Pain Reception Research Articles

Reactive Species Risk Assessment Using Optimized HET-CAM Safety Evaluation of Feed Gas-Modified Gas Plasma Technology and Anticancer Drugs.

Clinical therapies, including dermatology and oncology, require safe application. In vitro experiments allow only limited conclusions about in vivo effects, while animal studies in, e.g., rodents have ethical constraints at a large scale. Chicken embryos lack pain reception until day 15 postfertilization, making the in ovo model a suitable alternative to in vivo safety assessment. In addition, the hen's egg test on chorioallantoic membrane assay allows irritation potential analysis for topical treatments, but standardized analysis has been limited so far. Medical gas plasma is a topical, routine, approved dermatology treatment. Recent work suggests the potential of this technology in oncology. Its main mode of action is the release of various reactive species simultaneously. Intriguingly, varying plasma feed gas compositions generates customized reactive species profiles previously shown to be optimized for specific applications, such as skin cancer treatment. To support clinical implications, we developed a novel chicken embryo CAM scoring and study scheme and employed the model to analyze 16 different plasma feed gas settings generated by the atmospheric pressure plasmajet kINPen, along with common anticancer drugs (e.g., cisplatin) and physiological mediators (e.g., VEGF). Extensive gas- and liquid-phase plasma reactive species profiling was done and was found to have a surprisingly low correlation with irritation potential parameters. Despite markedly different reactive species patterns, feed gas-modulated kINPen plasma was equally tolerated compared to standard argon plasma. CAM irritation with gas plasmas but not anticancer agents was reversed 48 h after treatment, underlining the only temporary tissue effects of medical gas plasma. Our results indicate a safe therapeutic application of reactive species.

Analysis of the condition of the kallikrein-kinin system in the comorbid course of chronic pancreatitis and type 2 diabetes

Background. According to evidence, within approximately 8 years, 50% of patients with chronic pancreatitis (CP) develop endocrine and exocrine insufficiency, which manifests itself as steatorrhea, weight loss, diabetes mellitus (DM), and nutritional disorders. Diabetes mellitus aggravates these phenomena, which deepens the disorder of metabolic processes in CP, including protein metabolism. Protein structures include components of the kallikrein-kinin system (KKS), which plays a role in the regulation of vascular tone, diuresis, inflammation, coagulation, and pain reception. Due to repeated exacerbations of pancreatitis, there is fibrosis of the pancreas, which increases the risk of insulin resistance and the formation of type 2 diabetes. Thus, disorders of the kallikrein-kinin system and the progression of CP are interrelated, which makes it important to study the state of KKS in CP, especially in combination with type 2 diabetes mellitus. The purpose is to investigate the state of the kallikrein-kinin system in chronic pancreatitis in outpatients, depending on the presence of concomitant type 2 diabetes. Material and methods. 137 outpatients with CP with concomitant diabetes mellitus and without diabetes were studied: the main group— 112 patients with CP without exacerbation in combination with diabetes mellitus in a state of complete or subcompensation, and the comparison group— 25 patients with isolated CP. Evaluation of general and specific proteolysis (α1-proteinase inhibitor, α2-macroglobulin, kallikrein, kininase II activity, prekallikrein, plasma proteolytic activity) was performed by the method analysis using standard kits from BIOSERV ELISA. Results. There was a 20.8% increase in the level of the total proteolytic activity of plasma in the group of patients with comorbidity of CP and diabetes mellitus compared with the group with isolated CP, 51.9% increase in specific proteolysis (or kininogenesis)— the level of kallikrein (proteolysis enzyme), p<0.05. At the same time, a decrease in prekallikrein (inactive precursor of kallikrein) was found in the group of patients with comorbidity by 19.4% compared to that in isolated CP (p < 0.05). The presence of dissociation of protective parameters of kallikrein-kinin system is proved; increase by 9.4% of α1-proteinase inhibitor content at comorbidity of CP and DM2 in relation to that in isolated CP; decrease in the content of α2-macroglobulin in CP relative to the control group (p<0.05), which showed a decrease in the body’s compensatory capacity in CP, at the same time found an increase in α2-macroglobulin in the comorbidity of CP with DM2 by 49.5% relative to isolated CP; reduction of kininase-II activity in CP and diabetes mellitus by 14.5% relative to that in CP. Conclusions. Activation of KKS with multidirectional changes in KKS parameters and general and specific proteolysis in CP was noted. In CP and in the comorbid course with diabetes mellitus, further activation of proteolysis took place with the simultaneous inclusion of protective mechanisms for resolving inflammation and detoxification. Excessive production of kinins in concomitant DM2 has been proven, which weakened the body’s protective response.

NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC-Derived Nociceptor Maturation.

Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human induced pluripotent stem cell‐derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co‐sequencing, immunofluorescence staining, and qPCR validations, reveal expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerge as regulatory hubs for neurite outgrowth, synapse development, and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis reveals distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs).

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

Ecological Load and Balancing Selection in Circumboreal Barnacles.

Acorn barnacle adults experience environmental heterogeneity at various spatial scales of their circumboreal habitat, raising the question of how adaptation to high environmental variability is maintained in the face of strong juvenile dispersal and mortality. Here, we show that 4% of genes in the barnacle genome experience balancing selection across the entire range of the species. Many of these genes harbor mutations maintained across 2 My of evolution between the Pacific and Atlantic oceans. These genes are involved in ion regulation, pain reception, and heat tolerance, functions which are essential in highly variable ecosystems. The data also reveal complex population structure within and between basins, driven by the trans-Arctic interchange and the last glaciation. Divergence between Atlantic and Pacific populations is high, foreshadowing the onset of allopatric speciation, and suggesting that balancing selection is strong enough to maintain functional variation for millions of years in the face of complex demography.

Ganglion impar neurolysis in chronic perineal pain

<br><b>Background:</b> Chronic perineal pain (CPP)-mediated through ganglion impar produces complex symptoms causing functional impairment and chronic disabling persistent pain. Ganglion impar which is a solitary midline sympathetic ganglion receives afferent pain fibers from various pelvic organs mediates this type of nociception. A procedure such as ganglion impar block-neurolysis allows blockade of pain reception and mediation, providing effective pain relief. This study is aimed at analyzing the technical feasibility, safety, and efficacy of trans-sacrococcygeal ganglion impar neurolysis in patients with CPP at a rural territory care center. <b>Materials and Methods:</b> Twenty-six patients with CPP were prospectively evaluated for trans-sacrococcygeal ganglion impar neurolysis following good response to diagnostic block under fluoroscopy guidance. Numeric Pain Rating Scale (NPRS) score was observed at various time intervals. The number of attempts and procedural complications was also noted. <b>Results:</b> Out of 26 patients, 16 (62%) were of cancer origin and 10 (38%) cases of noncancer pain with a mean age of 53 and 34 years, respectively. The average NPRS score at 6 weeks in the cancer group was three, while the score was zero in noncancer group. NPRS score at presentation and at 8 weeks follow-up showed significant improvement in both groups. There was no more than a single attempt made to access ganglia through our approach. No procedural-related complications were found. <b>Conclusion:</b> Social burden of CPP, especially in the rural population, is neglected and may appear demanding both to patients and treating professionals. Transsacrococcygeal Ganglion impar block and neurolysis offer a safe, convenient day-care procedure without any significant complications. In cancerous origin of pain, neurolysis could be a good adjuvant.<br>

Neurotrophe Keratopathie

Corneal nerves are responsible for the reception of pain, temperature, and touch and are an important component of homeostasis of the ocular surface. When the corneal innervation is reduced, degenerative changes can be manifested as neurotrophic keratopathy (NK). In astage-dependent course, corneal epithelial alterations and ulcers may occur, even leading to perforation. The cause of NK is predominantly damage to the trigeminal nerve, which can be caused by infections (especially herpesviruses), metabolic diseases (such as diabetes mellitus) and by surgical procedures. The diagnosis of NK requires adetailed medical and ocular history, assessment of corneal sensitivity, and a complete ocular examination. The treatment of NK is oriented to the clinical severity and aims to restore the integrity of the ocular surface in order to prevent further disease progression.

