Desensitized State Research Articles

Cryo-EM structures of the human P2X1 receptor reveal subtype-specific architecture and antagonism by supramolecular ligand-binding

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.

End-to-end synaptic molecular communication with astrocytic feedback and generic three-state receptors

This paper investigates the mutual information of synaptic molecular communications using a realistic end-to-end synaptic model. In particular, we have considered the influence of astrocytes on neural signaling within the synaptic molecular communication. We have evaluated the average mutual information of the resultant tripartite synapse while considering realistic synaptic geometry that accounts for neurotransmitter reflections from the pre-synaptic and post-synaptic boundaries. The clearance of neurotransmitters from the synapse through diffusion and re-absorption by pre-synaptic terminal is also considered in the simulated model. Moreover, we have used a generic three-state model for postsynaptic receptors to include desensitization state of the receptors. The presented simulation results depict the effects of different pre-synaptic and post-synaptic parameters on the information transfer for a tripartite synaptic channel with three-state receptor model, which is more realistic than the commonly-used bipartite synaptic channel with the two-state receptor model.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy.

The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure-activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist 26a, featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC50 = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that 26a stabilizes the P2X3R in its desensitized state, acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent antiallodynic effects of 26a, thus presenting a novel, promising lead structure.

Engineering a human P2X2 receptor with altered ligand selectivity in yeast

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker’s yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity towards the ATP analog AMP-PNP. The F303Y A304 variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304 P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer’s disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Modulation of GluA2-γ5 synaptic complex desensitization, polyamine block and antiepileptic perampanel inhibition by auxiliary subunit cornichon-2.

Synaptic complexes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) with auxiliary subunits mediate most excitatory neurotransmission and can be targeted to treat neuropsychiatric and neurological disorders, including epilepsy. Here we present cryogenic-electron microscopy structures of rat GluA2 AMPAR complexes with inhibitory mouse γ5 and potentiating human cornichon-2 (CNIH2) auxiliary subunits. CNIH2 appears to destabilize the desensitized state of the complex by reducing the separation of the upper lobes in ligand-binding domain dimers. At the same time, CNIH2 stabilizes binding of polyamine spermidine to the selectivity filter of the closed ion channel. Nevertheless, CNIH2, and to a lesser extent γ5, attenuate polyamine block of the open channel and reduce the potency of the antiepileptic drug perampanel that inhibits the synaptic complex allosterically by binding to sites in the ion channel extracellular collar. These findings illustrate the fine-tuning of synaptic complex structure and function in an auxiliary subunit-dependent manner, which is critical for the study of brain region-specific neurotransmission and design of therapeutics for disease treatment.

Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization.

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

Molecular mechanism underlying desensitization of the proton-activated chloride channel PAC.

Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role in regulating the pH and size of organelles in the endocytic pathway, and is also involved in acid-induced cell death. However, how the PAC channel desensitizes is largely unknown. Here, we show by patch-clamp electrophysiological studies that PAC (also known as TMEM206/ASOR) undergoes pH-dependent desensitization upon prolonged acid exposure. Through structure-guided and comprehensive mutagenesis, we identified several residues critical for PAC desensitization, including histidine (H) 98, glutamic acid (E) 94, and aspartic acid (D) 91 at the extracellular extension of the transmembrane helix 1 (TM1), as well as E107, D109, and E250 at the extracellular domain (ECD)-transmembrane domain (TMD) interface. Structural analysis and molecular dynamic simulations revealed extensive interactions between residues at the TM1 extension and those at the ECD-TMD interface. These interactions likely facilitate PAC desensitization by stabilizing the desensitized conformation of TM1, which undergoes a characteristic rotational movement from the resting and activated states to the desensitized state. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for future investigation of its relevance in cellular physiology and disease.

Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryogenic electron microscopy (cryo-EM) structures of the human α7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures and their differential interactions with unresolved lipids and ligands remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to nonconductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded α7-receptor structures with ion-permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.

Differential Activation and Desensitization States Promoted by Noncanonical α7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential α7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human α7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of α7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.

Physiological Role of ATPase for GABAA Receptor Resensitization.

