Molecular determinants of ASIC1 modulation by divalent cations.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. ASIC gating is modulated by divalent cations as well as small molecules; however, the molecular determinants of gating modulation by divalent cations are not well understood. Previously, we identified two small molecules that bind to ASIC1a at a novel site in the acidic pocket and modulate ASIC1 gating in a manner broadly resembling divalent cations, raising the possibility that these small molecules may help to illuminate the molecular determinants of gating modulation by divalent cations. Here, we examined how these two groups of modulators might interact as well as mutational effects on ASIC1a gating and its modulation by divalent cations. Our results indicate that binding of divalent cations to an acidic pocket site plays a key role in gating modulation of the channel.

Molecular mechanism and structural basis of small-molecule modulation of the gating of acid-sensing ion channel 1.

Acid-sensing ion channels (ASICs) are proton-gated cation channels critical for neuronal functions. Studies of ASIC1, a major ASIC isoform and proton sensor, have identified acidic pocket, an extracellular region enriched in acidic residues, as a key participant in channel gating. While binding to this region by the venom peptide psalmotoxin modulates channel gating, molecular and structural mechanisms of ASIC gating modulation by small molecules are poorly understood. Here, combining functional, crystallographic, computational and mutational approaches, we show that two structurally distinct small molecules potently and allosterically inhibit channel activation and desensitization by binding at the acidic pocket and stabilizing the closed state of rat/chicken ASIC1. Our work identifies a previously unidentified binding site, elucidates a molecular mechanism of small molecule modulation of ASIC gating, and demonstrates directly the structural basis of such modulation, providing mechanistic and structural insight into ASIC gating, modulation and therapeutic targeting.

Development of phenotypic assays for identifying novel blockers of L-type calcium channels in neurons.

L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. To exploit the possibility of identifying mechanistically novel and/or neuro-selective blockers, we developed two phenotypic assays-a calcium flux-based primary screening assay and a patch clamp secondary assay, using rat primary cortical cultures. We screened a library comprised of 1278 known bioactive agents and successfully identified a majority of the potent LTCC-blocking drugs in the library. Significantly, we identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. As such, these phenotypic assays are robust and represent an important step towards identifying mechanistically novel and neuro-selective LTCC blockers.

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

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 < 10 nM). PcTx1 potentiates ASIC1a/2a by increasing the apparent affinity of channel activation for protons. As such, potentiation is the strongest at moderate pHs, diminishing with increasing proton concentrations. Our findings identify PcTx1 as a valuable tool for studying ASIC1a/2a function and contribute significantly to the understanding of the diverse and complex pharmacology of PcTx1.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.