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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5309fjev1 fj.05-5309fjev2 fj.05-5309fjev3 20/8/1221    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Locke, D. Articles by Harris, A. L. Search for Related Content PubMed PubMed Citation Articles by Locke, D. Articles by Harris, A. L.

Isoelectric points and post-translational modifications of connexin26 and connexin32

Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA

¹Correspondence: Department of Pharmacology and Physiology, New Jersey Medical School, 185 South Orange Ave., UMDNJ, Newark, New Jersey 07103, USA. E-mail: lockeda{at}umdnj.edu

SPECIFIC AIMS

The selectivity of gap junction hemichannels among biological signaling molecules (e.g., cyclic nucleotides, inositol phosphates) depends critically on the connexin composition of the channel. In previous work, we have shown that heteromeric connexin26/connexin32 (Cx26/Cx32) channels have a greater degree of selectivity among these second messengers than the corresponding homomeric Cx26 or homomeric Cx32 channels. Mechanistic insights about the molecular basis of the selectivity of the heteromeric channels (and its defects in the case of disease-causing mutations) require working with channels of defined isoform stoichiometry.

We set out to determine if Cx26 and Cx32 had different isoelectric points (pIs) in the hope of using isoelectric charge to fractionate heteromeric channels into defined isoform stoichiometries. After finding that the pIs of Cx26 and Cx32 were significantly different than predicted from amino acid sequence, we also explored the possibility that these connexins had substantial covalent post-translational modifications that could have profound functional consequences.

PRINCIPAL FINDINGS

1. The pIs of Cx26 and Cx32 are different, substantially more acidic than predicted, and allow homomeric hemichannels to be separated by differences in charge
Before the present work, the pIs of Cx26 and Cx32 were unknown. The pIs of Cx26 and Cx32 were determined by liquid-phase isoelectric focusing (IEF) of homomeric hemichannels in 80 mM n-octyl-ßbeta;-D-glucopyranoside nonionic detergent (OG). Rat (r) Cx26 and rCx32 were expressed in HeLa cells and tagged (_T) at their carboxyl-terminal with a hemagglutinin-(His-Asn)₆ sequence (i.e., Cx26_T or Cx32_T). This tag could be cleaved (i.e., Cx26_Tc or Cx32_Tc) from immunopurified protein leaving four extra amino acids (LVPR) at the carboxyl-terminal that are part of a thrombin cleavage site.

The predicted pIs of rCx26 and rCx32 are identical (pI9.20). However, the actual pI of Cx26 and Cx32 were different and, more surprisingly, substantially more acidic than predicted from sequence alone. After tag cleavage, the pI of Cx26_Tc was 5.82 ± 0.11_n=3 and the pI of Cx32_Tc was 6.33 ± 0.08_n=3 (mean±SD_trials). With the tag uncleaved, connexin pIs were more acidic; Cx26_T focused to pI 5.05 ± 0.13_n=7 and Cx32_T to pI 5.72 ± 0.12_n=7. On the basis of these differences in pI, homomeric Cx26 and homomeric Cx32 channels could be resolved separately (Fig. 1 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. IEF of homomeric channels. pIs of Cx26 or Cx32 expressed in HeLa cells as homomeric channels were determined by liquid-phase IEF. Connexins were HA(HN)6-tagged at CT (i.e., Cx26T or Cx32T). Tag cleavage left 4 residues (LVPR), part of a thrombin cleavage site (i.e., Cx26Tc or Cx32Tc). IEF was performed in 10 mM salt, 80 mM OG, and 4.0 M deionized urea. A pH 5–7 gradient was generated in a focusing core with 20 1 ml chambers using 1.8% w/v ampholytes. Ampholine solution of connexin was injected from the cathode (arrowhead). At equilibrium, fractions were collected by vacuum, and pH was determined. Not all fractions recovered are shown. For clarity, as fraction pH in adjacent chambers is unequally spaced, those with similar pH were combined; lanes contain protein from 3 focusing runs when 0.04 pH units acidic < X < 0.05 pH units basic at 0.10 pH unit intervals.

