Channel Desensitization Research Articles

Uncaria Rhynchophylla and hirsuteine as TRPV1 agonists inducing channel desensitization

Ethnopharmacological relevanceUncaria rhynchophylla (UR) is recognized for its therapeutic applications in treating hypertension and inflammation. However, the specific molecular mechanisms how UR and its bioactive constituents modulate inflammatory pathways remain unknown. This study investigates the effects of UR extract and its constituent, hirsuteine (HST), on TRPV1 channel modulation which is related to hypertension and inflammation. Materials and methodsElectrophysiological recordings and calcium imaging experiments were conducted to assess TRPV1 activation by UR extract and HST in HEK293T cells and sensory neurons. ResultsUR extract and HST activated TRPV1 in HEK293T cells, with repeated applications causing channel desensitization. HST application on TRPV1-positive sensory neurons significantly reduced electrical activity compared to capsaicin. ConclusionThis study demonstrated UR extract and HST are a novel TRPV1 agonists inducing channel desensitization and a potent agent for treatment of TRPV1 dependent pain relief.

Chemical shift assignments of the α-actinin C-terminal EF-hand domain bound to a cytosolic C0 domain of GluN1 (residues 841–865) from the NMDA receptor

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the brain are important for controlling neuronal excitability to promote synaptic plasticity. The cytoskeletal protein, α-actinin-1 (100 kDa, called ACTN1) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca2+-dependent desensitization of NMDAR channels. Mutations that disrupt NMDAR channel function are linked to Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for the C-terminal EF-hand domain of ACTN1 (residues 824–892, called ACTN_EF34) and ACTN_EF34 bound to the GluN1 C0 domain (BMRB numbers 52385 and 52386, respectively).

Dual Role of TRPV1 Channels in Cerebral Stroke: An Exploration from a Mechanistic and Therapeutic Perspective.

Transient receptor potential vanilloid subfamily member 1 (TRPV1) has been strongly implicated in the pathophysiology of cerebral stroke. However, the exact role and mechanism remain elusive. TPRV1 channels are exclusively present in the neurovascular system and involve many neuronal processes. Numerous experimental investigations have demonstrated that TRPV1 channel blockers or the lack of TRPV1 channels may prevent harmful inflammatory responses during ischemia-reperfusion injury, hence conferring neuroprotection. However, TRPV1 agonists such as capsaicin and some other non-specific TRPV1 activators may induce transient/slight degree of TRPV1 channel activation to confer neuroprotection through a variety of mechanisms, including hypothermia induction, improving vascular functions, inducing autophagy, preventing neuronal death, improving memory deficits, and inhibiting inflammation. Another factor in capsaicin-mediated neuroprotection could be the desensitization of TRPV1 channels. Based on the summarized evidence, it may be plausible to suggest that TPRV1 channels have a dual role in ischemia-reperfusion-induced cerebral injury, and thus, both agonists and antagonists may produce neuroprotection depending upon the dose and duration. The current review summarizes the dual function of TRPV1 in ischemia-reperfusion-induced cerebral injury models, explains its mechanism, and predicts the future.

Dynamic conformational changes of acid-sensing ion channels in different desensitizing conditions

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to fast synaptic transmission and have roles in fear conditioning and nociception. Apart from activation at low pH, ASIC1a also undergoes several types of desensitization, including acute desensitization, which terminates activation; steady-state desensitization, which occurs at sub-activating proton concentrations and limits subsequent activation; and tachyphylaxis, which results in a progressive decrease in response during a series of activations. Structural insights from a desensitized state of ASIC1 have provided great spatial detail, but dynamic insights into conformational changes in different desensitizing conditions are largely missing. Here, we use electrophysiology and voltage-clamp fluorometry to follow the functional changes of the pore along with conformational changes at several positions in the extracellular and upper transmembrane domain via cysteine-labeled fluorophores. Acute desensitization terminates activation in wild type, but introducing an N414K mutation in the β11-12 linker of mouse ASIC1a interfered with this process. The mutation also affected steady-state desensitization and led to pronounced tachyphylaxis. Although the extracellular domain of this mutant remained sensitive to pH and underwent pH-dependent conformational changes, these conformational changes did not necessarily lead to desensitization. N414K-containing channels also remained sensitive to a known peptide modulator that increases steady-state desensitization, indicating that the mutation only reduced, but not precluded, desensitization. Together, this study contributes to our understanding of the fundamental properties of ASIC1a desensitization, emphasizing the complex interplay between the conformational changes of the extracellular domain and the pore during channel activation and desensitization.

BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.

8-shogaol derived from dietary ginger alleviated acute and inflammatory pain by targeting TRPV1

Ginger, a well-known spice plant, has been used widely in medicinal preparations for pain relief. However, little is known about its analgesic components and the underlying mechanism. Here, we ascertained, the efficacy of ginger ingredient 8-Shogaol (8S), on inflammatory pain and tolerance induced by morphine, and probed the role of TRPV1 in its analgesic action using genetic and electrophysiology approaches. Results showed that 8S effectively reduced nociceptive behaviors of mice elicited by chemical stimuli, noxious heat as well as inflammation, and antagonized morphine analgesic tolerance independent on opioid receptor function. Genetic deletion of TRPV1 significantly abolished 8S’ analgesia action. Further calcium imaging and patch-clamp recording showed that 8S could specifically activate TRPV1 in TRPV1-expressing HEK293T cells and dorsal root ganglion (DRG) neurons. The increase of [Ca2+]i in DRG was primarily mediated through TRPV1. Mutational and computation studies revealed the key binding sites for the interactions between 8S and TRPV1 included Leu515, Leu670, Ile573, Phe587, Tyr511, and Phe591. Further studies showed that TRPV1 activation evoked by 8S resulted in channel desensitization both in vitro and in vivo, as may be attributed to TRPV1 degradation or TRPV1 withdrawal from the cell surface. Collectively, this work provides the first evidence for the attractive analgesia of 8S in inflammatory pain and morphine analgesic tolerance mediated by targeting pain-sensing TRPV1 channel. 8S from dietary ginger has potential as a candidate drug for the treatment of inflammatory pain.

Ca2+/calmodulin-mediated desensitization of glutamate receptors shapes plant systemic wound signalling and anti-herbivore defence.

Plants rely on systemic signalling mechanisms to establish whole-body defence in response to insect and nematode attacks. GLUTAMATE RECEPTOR-LIKE (GLR) genes have been implicated in long-distance transmission of wound signals to initiate the accumulation of the defence hormone jasmonate (JA) at undamaged distal sites. The systemic signalling entails the activation of Ca2+-permeable GLR channels by wound-released glutamate, triggering membrane depolarization and cytosolic Ca2+ influx throughout the whole plant. The systemic electrical and calcium signals rapidly dissipate to restore the resting state, partially due to desensitization of the GLR channels. Here we report the discovery of calmodulin-mediated, Ca2+-dependent desensitization of GLR channels, revealing a negative feedback loop in the orchestration of plant systemic wound responses. A CRISPR-engineered GLR3.3 allele with impaired desensitization showed prolonged systemic electrical signalling and Ca2+ waves, leading to enhanced plant defence against herbivores. Moreover, this Ca2+/calmodulin-mediated desensitization of GLR channels is a highly conserved mechanism in plants, providing a potential target for engineering anti-herbivore defence in crops.

The role of TRPA1 and TRPV1 in the perception of astringency.

Astringency, commonly described as a drying, roughening, and/or puckering sensation associated with polyphenol-rich foods affects their palatability. While the compounds eliciting astringency are known, its mechanism of action is debated. This study investigated the role of transient receptor potential (TRP) channels A1 and V1 in astringency perception. If TRP A1 or V1 have a functional role in astringency perception, then desensitizing these receptors should decrease perceived astringency. Thirty-seven panelists underwent unilateral lingual desensitization of TRP A1 and V1 channels using mustard oil and capsaicin, respectively. Panelists then evaluated four astringent stimuli: epicatechin (EC), epigallocatechin gallate (EGCG), tannic acid (TA), and potassium alum (Alum), via 2-AFC and intensity ratings. When TRPA1 receptors were desensitized on one half of the tongue via mustard oil, no significant differences were observed between the treated and untreated sides for both 2-AFC and intensity ratings. Similarly, when TRPV1 receptors were desensitized on one half of the tongue via capsaicin, no significant differences were observed between the treated and untreated sides for both 2-AFC and intensity ratings. These findings challenge the notion that TRP channels play a pivotal role in astringency perception.

