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Vol. 20, Issue 14, 3342-3352, July 15, 2009

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Gap Junction Turnover Is Achieved by the Internalization of Small Endocytic Double-Membrane Vesicles

Matthias M. Falk^*, Susan M. Baker^*,, Anna M. Gumpert^*,, Dominique Segretain, and Robert W. Buckheit, III^*

*Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015; and Institut National de la Santé et de la Recherche Médicale U895, Université Paris Descartes, 75006 Paris, France

Submitted April 9, 2009; Revised May 6, 2009; Accepted May 11, 2009
Monitoring Editor: Sandra L. Schmid

Double-membrane–spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains (0.05–0.5 µm in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles (0.18–0.27 µm in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1–5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.

Author contributions: M.M.F. designed the research; M.M.F., D. S., A.M.G., and R.W.B. performed the experiments; S.M.B. and A.M.G. analyzed the data; M.M.F. wrote the paper; and S.M.B. edited the manuscript.

These authors contributed equally to this work.

Address correspondence to: Matthias M. Falk (mfalk{at}lehigh.edu)

Abbreviations used: AGJ, annular gap junction; Cx, connexin; GFP, green fluorescent protein; GJ, gap junction; NM, nonjunctional membrane; PM, plasma membrane; wt, wild type.

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