A Novel Physically Guided Data Fusion Prediction Model for Micro-EDM Drilling.

Accurate prediction of Electro-Discharge Machining (EDM) results is crucial for industrial applications, aiming to achieve high-performance and cost-efficient machining. However, both the current physical model and the standard Artificial Neural Network (ANN) model exhibit inherent limitations, failing to fully meet the accurate requirements for predicting EDM machining results. In addition, Micro-EDM Drilling can lead to the distortion of the macroscopic shape of machining pits under different input conditions, rendering the use of only the volume of machining pits as the evaluation index insufficient to express the complete morphological information. In this study, we propose a novel hybrid prediction model that combines the strengths of both physical and data-driven models to simultaneously predict Material Removal Rate (MRR) and shape parameters. Our experiment demonstrates that the hybrid model achieves a maximum prediction error of 4.92% for MRR and 5.28% for shape parameters, showcasing excellent prediction accuracy and stability compared to the physical model and the standard ANN model.

Allosteric Site Mediates Inhibition of Tonic NMDA Receptor Activity by Low Dose Ketamine.

Ketamine, a general anesthetic, has rapid and sustained antidepressant effects when administered at lower doses. At anesthetic doses, ketamine causes a drastic reduction in excitatory transmission by lodging in the centrally located hydrophilic pore of the NMDA receptor, where it blocks ionic flow. In contrast, the molecular and cellular targets responsible for the antidepressant effects of ketamine remain controversial. Here, we report functional and structural evidence that, at nanomolar concentrations, ketamine interacts with membrane-accessible hydrophobic sites where it stabilizes desensitized receptors to cause an incomplete, voltage- and pH-dependent reduction in NMDA receptor activity. This allosteric mechanism spares brief receptor activations and reduces preferentially currents from tonically active receptors. The hydrophobic site is a promising target for safe and effective therapies against acute and chronic neurodegeneration.

A suicidal mechanism for the exquisite temperature sensitivity of TRPV1.

The vanilloid receptor TRPV1 is an exquisite nociceptive sensor of noxious heat, but its temperature-sensing mechanism is yet to define. Thermodynamics dictate that this channel must undergo an unusually energetic allosteric transition. Thus, it is of fundamental importance to measure directly the energetics of this transition in order to properly decipher its temperature-sensing mechanism. Previously, using submillisecond temperature jumps and patch-clamp recording, we estimated that the heat activation for TRPV1 opening incurs an enthalpy change on the order of 100 kcal/mol. Although this energy is on a scale unparalleled by other known biological receptors, the generally imperfect allosteric coupling in proteins implies that the actual amount of heat uptake driving the TRPV1 transition could be much larger. In this paper, we apply differential scanning calorimetry to directly monitor the heat flow in TRPV1 that accompanies its temperature-induced conformational transition. Our measurements show that heat invokes robust, complex thermal transitions in TRPV1 that include both channel opening and a partial protein unfolding transition and that these two processes are inherently coupled. Our findings support that irreversible protein unfolding, which is generally thought to be destructive to physiological function, is essential to TRPV1 thermal transduction and, possibly, to other strongly temperature-dependent processes in biology.

Complex functional phenotypes of NMDA receptor disease variants.

NMDA receptors have essential roles in the physiology of central excitatory synapses and their dysfunction causes severe neuropsychiatric symptoms. Recently, a series of genetic variants have been identified in patients, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we investigate the mechanism by which two GluN2A variants may be pathogenic. We use molecular dynamics simulation and single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic substitutions, P552R and F652V, affect receptor functions. We found that P552 and F652 interact during the receptors' normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engineering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R side chain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response. These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor primary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.

AC/DC Electric-Field-Assisted Growth of ZnO Nanowires for Gas Discharge.

Using ZnO nanowires as needle anodes in gas discharge is helpful for maintaining continuous discharge with a relatively low voltage. It is necessary that the ZnO nanowires are far enough apart to guarantee no electric field weakening and that the nanowire anodes are easy to assemble together with the discharging devices. An AC/DC electric-field-assisted wet chemical method is proposed in this paper. It was used to grow ZnO nanowires directly on discharging devices. The nanowires covered the whole electrode in the case in which only a DC field was applied. Moreover, the tips of the nanowires were scattered, similar to the results observed under the application of AC fields. The average distance between the tips of the highest nanowires was approximately equal to 4 µm, which almost meets the requirement of gas discharge. The research concerning growing ZnO nanowires directly on PCBs shown that, at the current time, ZnO nanowires on PCBs did not meet the requirements of gas discharge; however, in this study, the parameters regarding ZnO nanowire growth were established.