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.

The role of melatonin in regulating horticultural crop production under various abiotic stresses

Horticultural crops are very important component of our food chain, and their production is highly sensitive to different soil related abiotic stresses (SRAS). These stresses reduce plant growth and development by reducing seed germination (SG), seedling establishment, photosynthesis, reproduction, thus reduce fruit yield and quality. Nonetheless application of plant growth regulators has been shown a very promising technique to confer SRAS tolerance in horticultural crops. Melatonin (Mel)-a high conserved and stress regulatory molecule regulates a plethora of stress adaptive responses in plants. This paper summarizes the current state of knowledge of Mel involvement in regulating stress adaptive responses under SRAS. The major topic covered in this review included: (i) the effects of exogenous Mel application on seed germination, photosynthesis, flowering, and fruit yield under different SRAS, (ii) Mel crosstalk with stress regulatory mechanism to confer stress tolerance in horticultural crops. Latter topic further discussed the role of Mel in regulating (i) redox regulation, (ii) ionic homeostasis, and (iii) hormonal regulation. We show that Mel regulates stress adaptive responses and improves plant growth by allowing the direct scavenging of ROS, up-regulation of H+-ATPase activity and vacuolar sequestration of toxic ions in cytosol and activation of ROS-Ca2+ hub, which latter prevent the negative effects of ROS and membrane depolarization on the ROS-activated and voltage-activated ion channels. Such Mel mediated desensitization of ion channels may play a crucial role in reducing stress induced efflux of mineral ions such as K, Zn, Mg from cytosol, thereby improving plant growth. Moreover, Mel may operate in tandem with polyamines to regulate the biosynthesis of different phytohormones. However, studies are required to reveal the molecular identity of genes relating to Mel-polyamine tandem and to light signaling that could directly regulate phytohormone biosynthesis and the interaction of Mel with light signaling in regulating SG.

Resensitization of TRPV1 channels after the P2 receptor activation in sensory neurons of spinal ganglia in rats.

TRPV1 channels are responsible for detecting noxious stimuli such as heat (>43°C), acid, and capsaicin. P2 receptors are involved in numerous functions of the nervous system, including its modulation and specific response to the application of ATP. In our experiments, we investigated the dynamics of calcium transients in DRG neurons associated with TRPV1 channel desensitization and the effect of activation of P2 receptors on this process. We used DRG neurons from rats P7-8 after 1-2 days of culture to measure calcium transients by microfluorescence calcimetry using the fluorescent dye Fura-2 AM. We have shown that DRG neurons of small (d < 22 μm) and medium (d = 24-35 μm) sizes differ in TRPV1 expression. Thus, TRPV1 channels are mainly present in small nociceptive neurons (59% of the studied neurons). Short-term sequential application of the TRPV1 channel agonist capsaicin (100nM) leads to the desensitization of TRPV1 channels by the type of tachyphylaxis. We identified three types of sensory neurons based on responses to capsaicin: (1) desensitized 37.5%, (2) non-desensitized 34.4%, and (3) insensitive 23.4% to capsaicin. It has also been shown that P2 receptors are present in all types of neurons according to their size. So, the responses to ATP were different in different-sized neurons. Applying ATP (0.1 mM) to the intact cell membrane after the onset of tachyphylaxis caused recovery of calcium transients in response to the addition of capsaicin in these neurons. The amplitude of the capsaicin response after reconstitution with ATP was 161% of the previous minimal calcium transient in response to capsaicin. Significantly, the restoration of the amplitude of calcium transients under the ATP application is not associated with changes in the cytoplasmic pool of ATP because this molecule does not cross the intact cell membrane, thus, our results show the interaction between TRPV1 channels and P2 receptors. It is important to note that the restoration of the amplitude of calcium transients through TRPV1 channels after application of ATP was observed mainly in cells of 1-2 days of cultivation. Thus, the resensitization of capsaicin transients following P2 receptor activation may be associated with the regulation of the sensitivity of sensory neurons.

