title: Unified patch clamp protocol for the characterization of Pannexin 1 channels in isolated cells and acute brain slices.
abstract: In the central nervous system, Pannexin 1 (Panx1) channels are implicated in a variety of physiological and pathological conditions. One of the prerequisites to enlighten the role of Panx1 is the development and standardization of reliable methods. Here, we address the applicability of voltage clamp protocols to identify Panx1 channel mediated currents in neurons of acutely dissected brain slices. We improved an established protocol and report on a modified paradigm that robustly evokes Panx1 channel currents. Crucial advances are the use of physiologic ion gradient conditions and a preconditioning step of depolarizing membrane potential ramps of long duration. This new paradigm provides significant impact on membrane current generation at hypo- and depolarized holding potential steps post voltage ramp preconditioning in heterologous expression systems and primary hippocampal CA1 neurons of mouse brain slices in vitro. Finally, we demonstrate that under these conditions the analysis of tail currents elicited by repolarization of the cells from preconditioning holding potential depolarization permits an independent method to isolate Panx1 mediated channel activity. In summary, this study provides a comprehensive methodological improvement in the biophysical analysis of Panx1 channels with a particular focus on investigations under physiological conditions in complex tissues.
authors.affiliation: Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Gründken
authors.forename: Julian
authors.initials: J
authors.lastname: Hanske
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wengel
authors.forename: Wiebke
authors.initials: W
authors.lastname: Reuter
authors.forename: Amr
authors.initials: A
authors.lastname: Abdulazim
authors.forename: Valery I
authors.initials: VI
authors.lastname: Shestopalov
authors.forename: Rolf
authors.initials: R
authors.lastname: Dermietzel
authors.forename: Georg
authors.initials: G
authors.lastname: Zoidl
authors.forename: Nora
authors.initials: N
authors.lastname: Prochnow
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D056547:CA1 Region, Hippocampal / Q000378:metabolism; D002412:Cations / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D017630:Connexins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004558:Electric Stimulation; D005456:Fluorescent Dyes / Q000032:analysis; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D008164:Luminescent Proteins / Q000032:analysis; D015767:Mefloquine / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection; D029961:Zebrafish Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D017630:Connexins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D011993:Recombinant Fusion Proteins; D029961:Zebrafish Proteins; C544702:panx1a protein, zebrafish; C054562:yellow fluorescent protein, Bacteria; D000255:Adenosine Triphosphate; D015767:Mefloquine