TRPA1 channel contributes to myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1 ± 9.3 vs. 14.3 ± 9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3 ±8.4% and 42.2 ± 11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 237. Effect of a systemic postoperative cannabinoid receptor agonist on spine fusion in rat model

BACKGROUND CONTEXT Following spine surgery, most patients receive opioid medication for analgesia. Opioids have caused controversy in popular culture because of frequent overuse and negative side effects. Given the current opioid epidemic, alternative postoperative analgesic strategies are being explored. The endocannabinoid system, a known regulator of pain reception, has been shown to have anti-nociceptive activity in animal models of acute tissue and nerve injury. Likewise, endocannabinoids are important to bone homeostasis via activity at CB1 and CB2 receptors; increasing numbers of osteoblasts and limiting the development of osteoporosis. Like the FDA approved synthetic cannabinoid agonists currently used to treat cancer pain, WIN55.212-2 (WIN55) acts as a dual agonist at both CB1 and CB2 receptors. Our in vitro work has demonstrated that WIN55 has no negative effect on the osteogenic differentiation capacity of rat bone marrow stem cells (BMSC). Hence, WIN55 was examined as a postoperative pain analgesic in rats following posterolateral spinal fusion. PURPOSE To determine whether the dual cannabinoid receptor agonist, WIN55.212-2, has a quantifiable effect on spinal fusion when used for postoperative analgesia. METHODS Sprague Dawley rat BMSC was cultured under osteogenic conditions and treated with WIN55. Alkaline phosphatase (ALP) activity assay, matrix mineralization assay, and qPCR were performed. Forty-five female Sprague Dawley rats underwent L4-L5 PLF with placement of acellular-collagen sponge loaded with 1ug of recombinant bone morphogenetic protein-2. Experimental groups received either low dose (0.5mg/kg, n=15) or high dose (2.5mg/kg, n=15) WIN55, delivered postoperatively for 5 days via intraperitoneal injection. A control group (n=15) received intraperitoneal DMSO for 5 days postoperatively. All animals received routine intra-and postoperative pain control per protocol. Spine fusion was assessed using radiography, manual palpation-based fusion scoring, and microCT. For fusion scoring, an established scoring system was used whereby spines with an average score ≥1 were considered fused. RESULTS Early and late markers of osteogenic differentiation, ALP activity, and mineralization, were not inhibited by treatment of WIN55. mRNA expression of Alp and Run×2 was similar between DMSO and WIN55 treated cells. Rats treated with low dose WIN55 demonstrated the most robust fusion rate (93%) and mean fusion score (1.69). The high dose group had a fusion rate of 73% and a mean fusion score of 1.43. DMSO controls had the lowest fusion rate (60%) and score (1.17). The low dose group had a significantly greater fusion rate and mean score than the control group (pl0.05, pl0.05). No significant differences in fusion existed between the low dose and high dose groups. CONCLUSIONS The opioid crisis has prompted strong interest in the development of alternative strategies for pain management after spine procedures. However, clear evidence that alternative treatments have no negative impact on bone healing is necessary before consideration of their use. We have presented preclinical evidence that WIN55, a dual cannabinoid receptor agonist, does not adversely affect spinal fusion in a rat model. These results support the notion that cannabinoid receptor agonists may be used for postoperative analgesia following spine surgeries without risk of inhibiting bone healing. Continued analysis of microCT imaging will allow us to further quantify the effects of WIN55 on new bone growth.

The putative transient receptor potential channel protein encoded by the orf19.4805 gene is involved in cation sensitivity, antifungal tolerance, and filamentation in Candida albicans.