TRPA1 is involved in the inhibitory effect of Ke-teng-zi on allergic contact dermatitis via MAPK and JAK/STAT3 signaling pathways.

The seeds of Entada phaseoloides (Linn.) Merr. commonly named "Ke-teng-zi" is a traditional Chinese folk medicine and reported to treat dermatitis, spasm, and headache. However, the exact effect and the mechanism of Ke-teng-zi on the treatment of dermatitis is unclear. To elucidate the antipruritic effect and molecular mechanisms of Ke-teng-zi on the treatment of allergic contact dermatitis (ACD). The main components of the n-butanol fraction of 70% ethanol extract from Ke-teng-zi (abbreviated as KB) were analyzed by HPLC. The chloroquine (CQ)-induced acute itch and squaraine dibutyl ester (SADBE)-induced ACD chronic itch in mice was established, and the TNF-α/IFN-γ stimulated Human keratinocytes (HaCaT) were used to evaluate the antipruritic and anti-inflammatory effects of KB. Behavioral tests, lesion scoring, and histology were also examined. The expression levels of molecules in MAPK and JAK/STAT3 pathways, the mRNA levels of chemokines and cytokines in both the skin of ACD mice and the HaCaT cells were detected by western blot and qPCR. Furthermore, whole-cell patch-clamp recordings in TRPA1-tranfected HEK293T cells were used to elucidate the effect of KB on TRPA1 channels. TRPA1 siRNA was used to evaluate the role of TRPA1 in the anti-inflammatory effect of KB in keratinocytes. The main compounds in KB could bind to the active sites of TRPA1 mainly through hydrogen bond and hydrophobic bond interactions. KB could inhibit the scratching behavior in CQ-induced acute itch, and the inhibitory effect of KB was blocked by TRPA1 inhibitor HC-030031. In addition, KB significantly decreased the scratching bouts of ACD mice, reduced the skin lesion scores, mast cells degranulation, and epidermal thickening, inhibited the production of inflammatory chemokines/cytokines and CGRP, and down-regulated the levels of p-ERK1/2, p-p38, and p-STAT3, compared to the ACD mice. Moreover, continuous application of KB induced the desensitization of TRPA1 channels. Also, KB inhibited the expression of p-ERK1/2, p-p38, and p-STAT3, and down-regulated the expression of inflammatory chemokines and cytokines in vitro, which were reversed by the TRPA1 siRNA. KB alleviated the pruritus and skin inflammation in ACD mice through TRPA1 channels desensitization and down-regulation of intracellular MAPK and JAK/STAT3 signaling pathways. Our results suggested that Ke-teng-zi is a potential drug for the treatment of inflammatory skin diseases such as ACD.

Abdominal TRPV1 channel desensitization enhances stress-induced hyperthermia during social stress in rats.

In rats, stress-induced hyperthermia caused by social interaction depends on brown adipose tissue (BAT) thermogenesis and peripheral vasoconstriction. However, the peripheral mechanisms responsible for regulating the level of hyperthermia during social stress are still unknown. The transient receptor potential vanilloid 1 (TRPV1) subfamily, expressed in sensory and visceral neurons, can serve as a thermoreceptor. Here, we tested the hypothesis that the abdominal TRPV1 is essential in regulating stress-induced hyperthermia during social stress. Male Wistar rats received an intraperitoneal injection of Resiniferatoxin (RTX) - an ultra-potent capsaicin analog, (i.e., to desensitize the TRPV1 channels) or vehicle. Seven days later, we evaluated the effects of abdominal TRPV1 channels desensitization on core body temperature (CBT), brown adipose tissue (BAT) temperature, tail skin temperature, and heart rate (HR) of rats subjected to a social stress protocol. We found abdominal TRPV1 desensitization increased CBT and BAT temperature but did not change tail skin temperature and HR during rest. However, under social stress, we found that abdominal TRPV1 desensitization heightened the increase in CBT and BAT caused by stress. Also, it abolished the increase in tail skin temperature that occurs during and after social stress. TRPV1 desensitization also delayed the HR recovery after the exposure to the social stress. These results show that abdominal TRPV1 channels desensitization heightens stress-induced hyperthermia, causing heat dissipation during and after social stress, enabling optimal thermal control during social encounters.

