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Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells

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Abstract

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

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Abbreviations

aa:

Amino acids

ATP:

Adenosine triphosphate

bp:

Base pairs

CPC:

Cryopreparation chamber

DCE:

Dichloroethane

E:

Extraplasmic

ECL:

Electrochemiluminescence

EtBr:

Ethidium bromide

EYFP:

Enhanced yellow fluorescent protein

FCS:

Fetal calf serum

FRIL:

Freeze-fracture replica immunolabeling

GFP:

Green fluorescent protein

HEK:

Human embryonic kidney

IMP:

Intramembrane particle

LBB:

Labeling blocking buffer

LSM:

Laser scanning microscopy

P:

Protoplasmic

Panx1:

Pannexin 1

SDS:

Sodium dodecylsulfate

SEM:

Standard error of the mean

SD:

Standard deviation

SPB:

Soerensen’s phosphate buffer

SNR:

Signal-to-noise ratio

TEM:

Transmission electron microscope

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Acknowledgments

The authors wish to thank John E. Rash and Thomas Yasumura for their expert advice and continuous support. Thanks also to Alina Mattheis for the expertly drawn illustration. The authors thank Ann Soether for linguistic editing. The authors acknowledge financial support by the German Research Foundation and the Saarland University who funded the freeze-fracture unit and the LSM.

Declarations

The authors declare that they have no competing interests.

Author’s contributions

AB and AG carried out all molecular techniques, immunoblots and freeze-fracture procedures, participated in the design of the study, analyzed and documented the data. EK carried out the laser scanning microscopy. TT participated in the design of the study and in manuscript preparation. CM conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Saarland University, Building 61, 66421, Homburg/Saar, Germany

    Anja Beckmann, Alexander Grissmer, Thomas Tschernig & Carola Meier

  2. Department of Physiology, Saarland University, 66421, Homburg/Saar, Germany

    Elmar Krause

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  1. Anja Beckmann
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Correspondence to Carola Meier.

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Beckmann, A., Grissmer, A., Krause, E. et al. Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells. Cell Tissue Res 363, 751–763 (2016). https://doi.org/10.1007/s00441-015-2281-x

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  • DOI: https://doi.org/10.1007/s00441-015-2281-x

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