211 in mASIC1a) is suggested to play a key role in ASIC function. However, the precise contributions are difficult to dissect with conventional mutagenesis, as replacement of Lys211 with naturally occurring amino acids invariably changes multiple physico-chemical parameters. Here, we study the contribution of Lys211 to mASIC1a function using tandem protein trans-splicing (tPTS) to incorporate non-canonical lysine analogs. We conduct optimization efforts to improve splicing and functionally interrogate semisynthetic mASIC1a. In combination with molecular modeling, we show that Lys211 charge and side-chain length are crucial to activation and desensitization, thus emphasizing that tPTS can enable atomic-scale interrogations of membrane proteins in live cells.

Novel conorfamides from Conus austini venom modulate both nicotinic acetylcholine receptors and acid-sensing ion channels

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family.Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.

Inhalational anesthetics accelerate desensitization of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by extracellular acidification. Inhibiting ASICs is neuroprotective in mouse models of ischemic stroke. As inhalational anesthetics interact with many ion channels and as some of them have neuroprotective effects, we hypothesized that inhalational anesthetics modulate ASICs. We expressed different homo- and heteromeric ASICs heterologously in Xenopus oocytes. We co-applied with acidic pH the halogenated inhalational anesthetics sevoflurane, desflurane, and isoflurane and the noble gases xenon and argon at concentrations that are roughly equivalent to their minimal alveolar concentrations and analyzed their effect on current kinetics and amplitude. Sevoflurane, desflurane, and isoflurane as well as xenon and argon accelerated by a factor of ∼1.5 channel desensitization of the main ASICs of the central nervous system: homomeric ASIC1a and heteromeric ASIC1a/2a and ASIC1a/2b. Moreover, they decreased current amplitudes by ∼25%. For example, isoflurane accelerated desensitization of homomeric ASIC1a from 1.0 ± 0.4 s (mean ± SD) to 0.6 ± 0.2 s (n = 12; p = 0.0003) and decreased current amplitudes from 12.1 ± 7.5 μA to 9.3 ± 5.6 μA (n = 12; p = 0.0009). While inhalational anesthetics had similar effects on homomeric ASIC3, desensitization of ASIC1b was only accelerated by halogenated anesthetics but not noble gases; desensitization of homomeric ASIC2a was not modulated. In summary, we found a significant modulation of ASICs by different inhalational anesthetics. We conclude that ASICs should be considered as relevant targets of inhalation anesthetics.

ASIC3 fine-tunes bladder sensory signaling.

Acid-sensing ion channels (ASICs) are trimeric proton-activated, cation-selective neuronal channels that are considered to play important roles in mechanosensation and nociception. Here we investigated the role of ASIC3, a subunit primarily expressed in sensory neurons, in bladder sensory signaling and function. We found that extracellular acidification evokes a transient increase in current, consistent with the kinetics of activation and desensitization of ASICs, in ~25% of the bladder sensory neurons harvested from both wild-type (WT) and ASIC3 knockout (KO) mice. The absence of ASIC3 increased the magnitude of the peak evoked by extracellular acidification and reduced the rate of decay of the ASIC-like currents. These findings suggest that ASICs are assembled as heteromers and that the absence of ASIC3 alters the composition of these channels in bladder sensory neurons. Consistent with the notion that ASIC3 serves as a proton sensor, 59% of the bladder sensory neurons harvested from WT, but none from ASIC3 KO mice, fired action potentials in response to extracellular acidification. Studies of bladder function revealed that ASIC3 deletion reduces voiding volume and the pressure required to trigger micturition. In summary, our findings indicate that ASIC3 plays a role in the control of bladder function by modulating the response of afferents to filling.