Identification of a helix-turn-helix motif for high temperature dependence of vanilloid receptor TRPV2.

Pain and thermosensation rely on temperature-sensitive ion channels at peripheral nerve endings for transducing thermal cues into electrical signals. Members of the transient receptor potential (TRP) family are prominent candidates for temperature transducers in mammals. These thermal TRP channels possess an unprecedentedly steep temperature dependence, allowing them to discriminate small temperature variations. Thermodynamically, it is understood that the strong temperature sensitivity of the channel arises because opening of the channel undergoes reactions involving large enthalpy and entropy changes. However, the underlying molecular mechanisms have remained elusive. Here we investigated the molecular basis for heat activation of TRPV2, a thermal TRP channel in the vanilloid subfamily with the strongest temperature dependence among TRP channels. We unravel a minimum molecular region in the proximal N-terminus which dictates the slope temperature sensitivity of the channel. Structurally, the region comprises a helix-turn-helix motif and is positioned among the TRP helix from the C-terminus, the S2-S3 linker from the transmembrane domain and the ankyrin repeats from the distal N-terminus. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results support a pivotal role for the structural assembly around the TRP domain in the gating of thermal TRP channels by temperature. The findings also shed insight into how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors. KEY POINTS: The vanilloid receptor subtype 2 (TRPV2) is a heat-sensitive transient receptor potential (TRP) channel with the strongest temperature dependence among thermal TRP channels. The channel also has a high temperature activation threshold above 50°C which has rendered it difficult to study by conventional patch-clamp methods. Here we utilize fast laser temperature jumps to address the challenges of technical accessibility and explore the molecular basis underlying the high temperature dependence of the channel. We unravel a short helix-turn-helix motif in the proximal N-terminus, which controls the heat activation profile of the channel. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results provide insights on how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors.

Phosphorylated LASS2 inhibits prostate carcinogenesis via negative regulation of Wnt/ß-catenin signaling.

LASS2 is a novel tumor-suppressor gene and has been characterized as a ceramide synthase, which synthesizes very-long acyl chain ceramides. However, LASS2 function and pathway-related activity in prostate carcinogenesis are still largely unexplored. Here, we firstly report that LASS2 promotes ß-catenin degradation through physical interaction with STK38, SCYL2, and ATP6V0C via the ubiquitin-proteasome pathway, phosphorylation of LASS2 is essential for ß-catenin degradation, and serine residue 248 of LASS2 is illustrated to be a key phosphorylation site. Furthermore, we find that dephosphorylation of LASS2 at serine residue 248 significantly enhances prostate cancer cell growth and metastasis in vivo, indicating that phosphorylated LASS2 inhibits prostate carcinogenesis through negative regulation of Wnt/ß-catenin signaling. Thus, our findings implicate LASS2 as a potential biomarker and therapeutic target of prostate cancer.

Cross-subunit interactions that stabilize open states mediate gating in NMDA receptors.

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are currently under investigation. Very likely, the earliest steps include glutamate binding to glycine-bound receptors and subsequent constriction of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical pulling on transmembrane helices. Here, we used molecular and kinetic simulations and double-mutant cycle analyses to show that a direct chemical interaction between GluN1-I642 (on M3 helix) and GluN2A-L550 (on L1-M1 linker) stabilizes receptors after they have opened and thus represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence causes deficits in charge transfer by GluN1-I642L, a pathogenic human variant.

Patch-Clamp Combined with Fast Temperature Jumps to Study Thermal TRP Channels.

