ABSTRACT
Mutations in voltage-gated sodium channels are associated with altered pain perception in humans. Most of these mutations studied to date present with a direct and intuitive link between the altered electrophysiological function of the channel and the phenotype of the patient. In this study, we characterize a variant of Nav1.8, D1639N, which has been previously identified in a patient suffering from the chronic pain syndrome "small fiber neuropathy". Using a heterologous expression system and patch-clamp analysis, we show that Nav1.8/D1639N reduces current density without altering biophysical gating properties of Nav1.8. Therefore, the D1639N variant causes a loss-of-function of the Nav1.8 sodium channel in a patient suffering from chronic pain. Using immunocytochemistry and biochemical approaches, we show that Nav1.8/D1639N impairs trafficking of the channel to the cell membrane. Neither co-expression of ß1 or ß3 subunit, nor overnight incubation at 27 °C rescued current density of the D1639N variant. On the other hand, overnight incubation with lidocaine fully restored current density of Nav1.8/D1639N most likely by overcoming the trafficking defect, whereas phenytoin failed to do so. Since lidocaine rescues the loss-of-function of Nav1.8/D1639N, it may offer a future therapeutic option for the patient carrying this variant. These results demonstrate that the D1639N variant, identified in a patient suffering from chronic pain, causes loss-of-function of the channel due to impaired cell surface trafficking and that this trafficking defect can be rescued by lidocaine.
Subject(s)
Anesthetics, Local/pharmacology , Chronic Pain/genetics , Lidocaine/pharmacology , Loss of Function Mutation , NAV1.8 Voltage-Gated Sodium Channel/genetics , Action Potentials , Animals , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/physiology , Humans , NAV1.8 Voltage-Gated Sodium Channel/metabolism , Protein Transport/drug effects , XenopusABSTRACT
Effective and specific targeting of nanoparticles is of paramount importance in the fields of targeted therapeutics and diagnostics. In the current study, we investigated the targeting efficacy of nanoparticles that were functionalized with two angiogenesis-specific targeting ligands, an alpha(v)beta(3) integrin-specific and a galectin-1-specific peptide. We show in vitro, using optical techniques and MRI, that the dual-targeting approach produces synergistic targeting effects, causing a dramatically elevated uptake of nanoparticles as compared to single ligand targeting.
Subject(s)
Galectin 1/chemistry , Integrin alphaVbeta3/chemistry , Liposomes , Magnetic Resonance Imaging/methods , Nanotechnology/methods , Neovascularization, Pathologic , Angiogenesis Inhibitors/pharmacology , Cells, Cultured , Humans , Ligands , Magnetics , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Nanoparticles/chemistry , Peptides/chemistryABSTRACT
Noninvasive diagnostic imaging methods to establish the efficacy of angiostatic therapies are becoming increasingly important with the first Food and Drug Administration approvals of such agents. Magnetic resonance molecular imaging is an imaging technique that allows the visualization of pathological processes in vivo with a better spatial resolution as compared with nuclear methods, such as photon emission tomography and single photon emission computed tomography. In this study, we used alpha(v)beta3 targeted bimodal liposomes to quantitate angiogenesis in a tumor mouse model with magnetic resonance imaging (MRI) and to evaluate the therapeutic efficacy of the angiogenesis inhibitors anginex and endostatin. The MRI findings were validated with fluorescence microscopy and showed a very good correlation with the microvessel density. In conclusion, this study provides evidence that molecular MRI can be used to noninvasively measure the efficacy of angiogenesis inhibitors during the course of therapy.
