Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane α helices and a third cytoplasmic α helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.
This review summarizes recent findings on peripheral mechanisms underlying the generation and inhibition of pain. The focus is on events occurring in peripheral injured tissues that lead to the sensitization and excitation of primary afferent neurons, and on the modulation of such mechanisms. Primary afferent neurons are of particular interest from a therapeutic perspective because they are the initial generator of noxious impulses traveling towards relay stations in the spinal cord and the brain. Thus, if one finds ways to inhibit the sensitization and/or excitation of peripheral sensory neurons, subsequent central events such as wind-up, sensitization and plasticity may be prevented. Most importantly, if agents are found that selectively modulate primary afferent function and do not cross the blood-brain-barrier, centrally mediated untoward side effects of conventional analgesics (e.g. opioids, anticonvulsants) may be avoided. This article begins with the peripheral actions of opioids, turns to a discussion of the effects of adrenergic co-adjuvants, and then moves on to a discussion of pro-inflammatory mechanisms focusing on TRP channels and nerve growth factor, their signaling pathways and arising therapeutic perspectives.
Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.
Holt-Oram syndrome (HOS) is an autosomal dominant hearthand syndrome characterized by congenital heart disease (CHD) and upper limb deformity, and caused by mutations in the TBX5 gene. To date, the sensitivity of TBX5 genetic testing for HOS has been unclear. We now report mutational analyses of a nongenetically selected population of 54 unrelated individuals who were consecutively referred to our center with a clinical diagnosis of HOS. TBX5 mutational analyses were performed in all individuals, and clinical histories and findings were reviewed for each patient without reference to the genotypes. Twenty-six percent of the complete cohort was shown to have mutations of the TBX5 gene. However, among those subjects for whom clinical review demonstrated that their presentations met strict diagnostic criteria for HOS, TBX5 mutations were identified in 74%. No mutations were identified in those subjects who did not meet these criteria. Thus, these studies validate our clinical diagnostic criteria for HOS including an absolute requirement for preaxial radial ray upper limb malformation. Accordingly, TBX5 genotyping has high sensitivity and specificity for HOS if stringent diagnostic criteria are used in assigning the clinical diagnosis. HOS is the most common of the heart-hand syndromes. It segregates in an autosomal dominant fashion and is estimated to occur in at least 1/100,000 live births (1). HOS is characterized by upper limb anomalies involving the preaxial radial ray and CHD (2,3). Upper limb deformity may be bilateral but asymmetric or even unilateral. The most common forms of CHD associated with HOS are ASD, usually of the ostium secundum variety, and VSD, usually occurring in the muscular trabeculated septum. Cardiac conduction disease may also occur, regardless of the presence or absence of structural cardiac disease (3
BACKGROUND AND PURPOSE Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid‐sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons. EXPERIMENTAL APPROACH To examine the contribution of ASIC3 to the development and maintenance of muscle pain and inflammatory pain, we studied the in vivo efficacy of a selective ASIC3 inhibitor, APETx2, in rats. KEY RESULTS Administration of APETx2 into the gastrocnemius muscle prior to the administration of low pH saline prevented the development of mechanical hypersensitivity, whereas APETx2 administration following low‐pH saline was ineffective in reversing hypersensitivity. The prevention of mechanical hypersensitivity produced by acid administration was observed whether APETx2 was applied via i.m. or i.t. routes. In the complete Freund's adjuvant (CFA) inflammatory pain model, local administration of APETx2 resulted in a potent and complete reversal of established mechanical hypersensitivity, whereas i.t. application of APETx2 was ineffective. CONCLUSIONS AND IMPLICATIONS ASIC3 contributed to the development of mechanical hypersensitivity in the acid‐induced muscle pain model, whereas ASIC3 contributed to the maintenance of mechanical hypersensitivity in the CFA inflammatory pain model. The contribution of ASIC3 to established hypersensitivity associated with inflammation suggests that this channel may be an effective analgesic target for inflammatory pain states.
The structures of an increasing number of channels and other alpha-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family. In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins. In the case of channels, alpha-helices create the sealed barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes. The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle. The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity. Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.
Introduction: There is an unmet medical need for pruritus associated with chronic kidney disease, a distressing complication characterized by generalized and persistent itch affecting 20% to 40% of patients undergoing hemodialysis. Here we report the results of a phase 2 trial evaluating the efficacy and safety of a novel peripherally restricted kappa opioid receptor agonist, difelikefalin, in adult patients undergoing hemodialysis with pruritus. Methods: In this study, 174 hemodialysis patients with moderate-to-severe pruritus were randomly assigned to receive difelikefalin (0.5, 1.0, or 1.5 mg/kg) or placebo intravenously thrice weekly after each hemodialysis session for 8 weeks in a double-blind, controlled trial. The primary endpoint was the change from baseline at week 8 in the weekly mean of the 24-hour Worst Itching Intensity Numerical Rating Scale score. The secondary efficacy endpoint was the change in itch-related quality of life measured by the Skindex-10 questionnaire. Other endpoints included safety, sleep quality, and additional measures including the 5-D itch scale. Results: A significant reduction from baseline in itch intensity scores at week 8 favored all difelikefalin doses combined versus placebo (P ¼ 0.002). Difelikefalin also showed improvement over placebo in Skindex-10, 5-D itch, and sleep disturbance scores (P # 0.005). Overall, 78% of patients receiving difelikefalin reported treatment-emergent adverse events versus 42% of patients given placebo, with diarrhea, dizziness, nausea, somnolence, and fall being the most frequent ($5%). Conclusion: In this trial, difelikefalin effectively reduced itching intensity and improved sleep and itchrelated quality of life.
The voltage-gated K ؉ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10 4 to 5 ؋ 10 5 functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a ϳ2-kDa coresugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of ϳ64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the ϳ64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of ϳ65 ؋ 65 Å. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K ؉ -selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.
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