γ-Aminobutyric acid type A receptors (GABAARs) mediate primarily inhibitory synaptic transmission in the central nervous system. Following fast-paced activation, which provides the selective flow of mainly chloride (Cl−) and less bicarbonate (HCO3−) ions via the pore, these receptors undergo desensitization that is paradoxically prevented by the process of their recovery, referred to as resensitization. To clarify the mechanism of resensitization, we used the cortical synaptoneurosomes from the rat brain and HEK 293FT cells. Here, we describe the effect of γ-phosphate analogues (γPAs) that mimic various states of ATP hydrolysis on GABAAR-mediated Cl− and HCO3− fluxes in response to the first and repeated application of the agonist. We found that depending on the presence of bicarbonate, opened and desensitized states of the wild or chimeric GABAARs had different sensitivities to γPAs. This study presents the evidence that recovery of neuronal Cl− and HCO3− concentrations after desensitization is accompanied by a change in the intracellular ATP concentration via ATPase performance. The transition between the desensitization and resensitization states was linked to changes in both conformation and phosphorylation. In addition, the chimeric β3 isoform did not exhibit the desensitization of the GABAAR-mediated Cl− influx but only the resensitization. These observations lend a new physiological significance to the β3 subunit in the manifestation of GABAAR resensitization.

Differential interactions of closed, open, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that critically modulates neuronal excitability, largely by importing Ca2+ ions. Similar to other channels in this family, agonist binding to the α7 receptor promotes the transition from a closed state to an open state, then rapidly to a desensitized state. Recent cryo-electron microscopy structures of the human α7 receptor in lipid nanodiscs were reported in multiple conformations, selectively stabilized by inhibitory, activating, or potentiating compounds.

Perspective on the Relationship between GABAA Receptor Activity and the Apparent Potency of an Inhibitor.

Background: In electrophysiological experiments, inhibition of a receptor-channel, such as the GABAA receptor, is measured by co-applying an agonist producing a predefined control response with an inhibitor to calculate the fraction of the control response remaining in the presence of the inhibitor. The properties of the inhibitor are determined by fitting the inhibition concentration-response relationship to the Hill equation to estimate the midpoint (IC50) of the inhibition curve. Objective: We sought to estimate sensitivity of the fitted IC50 to the level of activity of the control response. Methods: The inhibition concentration-response relationships were calculated for models with distinct mechanisms of inhibition. In Model I, the inhibitor acts allosterically to stabilize the resting state of the receptor. In Model II, the inhibitor competes with the agonist for a shared binding site. In Model III, the inhibitor stabilizes the desensitized state. Results: The simulations indicate that the fitted IC50 of the inhibition curve is sensitive to the degree of activity of the control response. In Models I and II, the IC50 of inhibition was increased as the probability of being in the active state (PA) of the control response increased. In Model III, the IC50 of inhibition was reduced at higher PA. Conclusion: We infer that the apparent potency of an inhibitor depends on the PA of the control response. While the calculations were carried out using the activation and inhibition properties that are representative of the GABAA receptor, the principles and conclusions apply to a wide variety of receptor-channels.

The Sulfated Steroids Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate Inhibit the α1β3γ2L GABAA Receptor by Stabilizing a Novel Nonconducting State.

The GABAA receptor is inhibited by the endogenous sulfated steroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). It has been proposed in previous work that these steroids act by enhancing desensitization of the receptor. Here, we have investigated the modulatory effects of the steroids on the human α1β3γ2L GABAA receptor. Using electrophysiology and quantitative model-based data analysis, we show that exposure to the steroid promotes occupancy of a nonconducting state that retains high affinity to the transmitter but whose properties differ from those of the classic, transmitter-induced desensitized state. From the analysis of the inhibitory actions of two combined steroids, we infer that PS and DHEAS act through shared or overlapping binding sites. SIGNIFICANCE STATEMENT: Previous work has proposed that sulfated neurosteroids inhibit the GABAA receptor by enhancing the rate of entry into the desensitized state. This study shows that the inhibitory steroids pregnenolone sulfate and dehydroepiandrosterone sulfate act through a common interaction site by stabilizing a distinct nonconducting state.

Molecular Investigation of Chicken Acid-Sensing Ion Channel 1 β11-12 Linker Isomerization and Channel Kinetics.

