Browsing by Author "Rojas, Maximiliano"

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    A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII
    (2021) Lopez, Ximena; Palacios-Prado, Nicolas; Guiza, Juan; Escamilla, Rosalba; Fernandez, Paola; Vega, Jose L.; Rojas, Maximiliano; Marquez-Miranda, Valeria; Chamorro, Eduardo; Cardenas, Ana M.; Maldifassi, Maria Constanza; Martinez, Agustin D.; Duarte, Yorley; Gonzalez-Nilo, Fernando D.; Saez, Juan C.
    Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation- measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1EGFP and rPanx1S394D-EGFP channels showed current at all voltages between +/- 100 mV, similar single channel currents with outward rectification, and unitary conductance (similar to 30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
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    Different Classes of Antidepressants Inhibit the Rat α7 Nicotinic Acetylcholine Receptor by Interacting within the Ion Channel: A Functional and Structural Study
    (2021) Duarte, Yorley; Rojas, Maximiliano; Canan, Jonathan; Perez, Edwin G.; Gonzalez-Nilo, Fernando; Garcia-Colunga, Jesus
    Several antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat alpha 7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, I-Ch, which activates alpha 7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat alpha 7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited I-Ch with the order: norfluoxetine similar to mirtazapine similar to imipramine < bupropion similar to fluoxetine similar to venlafaxine similar to escitalopram. The constructed homology model of the rat alpha 7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the alpha 7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of I-Ch and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the alpha 7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.
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    Endogenous pannexin1 channels form functional intercellular cell-cell channels with characteristic voltage-dependent properties
    (2022) Palacios-Prado, Nicolas; Soto, Paola A.; Lopez, Ximena; Choi, Eun Ju; Marquez-Miranda, Valeria; Rojas, Maximiliano; Duarte, Yorley; Lee, Jinu; Gonzalez-Nilo, Fernando D.; Saez, Juan C.
    The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exoge-nous human pannexin1 (hPanx1) cell-cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell-cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single -channel conductance of -175 pS, with a substate of -35 pS; the latter showed a pecu-liar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell-cell channels were also iden-tified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfer-ing RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell-cell cou-pling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibi-tor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the chan-nel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell-cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell-cell channels in different cell types might require special attention.