RESUMO
Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, "noncanonical" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.
Assuntos
Conexinas , Canais Iônicos , Humanos , Conexinas/genética , Conexinas/metabolismo , Células HeLa , Junções Comunicantes/metabolismo , DNA/genéticaRESUMO
Connexin (Cxs) hemichannels participate in several physiological and pathological processes, but the molecular mechanisms that control their gating remain elusive. We aimed at determining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in "permanently closed hemichannels", which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.