Transient receptor potential (TRP) channels, an ancient family of cation channels, are highly conserved in eukaryotes and play various physiological functions, ranging from sensation of ion homeostasis to reception of pain and vision. Calcium-permeable TRP channels have been identified from the plant Arabidopsis thaliana (AtCsc1) and the budding yeast Saccharomyces cerevisiae (ScCsc1). In this study, we characterized the functions of the Csc1 homolog, orf19.4805, in Candida albicans. Orf19.4805 is a protein of 866 amino acids and 11 transmembrane domains, which shares 49% identity (69% similarity) in amino acid sequence with ScRsn1. Here, we demonstrate that deletion of the orf19.4805 gene causes C. albicans cells to be sensitive to SDS (sodium dodecyl sulfate) and antifungal drugs, and tolerance to zinc, manganese, and cadmium ions. Candida albicans cells lacking orf19.4805 show a defect in filamentation in vitro. Therefore, orf19.4805 is involved in the regulation of cation homeostasis and filamentation in C. albicans.

C7 Ketamine for Post-operative Pain: Does It Affect Length of Stay in the PACU?

Enhanced Recovery After Surgery (ERAS) protocols aim to improve perioperative outcomes by using multimodal analgesia to reduce opioid use. Ketamine is a unique multimodal adjunct which modulates sensory pain reception while minimizing effects on the central respiratory drive. Common misconceptions about ketamine often prevent its use in the post-anesthesia care unit (PACU) setting, including delayed emergence from anesthesia and longer recovery time. The purpose of this review was to examine the use of ketamine in the PACU setting as a standard component of an ERAS protocol and to determine its effect on PACU length of stay.

Dual-Modal Tactile Perception and Exploration

Tactile sensing is required for human-like control with robotic manipulators. Multimodality is an essential component for these tactile sensors, for robots to achieve both the perceptual accuracy required for precise control, as well as the robustness to maintain a stable grasp without causing damage to the object or the robot itself. In this study, we present a cheap, 3D-printed, compliant, dual-modal, optical tactile sensor that is capable of both high (temporal) speed sensing, analogous to pain reception in humans and high (spatial) resolution sensing, analogous to the sensing provided by Merkel cell complexes in the human fingertip. We apply three tasks for testing the sensing capabilities in both modes; first, a depth modulation task, requiring the robot to follow a target trajectory using the high-speed mode; second, a high-resolution perception task, where the sensor perceives angle and radial position relative to an object edge; and third, a tactile exploration task, where the robot uses the high-resolution mode to perceive an edge and subsequently follow the object contour. The robot is capable of modulating contact depth using the high-speed mode, high accuracy in the perception task, and accurate control using the high-resolution mode.

Vitamin D concentration effect in health care and sport performance

The molecular structure of Vitamin D is closely allied to that of classical steroids such as cholesterol. Technically Vitamin D is a seco-steroid hormone. Vitamin D3 (cholecalciferol) is normally produced by exposure to sunlight of the precursor (7-dehydrocholesterol), present in the skin. Vitamin D supplementation vary between 200 IU to 1000 IU. Serum levels of 25(OH)D are generally consider as a indicator of Vitamin D status.Vitamin D plays an essential role in healthcare, related not only with developing and maintaining a healthy skeleton. Its adequate supplementation reduce even the risk of caries and oral infections. Furthermore Vitamin D as a steroid hormone, modulates many gene transcription and has anti-inflammatory and cardiovascular protective effect. Uncertainly low serum levels of 25(OH)D are associated with misbalance in lipid profile and dyslipidemia, sacropenia and muscle weakness. Its insufficiency is also a risk factor for enhanced reception of pain, risk of type 2 diabetes, and often falls occurrences.Adequate Vitamin D status is protective against musculoskeletal disorders, infection disease, depression, diabetes mellitus, autoimmune diseases and neurocognitive dysfunctions.In sport activities and athletic population adequate serum levels of 25(OH)D increase muscle strength, and physical performance, and should be monitored.