Patch-clamp recording combined with biophysical modeling and mutagenic perturbations provides an effective means to study structural functions of ion channels. The methodology has been successful for studying ligand- or voltage-gated channels and brought about much of the knowledge we know today on how ligand or voltage gates an ion channel. The approach, when applied to thermal channels, however, has faced unique challenges. For one problem, thermal channels can operate at high temperatures, and for these channels, prolonged temperature stimulation incurs excessive thermal stress to destabilize patches. For another problem, conventional temperature controls are slow and limit the attainment of high resolution data such as time-resolved activations of thermal channels. Due to these issues, thermal channels have been less accessible to biophysical studies at mechanistic levels. In this chapter we address the problems and demonstrate fast temperature controls enabling recording of time-resolved responses of thermal channels at high temperatures.

A novel case of endometrial dedifferentiated adenocarcinoma associated with MLH1 promotor hypermethylation and microsatellite instability.

Endometrial dedifferentiated carcinoma is a rare, malignant tumor whose molecular alterations have not been clarified yet. We report a novel case of a 61-year old woman who presented with irregular vaginal bleeding after menopause and a 3 cm uterus mass. Histology revealed endometrial dedifferentiated adenocarcinoma, a rare subtype comprised of undifferentiated adenocarcinoma. The patient still survived 1 year after surgery without chemotherapy and radiotherapy. Immunohistochemistry revealed loss of MLH1/PMS2 expression and retained MSH2/MSH6 expression. Consistently, microsatellite instability was detected indicative of high microsatellite instability (MSI-H). No BRAF V600E, KRAS and POLE mutations were identified. Remarkably, the promoter regions of mutL homolog 1(MLH1) were methylated. Furthermore, several tumor cells were PD-L1 positive in this case with a concentration at the infiltrating tumor edge indicating MSI-H in endometrial dedifferentiated adenocarcinoma is a potential predictive factor for response to immunotherapy targeting the PD-1 or its ligand PD-L1.

Silencing of vacuolar ATPase c subunit ATP6V0C inhibits the invasion of prostate cancer cells through a LASS2/TMSG1-independent manner.

Vacuolar ATPase (V-ATPase), widespread in eukaryotic cells, is extensively expressed in many highly metastatic tumors, of which the V-ATPase c subunit ATP6V0C is particularly associated with the invasion and metastasis of cancer. ATP6V0C was directly found to interact with LASS2/TMSG1 which is a new tumor metastasis inhibitory gene identified by our laboratory in 1999. In order to study the role of ATP6V0C, we generated small interference RNA (siRNA) targeting ATP6V0C and investigated its function on the invasion of human prostate cancer cell line PC-3M-1E8 with high metastatic potential and its interplay with LASS2/TMSG1. We found that the expression of ATP6V0C was higher in prostate cancer cell lines PC-3M-1E8 and PC-3M with high metastatic potential than that from cell lines PC-3M-2B4 and PC-3 with low metastatic potential, indicating that ATP6V0C enhanced metastatic capacity in prostate cancer cells. Furthermore, silencing of ATP6V0C in PC-3M-1E8 cells inhibited V-ATPase activity (by ~5-fold), decreased extracellular hydrogen ion concentration and successively decreased activation of secreted MMP-9 (by ~3.6-fold), which coincided with the inhibition of cell migration and invasion in vitro, as well as a marked decrease in the expression of LASS2/TMSG1 probably through positive feedback. Thus we concluded that silencing of the ATP6V0C gene effectively suppressed the migration and invasion of prostate carcinoma cells through the inhibition of the function of V-ATPase, not through a LASS2/TMSG1-dependent manner. Therefore ATP6V0C inhibitors are promising therapeutic targets for advanced prostate cancer.

Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3.

Thermal transient receptor potential (TRP) channels, a group of ion channels from the transient receptor potential family, play important functions in pain and thermal sensation. These channels are directly activated by temperature and possess strong temperature dependence. Furthermore, their temperature sensitivity can be highly dynamic and use-dependent. For example, the vanilloid receptor transient receptor potential 3 (TRPV3), which has been implicated as a warmth detector, becomes responsive to warm temperatures only after intensive stimulation. Upon initial activation, the channel exhibits a high-temperature threshold in the noxious temperature range above 50 °C. This use dependence of heat sensitivity thus provides a mechanism for sensitization of thermal channels. However, how the channels acquire the use dependence remains unknown. Here, by comparative studies of chimeric channels between use-dependent and use-independent homologs, we have determined the molecular basis that underlies the use dependence of temperature sensitivity of TRPV3. Remarkably, the restoration of a single residue that is apparently missing in the use-dependent homologs could largely eliminate the use dependence of heat sensitivity of TRPV3. The location of the region suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain.

