Hernandez Gonzalez, Victor Hugo (2008) Biophysical analysis of gap-junction channels involved in congenital diseases. [Tesi di dottorato]
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Mutations in connexin genes have been linked to a variety of human diseases, including cardiovascular anomalies, peripheral neuropathies, deafness, skin disorders and cataracts. In connexin-based gap junction channels, each cell contributes a hexamer of connexins forming half a channel, or connexon, which, in the narrow extracellular cleft, interacts with and aligns to another connexon from the adjacent cell. Several endogenous ions and low molecular weight species have been shown to cross gap junction channels, including all current- arrying anions and cations, glycolytic intermediates, vitamins, amino acids, nucleotides, as well as some of the more important second messengers involved in cell signaling, such as InsP3 and cAMP. InsP3 can be considered a global messenger molecule. InsP3 molecules diffuse throughout the cell nearly unbuffered with a diffusion coefficient of 280 µm2/s and lifetime up to 60s in the cytoplasm depending on cell type, interact with specific receptors (InsP3R) present in the endoplasmic reticulum and Ca2+ is liberated, raising its concentration in the cytosol. Similar to InsP3, cAMP is a ubiquitous intracellular
second messenger that affects cell physiology by directly interacting with effector molecules, including cAMP-dependent protein kinases (PKA), cyclic nucleotide-gated ion channels (CNG channels), hyperpolarization activated channels and the guanine exchange factor EPAC. A variety of specific functions have been proposed for the intercellular transfer of ions and endogenous solutes through gap junction channels, yet the procedures that have gained wide acceptance in assaying the molecular permeability of connexins are dependent on the introduction into living cells of exogenous markers which are then traced in their individual intercellular movements.
Direct measurement of endogenous messengers' transit has been so far problematic mostly due to lack of selective reporters. For example, to compare the transfer rate of cAMP through gap junction channels formed by different connexins, CFTR-mediated chloride currents, Ca2+ currents through CNG channels and Ca2+ imaging have been utilized as sensors for cAMP. Defective permeation of cAMP through gap junctions between adjacent cytoplasmic loops of myelinating Schwann cells has been hypothesized to underlie certain forms of CMTX disease. Also the transfer of InsP3 has been detected indirectly, using Ca2+ imaging as the readout for InsP3 dynamics. InsP3 permeability defects detected by a Ca2+ reporter system in supporting cells of the auditory sensory epithelium, in which Cx26 and Cx30 are expressed at high levels, have been recently implicated in genetic deafness. In an effort to develop direct, quantitative and reproducible means to monitor the flux of cAMP or InsP3 through recombinant connexin channels, we used novel ratiometric fluorescent biosensors that exploit the phenomenon of FRET for the quantitative monitoring of second messenger concentrations in single living cells in real time. This approach may have a general impact as it provides fast and reliable estimates of connexin permeability to second messengers and permits to investigate their role in the physiology and pathology of cell-cell communication.
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