Chemical shift assignments of calmodulin bound to a cytosolic domain of GluN2A (residues 1004-1024) from the NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841-865) and GluN2 (residues 1004-1024) that may play a role in the Ca2+-dependent desensitization of NMDAR channels. Mutations that disrupt Ca2+-dependent desensitization of NMDARs are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca2+-saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821).

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.

Charged residues in the GABA-a receptor intracellular domain impact channel activation and desensitization.

Opening and closing of GABA receptor (GABAR) channels regulate inhibitory signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. Although GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, little is understood about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR homology model based on the 5HT-type-3 receptor, which revealed a helix (MX) at the C-terminal end of M3 that is lysine rich in the α1 (6 lysines) and γ2 (8 lysines) subunits. Charged residues in the α and γ subunits were mutated to alanine. Effects on GABA EC50 were measured using two-electrode voltage clamping of oocytes expressing α1β2γ2L WT and mutant GABARs. αK327A significantly increased GABA EC50 6-fold in comparison to WT and significantly reduced GABAR macroscopic desensitization. Extent of current desensitization after 10 seconds in saturating GABA (10 mM) was 6.86 ± 2.28% (n=7) versus WT (23.7 ± 7.29%, n=10). Neutralizing charge at αK328, K330 and K335 had no functional effects. γK328A, γ2K330A and γ2K332A significantly increased GABA EC50 by 2 to 3 fold. γ2K330A reduced GABAR desensitization (extent after 10 seconds 7.36 ± 2.37%, n=2). Our data indicate structural perturbations in the α and γ MX regions of the GABAR ICD alter GABAR channel activation and desensitization. Interestingly, in our GABAR structural model, α1R312 (in MX) interacts with α1R249 (M1-M2 linker). Single charge reversals at these positions increased GABA EC50 and the rate of GABA desensitization. A charge swap (R312E - E249R) rescued receptor function to WT suggesting a functional electrostatic interaction and that these residues form a salt bridge, providing support for our homology model.

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

Opened by acidic pH, the Proton-Activated Chloride (PAC) channel has been recorded in a variety of cell types. Although its molecular identify has only recently been identified, the PAC channel has established important roles in acid-induced cell death, endosomal acidification, and macropinosome shrinkage. Previous cryo-EM studies of the PAC channel reported a proton-bound non-conducting structure at low pH, suggestive that PAC undergoes desensitization - a process common to ligand-gated ion channels. Comparison of the proposed open and desensitized structures demonstrated significant rotational movement of the transmembrane helix 1 (TM1), switching its interaction partner to a neighboring subunit. However, properties and underlying mechanisms underlying PAC channel desensitization remain to be largely unknown. In this study, we demonstrate that PAC undergoes pH-dependent desensitization upon prolonged acid exposure with patch-clamp electrophysiology. Additional studies with structure-guided mutagenesis 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 TM1, as well as E107, D109, and E250 at the interface of the extracellular domain (ECD) and transmembrane domain (TMD). 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. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for further investigation of its relevance in cellular physiology and disease.

Nicotinic acetylcholine receptor structural pharmacology and gating transitions.

Our lab focuses on using structural and functional approaches to understand signaling by ligand-gated ion channels in the nervous system. A major emphasis is on understanding nicotinic receptor pharmacology from a structural perspective. Here I will present some of our recent work using the muscle-type nicotinic receptor from the Torpedo ray as a structurally tractable reference for the human receptor at the neuromuscular junction. I will focus on how toxins from animals and plants, as well as drugs used in the clinic, act on the receptor, and how we can use this information to understand state transitions that underlie ion channel gating. These studies have revealed a new class of allosteric site at the junction of the extracellular and transmembrane domains that small molecules can leverage to modulate channel desensitization. Our findings suggest that stabilizing a desensitized-like non-conducting state, through this allosteric site, is an important alternative mechanism for antagonizing the receptor.

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.

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

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