CNV1014802 Rescues the Paroxysmal Extreme Pain Disorders Nav1.7 Mutants by Restoring Impaired Inactivation

Voltage-gated sodium channel subtype Nav1.7 plays a critical role in pain reception and represents a promising target for pain relief. CNV1014802, a selective Nav1.7 inhibitor developed by Convergence Pharmaceuticals, has recently completed its phase II clinical trial in the treatment of trigeminal neuralgia. However, the inhibitory features and other potential applications of CNV1014802 remain elusive. Firstly, the effects of CNV on kinetics of Nav1.7 were characterized using whole-cell patch clamp. We find that holding potentials predominantly determines its inhibition potency on Nav1.7 channels. The IC50 values are 1.77 ± 0.28 µM and 71.66 ± 11.93 µM, respectively, when holding potentials are set at −70 mV or −120 mV. Moreover, CNV1014802 significantly hyperpolarizes channel inactivation whereas does not affect the channel activation. In addition, CNV1014802 accelerates the inactivation onset and delays the recovery from inactivation. Finally, CNV1014802 is capable of rescuing Nav1.7 mutations casual of paroxysmal extreme pain disorders (PEPD) by restoring their impaired inactivation to that of wild-type channels. Our study indicates that CNV1014802 may benefit hereditary painful disorder patients with Nav1.7 mutations impairing inactivation.Keywords: Nav1.7, CNV1014802, Inactivation, Paroxysmal extreme pain disorders

P29. Transversal spinal cord lesion after electrical injury

Case A 37 year old electrician presented with pain and paresthesia after electrical injury. The patient reported that he was removing a heating system at work. Because of an incorrect electrical installation a short circuit was built up by the patient holding the heater in both hands. The 230 voltage flowed through both arms. Because of a defect fuse the electric flow was not interrupted. During and a short time after the accident the patient reported strong pain in both arms and between the scapulae. Initially the symptoms were regressive. With a delay of 1 week the pain restarted and paresthesia in both arms surfaced. The patient presented himself to an emergency department of a local clinic. He was admitted and within one more day a motoric syndrome with a tetraparesis and passive incontinence developed. The patient was sent to a local neurological clinic for further diagnostic investigation. MRI of the spinal cord showed a correlating lesion C 4/5–C5/6. Functional deficits resulted in a sensomotorical transverse section down from C6 with focus on both arms. Although specific diagnostic, including ultrasound and vasculitis screening, was negative the lesion was suspected to be a spinal ischemia. Following treatment was done at the Department of Neurological Rehabilitation. The patient’s history was reevaluated and the diagnosis was revealed as a symptomatic lesion after electrical injury. Clinical and electrophysiological investigations were performed at the Department of Neurology. Quantitative sensoric testing (QST) showed pathological results for temperature and pain reception. Additional, a reduction of the density of intraepidermal nerve fiber could be seen in biopsy of affected skin area. After a year of intensive neurorehabilitation most symptoms recovered, except mild paresthesia, erectile dysfunction and reduced fine motor skills. Conclusion Electrical injury is a rare cause of spinal cord lesion. While diagnostic and therapy is focused mostly on acute symptoms, also delayed complications have to be considered.

Post-Translational Modifications of TRP Channels

Transient receptor potential (TRP) channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

The zebrafish as a model for nociception studies

Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely, the use of Drosophila and Caenorhabditis elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model.

