Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC

doi:10.1073/pnas.0603154104

Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC

*Department of Neuroscience and Cell Biology,
^†Division of Nephrology and Hypertension, Department of Internal Medicine, and
^‡Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0437

Edited by Michael V. L. Bennett, Albert Einstein College of Medicine, Bronx, NY, and approved January 8, 2007 (received for review April 18, 2006)

Abstract

Gap-junctional channels, permeable to large hydrophilic solutes of up to M _r ≈ 1,000, are responsible for cell-to-cell communication. Phosphorylation of connexin 43 (Cx43) by PKC abolishes the permeability of gap-junctional channels and hemichannels to large hydrophilic solutes, but not to small inorganic ions. Here, we report on a methodology to produce purified hemichannels of controlled subunit composition and apply it to the generation of hemichannels with variable number of PKC-phosphorylated subunits. The subunit composition was determined by luminescence resonance energy transfer. We show that all Cx43 subunits in the hemichannel hexamer have to be phosphorylated to abolish sucrose (M _r 342) permeability. We also show that the hemichannel pores with all subunits phosphorylated by PKC have a sizable diameter, allowing for permeation of the small hydrophilic solute ethyleneglycol (M _r 62). These results indicate that phosphorylation of Cx43 by PKC alters the hemichannel size selectivity and explain why PKC activity affects dye transfer between cells without consistent effects on electrical communication.

Footnotes

^§To whom correspondence should be addressed. E-mail: galtenbe{at}utmb.edu
Author contributions: X.B. and S.C.L. contributed equally to this work; L.R. and G.A.A. designed research; X.B., S.C.L., and G.A.A. performed research; X.B., S.C.L., L.R., and G.A.A. analyzed data; and L.R. and G.A.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0603154104/DC1.
Abbreviations:

Cx43,

connexin 43;

Cx43-dP,

dephosphorylated Cx43;

Cx43-P,

PKC-phosphorylated Cx43;

decylmaltoside,

n-decyl-β-d-maltopyranoside;

DTPA,

diethylenetriaminepentaacetate;

EMCH,

ε-maleimidocaproic acid hydrazide;

LRET,

luminescence resonance energy transfer.
Freely available online through the PNAS open access option.