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

Abstract

Gap-junctional channels, permeable to large hydrophilic solutes of up to M(r) approximately 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.

Fig. 1.

Effects of PKC-mediated phosphorylation of hemichannels on their permeability to hydrophilic solutes. The proteoliposomes were loaded with radiolabeled probes, and the percent retention of the permeability probes was measured after gel filtration. Studies were performed in proteoliposomes containing hemichannels formed by fully dephosphorylated Cx43 (Cx43-dP) or Cx43 fully phosphorylated by PKC (all six Ser-368 residues phosphorylated, Cx43-P). Values were normalized to the amount of probe retained by liposomes formed by Cx43-P (statistically indistinguishable from the value measured in liposomes without hemichannels, 112 ± 9%, n = 7), after subtraction of the background measured in DMSO-permeabilized liposomes. Ethyleneglycol values were normalized to those in liposomes without hemichannels. The average number of hemichannels per liposome was 2.3. Data are means ± SEM of four to seven experiments. ∗, P < 0.05 compared with proteoliposomes containing Cx43-dP hemichannels.

Fig. 2.

Gel-filtration chromatography of hemichannel mixtures. Purified Cx43 and Cx43-EGFP gently mixed overnight at a 4:2 molar ratio, at 4°C, were analyzed by gel filtration in 0.3% decylmaltoside, 150 mM NaCl, 0.1 mM EDTA, and 10 mM Hepes/NaOH, pH 7.5. Ferritin (440 kDa, labeled 2) and aldolase (158 kDa, labeled 4) or thyroglobulin (669 kDa, labeled 1), catalase (232 kDa, labeled 3), and aldolase (158 kDa, labeled 4) were mixed with Cx43 and Cx43-EGFP, respectively, before injection into the FPLC system. Absorbance was measured at 280 nm (A₂₈₀) and normalized to the hexamer peak values.

Fig. 3.

LRET between Tb³⁺- and fluorescein-labeled Cx43 subunits in hemichannels. Purified WT Cx43 solubilized in 0.3% decylmaltoside was labeled with either fluorescein maleimide or Tb³⁺-DTPA-cs124-EMCH, by incubation for 2 h at 4°C with a 10-fold molar excess of the thiol reagents. Unreacted labels were removed by gel filtration, and unlabeled and labeled proteins were mixed in varying proportions and incubated for at least 2 h before analysis. (A) Steady-state fluorescence emission spectra (excitation at 490 nm). Data are normalized to the peak value of fully fluorescein-labeled preparation. (B) Gated emission spectra (60-μs delay after a 337-nm, 1-ns pulse from a nitrogen laser). Control experiments showed that at the concentrations present during Cx43 labeling there is no significant LRET between free Tb³⁺ and free fluorescein after gel filtration. Data were normalized to the peak value of 1 Tb/5 fluorescein-labeled preparation. Traces are representative from four independent experiments.

Fig. 4.

Determination of hemichannel composition. (A) Sensitized fluorescence emission (60-μs delay after pulse), normalized as described in Fig. 3B. (B) Time course of sensitized fluorescence emission (520 ± 20 nm bandpass filter, red). (Inset) Donor Tb³⁺ emission (540 ± 20 nm filter, blue trace) and sensitized emission (red trace) lifetimes. Data in A and B correspond to typical traces that were obtained by using Cx43 solubilized in decylmaltoside at a concentration >1.5 mg/ml. (C) Sensitized fluorescence emission as a function of the average number of fluorescein-labeled connexins per hemichannel. Data were normalized to the peak value of 1 Tb/5.5 fluorescein-labeled preparation and are presented as means ± SEM of seven to nine experiments. Cx43 hemichannels were reconstituted at a ratio of 0.8 hemichannels per liposome (20 μg protein per measurement). Similar results were obtained in detergent-solubilized Cx43 at >1.5 mg/ml (data not shown), where essentially all Cx43 subunits form hemichannels (see Fig. 2). All values are statistically different from the previous one (P < 0.001), except for that at the 3/3 ratio. For additional details see Fig. 3.

Fig. 5.

Effects of the number of PKC-phosphorylated subunits per hemichannel on sucrose permeability. Effects of varying Cx34-dP/Cx43-P average ratios on the percentage of sucrose retained in proteoliposomes preloaded with the radiolabeled probe are shown. Values were normalized as described in Fig. 1. The average number of hemichannels per liposome was 0.8, and data are means ± SEM of four to seven experiments. The lines represent the percentage probe retained, expected if the number of Cx43-P subunits necessary to render the hemichannels impermeable to sucrose are ≥1 (gray, short dash), ≥2 (gray, long dash), ≥3 (gray, solid), ≥4 (blue), ≥5 (red), or 6 (black). The lines were obtained by joining with spline lines the values, calculated from the binomial distribution, for each Cx43-dP/Cx43-P mixture.