RESUMO
Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.
Assuntos
Conexina 26/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Animais , Conexina 26/genética , Conexinas/genética , Junções Comunicantes/genética , Células HeLa , Humanos , Técnicas de Patch-Clamp , Xenopus laevisRESUMO
In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.
Assuntos
Doenças Desmielinizantes/genética , Neuregulina-1/genética , Comunicação Parácrina , Células de Schwann/metabolismo , Nervo Sural/metabolismo , Animais , Animais Geneticamente Modificados , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Atividade Motora , Proteínas da Mielina/genética , Neuregulina-1/metabolismo , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/metabolismo , Neurite Autoimune Experimental/patologia , Neuroglia/metabolismo , Ratos , Receptor ErbB-2/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/lesões , Transdução de Sinais , Nervo Sural/ultraestrutura , Nervo TibialRESUMO
Michael W. Sereda was incorrectly associated with the Department of Cellular Neurophysiology, Hanover Medical School, Carl-Neuberg-Str. 1, 30625 Hanover, Germany. The correct affiliations for Michael W. Sereda are Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany and Department of Clinical Neurophysiology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.