Structure and analysis of nanobody binding to the human ASIC1a ion channel

Wu, Yangyu; Chen, Zhuyuan; Sigworth, Fred J.; Canessa, Cecilia M.

doi:10.7554/elife.67115

Cited by 17 publications

(9 citation statements)

References 44 publications

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“…Interestingly, two recent structures of ASIC1a from both chicken and human show endogenous lipid present between TM1 and TM2 of the same subunit immediately adjacent to our putative binding site ( Yoder and Gouaux, 2020 ; Wu et al, 2021 ). Complicating our hypothesis, R64 points into the pore of the channel in the solved structures ( Jasti et al, 2007 ; Baconguis et al, 2014 ; Yoder et al, 2018 ).…”

Section: Discussionmentioning

confidence: 86%

Structural determinants of acid-sensing ion channel potentiation by single chain lipids

Klipp

Bankston

2022

Journal of General Physiology

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Acid-sensing ion channels (ASICs) are sensitized to activation by inflammatory mediators such as the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). Previous work has shown that AA can potentiate ASIC currents at subsaturating proton concentrations, but the structural mechanisms of this change in gating are not understood. Here we show that PUFAs cause multiple gating changes in ASIC3, including shifting the pH dependence of activation, slowing the rate of desensitization, and increasing the current even at a saturating pH. The impact on gating depends on the nature of both the head and tail of the lipid, with the head group structure primarily determining the magnitude of the effect on the channel. An N-acyl amino acid (NAAA), arachidonyl glycine (AG), is such a strong regulator that it can act as a ligand at neutral pH. Mutation of an arginine in the outer segment of TM1 (R64) eliminated the effect of docosahexaenoic acid (DHA) even at high concentrations, suggesting a potential interaction site for the lipid on the channel. Our results suggest a model in which PUFAs bind to ASICs via both their tail group and an electrostatic interaction between the negatively charged PUFA head group and the positively charged arginine side chain. These data provide the first look at the structural features of lipids that are important for modulating ASICs and suggest a potential binding site for PUFAs on the channel.

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Section: Discussionmentioning

confidence: 86%

Structural determinants of acid-sensing ion channel potentiation by single chain lipids

Klipp

Bankston

2022

Journal of General Physiology

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“…Interestingly, two recent structures of ASIC1a from both chicken and human shows endogenous lipid present between TM1 and TM2 of the same subunit immediately adjacent to our putative binding site 21,39 . Complicating our hypothesis, R64 points into the pore of the channel in the solved structures [22][23][24] .…”

Section: Discussionmentioning

confidence: 83%

Structural determinants of Acid-sensing ion channel potentiation by single chain lipids

Klipp

Bankston

2021

Preprint

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Acid-sensing ion channels (ASICs) are thought to be endogenous sensors of acidic pain in inflammatory pathways. It has previously been demonstrated that arachidonic acid (AA), a pain and inflammation promoting molecule, potentiates ASICs. However, a mechanistic understanding of how AA regulates ASICs is lacking. Furthermore, little is known regarding modulation by other polyunsaturated fatty acids (PUFAs). Here we show that PUFAs stabilize the open state of the channel by shifting the pH dependence of activation to more alkaline values, increasing max conductance, and slowing channel desensitization. We examine the effects of 35 PUFAs/PUFA derivatives and show that ASICs can be more strongly potentiated by these lipids than was originally seen for AA. In fact, arachidonoyl glycine (AG) can act as a ligand and activate the channel in the absence of acidic pH. We find that the strength of potentiation is critically dependent upon a negatively charged PUFA head group as well as both the length and the number of doubles bonds in the acyl tail. PUFA-induced shifts in the pH dependence of activation could be eliminated upon mutation of a highly conserved, positively charged arginine in the outer segment of TM1 (R64). Combined our results suggest a hypothesis whereby an electrostatic interaction between the charged PUFA head group and the positively charged arginine side chain potentiates ASIC currents by stabilizing the open state of the channel. This work uncovers a novel putative lipid binding site on ASICs and provides the structural basis for future development of compounds targeting ASICs.