Structures of the trimeric acid-sensing ion channel have been solved in the resting, toxin-bound open and desensitized states. Within the extracellular domain, there is little difference between the toxin-bound open state and the desensitized state. The main exception is that a loop connecting the 11th and 12th β-strand, just two amino acid residues long, undergoes a significant and functionally critical re-orientation or flipping between the open and desensitized conformations. Here we investigate how specific interactions within the surrounding area influence linker stability in the “flipped” desensitized state using all-atom molecular dynamics simulations. An inherent challenge is bringing the relatively slow channel desensitization and recovery processes (in the milliseconds to seconds) within the time window of all-atom simulations (hundreds of nanoseconds). To accelerate channel behavior, we first identified the channel mutations at either the Leu414 or Asn415 position with the fastest recovery kinetics followed by molecular dynamics simulations of these mutants in a deprotonated state, accelerating recovery. By mutating one residue in the loop and examining the evolution of interactions in the neighbor, we identified a novel electrostatic interaction and validated prior important interactions. Subsequent functional analysis corroborates these findings, shedding light on the molecular factors controlling proton-mediated transitions between functional states of the channel. Together, these data suggest that the flipped loop in the desensitized state is stabilized by interactions from surrounding regions keeping both L414 and N415 in place. Interestingly, very few mutations in the loop allow for equivalent channel kinetics and desensitized state stability. The high degree of sequence conservation in this region therefore indicates that the stability of the ASIC desensitized state is under strong selective pressure and underlines the physiological importance of desensitization.

The Influence of AA29504 on GABAA Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity

The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses.

Nanoparticles in allergen immunotherapy.

Allergen immunotherapy is the only recognized causal treatment for allergic disease that modulates the immune system toward a tolerogenic or desensitized state. Allergens or their derivative preparations are formulated with adjuvants of different origin and having diverse immunological functions, such as prolonged tissue release and specific immunomodulatory properties. In the last 2 decades, thanks to developments in the field of nanotechnology, more biosafe nanoscale materials have become available for use as pharmaceutical adjuvants in medical research. Nanomaterials possess unique and versatile properties which can be employed to develop drug carriers with safer profiles, better stability in physiological conditions and immunomodulatory properties. Nanoparticles can have an adjuvant effect per se or also when they are packed in structures whose physical-chemical properties can be handled in a way that also influences its release dynamics. In particular, it has been suggested that nanoparticle preparations can be put in complexes or loaded with allergens or allergenic extracts, opening the way to innovative paradigms. In this review, we analyze allergen/nanoparticle properties in terms of cytotoxicity, stability and immunogenic reaction in in-vitro and animal systems.

Urinary prostaglandin D2 metabolite appears to be a useful biomarker for evaluating the status of egg oral immunotherapy in children

Food allergy (FA) is an increasingly prevalent global health problem.1 Previously, the only management for FA was the avoidance of food allergens. However, oral immunotherapy (OIT) has become increasingly popular as a potential treatment option for FA.2,3 Recently, a product for peanut OIT, Palforzia (Aimmune Therapeutics, Brisbane, Calif), was approved by the US Food and Drug Administration for use in children aged 4 to 17 years. OIT involves the continued daily consumption of a progressively escalating dose of food allergen, which can induce a state of desensitization or tolerance in patients with FA after a maintenance period ranging from months to years.

A detailed electrochemical analysis of sensitized AISI 410 martensitic stainless steel

In the present work, the effect of sensitization heat treatment on the intergranular corrosion (IGC) behavior of AISI 410 martensitic stainless steel was investigated using potentiodynamic electrochemical impedance spectroscopy (PDEIS) in conjunction with conventional methods including acid etching test, anodic potentiodynamic polarization, double-loop electrochemical potentiodynamic reactivation (DLEPR) and electrochemical noise (EN). Microstructural studies by scanning electron microscopy revealed that increasing tempering time up to 2h would be followed by more severe localized attack, but desensitization state attributed to specimens undergone prolonged tempering cannot be interpreted based on the standard test method. Susceptibility to IGC via DLEPR experiments indicates differences in the degree of sensitization (DOS) of specimens with varying tempering time, revealing a healing effect arising from prolonged tempering. The EN results elucidated the alloy tendency for active dissolution, but no obvious correlation can be established between the EN results and IGC behavior. The PDEIS data clarified that the charge transfer resistance values related to the AC responses in the middle of transpassive potential region can be utilized to investigate and compare the IGC susceptibility of the alloy. Moreover, the surface morphology of corroded samples under such experimental conditions can be used to qualitatively compare the severity of intergranular attack.