Probing TRPV1 Structure with Limited Proteolysis

The transient receptor potential (TRP) family of ion channels is a large class of ion channels that is involved in various biological functions. These ion channels are activated by a wide range of stimuli but the general membrane topology and permeability to cations are common features.This study focuses mainly on one of the members, TRPV1, which responds to noxious heat and ligands like protons and capsaicin. This ion channel is situated in nociceptive sensory neurons and thereby upon activation; TRPV1 causes a sensation of pain. TRPV1 has been the subject of intense research due to this involvement in pain reception although much is left to be discovered regarding its detailed structure and the regulatory mechanisms during desensitization and tachyphylaxis.This study utilizes the principle of limited proteolysis as a way to attain information about surface exposed regions of TRPV1. Since flexible, exposed regions of a protein are more prone to proteolysis, information regarding flexible and dense regions of a protein can be obtained if proteolysis is performed at different time scales. Exposed regions often correlates with ligand binding sites making limited proteolysis a usable tool for fast screening of possible binding sites.The study is performed by immobilizing membrane vesicles containing the protein in a flow cell, subsequently exposing them to trypsin and finally analyzing eluted peptides with mass spectrometry. A protocol using sequential digestions rather than standard batch digestions proved to yield higher sequence coverage.

Activity of TRPV1 Channels in Primary Nociceptive Neurons of Rats: Modulation by Changes in Intracellular Calcium Level

In rat neurons of the dorsal root ganglia (DRG) with mid- (35 to 25 μm) and small-sized (less than 25 μm) somata, we studied calcium transients induced by application of capsaicin (selective agonist of TRPV1 channels) under conditions of the development of other calcium transients caused by preliminary depolarization of the plasma membrane of these neurons. The above transients in rat DRG neurons were measured using the calcium-sensitive fluorescent dye Fura 2/AM. At delays of 3, 7, and 10 sec with respect to the beginning of preliminary potassium depolarization, the amplitudes of capsaicin-induced responses were smaller, as compared with the control, on average, by 26.8, 22.1, and 4.5%, respectively, in the population of mid-sized neurons and by 35.3, 21.1, and 22.4% in small neurons. Under such conditions, we observed noticeable delays of reactions to applications of capsaicin and a certain decrease in the level of intracellular calcium at the moment of beginning of development of these reactions with respect to the corresponding values in isolated depolarization-induced transients. We conclude that excitation of primary nociceptive neurons and activation of voltage-operated calcium channels result in noticeable modulation of the activity of TRPV1 channels and change their role during pain reception.

Nav Channel Mechanosensitivity: Activation and Inactivation Accelerate Reversibly with Stretch

Voltage-gated sodium channels (Nav) are modulated by many bilayer mechanical amphiphiles, but whether, like other voltage-gated channels (Kv, HCN, Cav), they respond to physical bilayer deformations is unknown. We expressed human heart Nav1.5 pore α-subunit in oocytes (where, unlike αNav1.4, αNav1.5 exhibits normal kinetics) and measured small macroscopic currents in cell-attached patches. Pipette pressure was used to reversibly stretch the membrane for comparison of I Na( t) before, during, and after stretch. At all voltages, and in a dose-dependent fashion, stretch accelerated the I Na( t) time course. The sign of membrane curvature was not relevant. Typical stretch stimuli reversibly accelerated both activation and inactivation by ∼1.4-fold; normalization of peak I Na( t) followed by temporal scaling (∼1.30- to 1.85-fold) resulted in full overlap of the stretch/no-stretch traces. Evidently the rate-limiting outward voltage sensor motion in the Nav1.5 activation path (as in Kv1) accelerated with stretch. Stretch-accelerated inactivation occurred even with activation saturated, so an independently stretch-modulated inactivation transition is also a possibility. Since Nav1.5 channel-stretch modulation was both reliable and reversible, and required stretch stimuli no more intense than what typically activates putative mechanotransducer channels (e.g., stretch-activated TRPC1-based currents), Nav channels join the ranks of putative mechanotransducers. It is noteworthy that at voltages near the activation threshold, moderate stretch increased the peak I Na amplitude ∼1.5-fold. It will be important to determine whether stretch-modulated Nav current contributes to cardiac arrhythmias, to mechanosensory responses in interstitial cells of Cajal, to touch receptor responses, and to neuropathic (i.e., hypermechanosensitive) and/or normal pain reception.