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“…Furthermore, the molecular knowledge of ASIC gating and interaction with ASIC-targeting toxin inhibitors now allows to design new molecules like C5b and to predict their pharmacological potential and possibly their therapeutic relevance. Other putative applications of these peptides are also emerging, such as the design of a fusion protein incorporating an alpaca-derived nanobody targeting hASIC1a and the peptide toxin PcTx1 to achieve potent and, contrary to PcTx1 alone, more stable inhibition of ASIC1a (~84–87% current inhibition), improving the potency of PcTx1 and its potential applications [ 16 ].…”

Section: Discussionmentioning

confidence: 99%

“…An “acidic pocket” containing several pairs of acidic amino acids is present at the interface of each subunit and was proposed to be one of the pH sensors of the channel, whereas cations may access the ion channel by lateral fenestrations, then moving into a broad extracellular vestibule just above the inactivation gate and the selectivity filter (i.e., the structural element in the narrowest part of the pore that determines ionic selectivity) ( Figure 1 D) [ 12 , 15 ]. The most noticeable structural difference between human (h) ASIC1a (hASIC1a) and cASIC1 is a longer loop that extends down from the α4-helix to the tip of the thumb, due to two extra amino acids (D298 and L299) absent in all other ASIC isoforms [ 16 ].…”

Section: Molecular and Functional Properties Of Asicsmentioning

confidence: 99%

“…The structures of the three conformational states involved in H + -dependent gating of homotrimeric cASIC1 were solved by X-ray crystallography and Cryo-EM: resting state [ 50 ] ( Figure 3 A), open state [ 51 ] ( Figure 3 B) and desensitized state [ 12 ] ( Figure 3 C). Cryo-EM structures of the hASIC1a in its closed state have also been solved, in complex with the toxin mambalgin-1 [ 52 ], and in complex with a specific nanobody Nb.C1 [ 16 ]. At the interface of each subunit of the trimeric channel, an acidic pocket is formed by intra-subunit contacts between the thumb, the β-ball and the finger domains, together with residues from the palm domain on the adjacent subunit ( Figure 1 A,D and Figure 3 ) [ 12 , 50 ].…”

Section: Molecular and Functional Properties Of Asicsmentioning

confidence: 99%

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Mechanisms of Action of the Peptide Toxins Targeting Human and Rodent Acid-Sensing Ion Channels and Relevance to Their In Vivo Analgesic Effects

Verkest

Salinas

Diochot

et al. 2022

Toxins

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Acid-sensing ion channels (ASICs) are voltage-independent H+-gated cation channels largely expressed in the nervous system of rodents and humans. At least six isoforms (ASIC1a, 1b, 2a, 2b, 3 and 4) associate into homotrimers or heterotrimers to form functional channels with highly pH-dependent gating properties. This review provides an update on the pharmacological profiles of animal peptide toxins targeting ASICs, including PcTx1 from tarantula and related spider toxins, APETx2 and APETx-like peptides from sea anemone, and mambalgin from snake, as well as the dimeric protein snake toxin MitTx that have all been instrumental to understanding the structure and the pH-dependent gating of rodent and human cloned ASICs and to study the physiological and pathological roles of native ASICs in vitro and in vivo. ASICs are expressed all along the pain pathways and the pharmacological data clearly support a role for these channels in pain. ASIC-targeting peptide toxins interfere with ASIC gating by complex and pH-dependent mechanisms sometimes leading to opposite effects. However, these dual pH-dependent effects of ASIC-inhibiting toxins (PcTx1, mambalgin and APETx2) are fully compatible with, and even support, their analgesic effects in vivo, both in the central and the peripheral nervous system, as well as potential effects in humans.

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Structure and analysis of nanobody binding to the human ASIC1a ion channel

Cited by 17 publications

References 44 publications

Structural determinants of acid-sensing ion channel potentiation by single chain lipids

Structural determinants of acid-sensing ion channel potentiation by single chain lipids

Structural determinants of Acid-sensing ion channel potentiation by single chain lipids

Mechanisms of Action of the Peptide Toxins Targeting Human and Rodent Acid-Sensing Ion Channels and Relevance to Their In Vivo Analgesic Effects

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