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2.

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia.

The Xenopus tropicalis orthologue of TRPV3 is heat sensitive.

Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating.

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.

LASS2/TMSG1 inhibits growth and invasion of breast cancer cell in vitro through regulation of vacuolar ATPase activity.

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2)/tumor metastasis suppressor gene 1 (TMSG1) was a novel tumor metastasis-related gene identified using messenger RNA differential display from non-metastatic human prostate cancer cell variants. The mechanism of LASS2/TMSG1 inhibiting tumor invasion metastasis in breast cancer cells had not been well investigated. In the present study, a full length of 1.2 kb LASS2/TMSG1 complementary DNA (cDNA) coding for a protein of 380 amino acids was cloned. PcDNA3 eukaryotic expression plasmids of LASS2/TMSG1 were constructed and transfected into human breast cancer cell line MCF-7 by lipofectin transfection method. And, the biological effects were observed comparing with control groups. As the result, LASS2/TMSG1 inhibited cell growth in vitro by increasing apoptosis and changing cell cycle distribution. Furthermore, the vacuolar ATPase (V-ATPase) activity and extracellular hydrogen ion concentration were significantly decreased and the activity of secreted matrix metalloproteinase-2 (MMP-2) was downregulated in MCF-7 cells overexpressing LASS2/TMSG1 compared with the controls. Therefore, LASS2/TMSG1 may inhibit growth and invasion of breast cancer cell in vitro through decreasing V-ATPase activity and extracellular hydrogen ion concentration and inactivating secreted MMP-2. The findings provided the evidence that the LASS2/TMSG1 gene had tumor growth and invasion suppressor function in human breast cancer cell and may provide a promising target for cancer metastasis diagnosis and therapy.

Silencing of LASS2/TMSG1 enhances invasion and metastasis capacity of prostate cancer cell.

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), was firstly cloned by our laboratory in 1999. However, its antitumor molecular mechanisms are still unclear. LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar H(+) ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. In this study, we explored the effect of small hairpin RNA (shRNA) targeting LASS2/TMSG1 on the invasion and metastasis of human prostate carcinoma cell line PC-3M-2B4 with low metastatic potential and its functional interaction with V-ATPase. Silencing of LASS2/TMSG1 gene in PC-3M-2B4 cells increased V-ATPase activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2 and MMP-9, which coincided with enhancing cell proliferation, cell survival, and cell invasion in vitro, as well as acceleration of prostate cancer (PCA) growth and lymph node metastases in vivo. Thus we concluded that silencing of LASS2/TMSG1 enhances invasion and metastasis of PCA cell through increase of V-ATPase activity. These results establish LASS2/TMSG1 as a promising therapeutic target for advanced PCA.

TRPV1 channels are functionally coupled with BK(mSlo1) channels in rat dorsal root ganglion (DRG) neurons.

The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca(2+)). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca(2+) influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 µM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system.

TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids.

The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels.

Hysteresis of gating underlines sensitization of TRPV3 channels.

Vanilloid receptors of the transient receptor potential family have functions in thermal sensation and nociception. Among them, transient receptor potential vanilloid (TRPV)3 displays a unique property by which the repeated stimulation causes successive increases in its activity. The property has been known as sensitization and is observed in both native cells and cells heterologously expressing TRPV3. Transient increases in intracellular calcium levels have been implicated to play a key role in this process by mediating interaction of calmodulin with the channel. In support of the mechanism, BAPTA, a fast calcium chelator, accelerates the sensitization, whereas the slow chelator EGTA is ineffectual. Here, we show that the sensitization of TRPV3 also occurred independently of Ca(2+). It was observed in both inside-out and outside-out membrane patches. BAPTA, but not EGTA, has a direct potentiation effect on channel activation. Analogues of BAPTA lacking Ca(2+)-buffering capability were similarly effective. The stimulation-induced sensitization and the potentiation by BAPTA are distinguishable in reversibility. We conclude that the sensitization of TRPV3 is intrinsic to the channel itself and occurs as a result of hysteresis of channel gating. BAPTA accelerates the sensitization process by potentiating the gating of the channel.