Single Cysteines in the Extracellular and Transmembrane Regions Modulate Pannexin 1 Channel Function
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Pannexins form high-conductance ion channels in the membranes of many vertebrate cells. Functionally, they have been associated with multiple functional pathways like the propagation of calcium waves, ATP release, responses to ischemic conditions and apoptosis. In contrast to accumulating details which uncovered their functions, the molecular mechanisms for pannexin channel regulation and activation are hardly understood. To further elucidate regulatory mechanisms, we substituted cysteine residues, expected key elements for channel function, in extracellular and transmembrane regions of Pannexin 1 (Panx1). Most apparently, substitution of the transmembrane cysteine C40 resulted in constitutively open channels with profoundly increased activity. Hence, Xenopus laevis oocytes injected with corresponding cRNA showed strongly impaired viability, anomalous dye uptake and greatly increased whole-cell conductivity. All changes induced by C40 substitution were significantly reduced by the Panx1 channel blocker carbenoxolone, indicating that channel activity of the mutated Panx1 had been affected. In contrast, no changes occurred after substitution of the two other transmembrane cysteines, C215 and C227, in terms of channel conductivity. Finally, substitution of any of the four extracellular cysteines resulted in complete loss of channel function in both X. laevis oocytes and transfected N2A cells. From this, we conclude that cysteine residues of Panx1 reveal differential functional profiles for channel activation and drug sensitivity.
KeywordsMembrane protein Site-directed mutagenesis Hemichannel Pannexin Channel activity
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