2. The pIs of the homomeric channels bracket the pI of heteromeric Cx26/Cx32 channels
We speculated that heteromeric channels would show intermediate pIs, depending on isoform stoichiometry. Only one isoform in the heteromers was tagged (i.e., Cx26/Cx32_T or Cx32/Cx26_T). Heteromeric Cx26/Cx32 channels had a range of pIs between those of the corresponding tagged and/or tag-cleaved homomeric channels. That is, Cx32/Cx26_T channels focused between pIs 5.17 and 6.31_n=8, the observed pIs of homomeric Cx32_Tc and homomeric rCx26_T channels, respectively. The Cx26/Cx32_T channels distributed between pIs 5.56 and 6.08_n=3, the observed pIs of homomeric Cx26_Tc and homomeric Cx32_T channels, respectively. After tag cleavage, Cx26/Cx32_Tc and/or Cx32/Cx26_Tc channels focused between pIs 5.50 and 6.30_n=3, the observed pIs of homomeric Cx26_Tc and homomeric Cx32_Tc channels, respectively. It was notable that quaternary structure of the channels was retained during the focusing, since both connexins were found in all heteromeric pI fractions.

3. The pIs of Cx26 and Cx32 from rodent liver and expressed in HeLa cells are similar
Previous biochemical and functional studies have shown that hemichannels from mouse liver are heteromeric Cx26/Cx32 and from rat liver are homomeric Cx32. The predicted pI of mouse (m) Cx26 is 9.22 rather than 9.20 (rCx26); two amino acids are different, one (_ratY68_mouseH68) replacing negative with positive charge. The predicted pI of rCx32 (9.20) is identical to mCx32. By liquid-phase IEF, mCx26/Cx32 channels focused between pI 5.73 and 6.49_n=8. Heteromeric mCx26/Cx32 channels were therefore slightly more basic than HeLa Cx26/Cx32_Tc and/or HeLa Cx32/Cx26_Tc channels or rCx26/Cx32 channels in rat livers, consistent with mCx26 being slightly more basic than rCx26. Rat liver homomeric Cx32 channels resolved narrowly at pI 6.22 ± 0.18_n=6 and were more basic than rCx26/Cx32 channels because they contained no Cx26, which has a more acidic pI. Homomeric rCx32 channels were also slightly more acidic than HeLa Cx32_Tc homomeric channels. This was consistent with the LVPR sequence that remains after tag cleavage adding 0.10 pH units basic charge (Fig. 2 ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Schematic shows pI differences of Cx26 and Cx32, which allowed homomeric and heteromeric channels to be separated by isoform content, pointing to significant post-translational modification of connexin. Several new post-translational modifications of Cx26 and Cx32 were identified, and differences in post-translational processing of the same isoform in liver and HeLa cells were also observed. These modifications may be instrumental in guiding and understanding novel molecular mechanisms of connexin regulation and disease.

Unfortunately, it was impossible to achieve sufficient resolution in these IEF studies to resolve individual heteromeric channel stoichiometries. However, the acidic shift of the pIs likely reflected substantial and acidic post-translation modifications of both proteins. Before this work, no post-translational modifications of Cx26 have been previously described, and only phosphorylation has been described for Cx32.

4. Substantial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry sequence coverage of connexin protein
The usual candidate for an acidic shift of pI is glycosylation, but connexins are not glycosylated. We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis to these proteins. For membrane proteins, such analyses can be difficult due to proteolytic resistance, the low ionization ability of hydrophobic transmembrane domains, and the difficulty of detecting the low abundance peptides in complex spectra.

Enzymatic digestions of Cx26 and Cx32 from HeLa cells and mouse liver were carried out with trypsin and/or endoproteinase-GluC (Endo-GluC) to maximize the sequence coverage. Peptides from each digest were fractionated by stepwise elution from reverse-phase beads with increasing concentrations of organic solvent. Spectra were acquired from each elution, and mass/charge (m/z) peaks from all elution steps of a single digest were combined for sequence analysis. In this way, coverage values obtained were above the typical sequence coverage for membrane proteins. Submission of m/z values to the ProFound fingerprint database led to the unambiguous verification of Cx26 or Cx32 in the different digests. Sequence coverage by trypsin digest of mCx26 was 43% and of HeLa rCx26 was 70%. Endo-GluC digest coverage of HeLa rCx26 was 17%, with no additional coverage of mCx26; combined coverage for Cx26 was 82%. Sequence coverage by trypsin digest of mCx32 was 61%, and HeLa rCx32 was 47%, with Endo-GluC digest providing 18% coverage of both isoforms; combined coverage for Cx32 was 66%.

5. Post-translational modifications of Cx26 and Cx32 contributing to the observed acidic pIs
With the use of MALDI-TOF-MS, we found that both connexins were differentially modified and furthermore that the post-translational modifications differ when each isoform is expressed in native tissue (liver) or in a commonly used heterologous expression system (HeLa cells). We found phosphorylation sites on Cx32 that were previously described and propose several new sites for phosphorylation and new post-translational modifications of Cx32. We also describe the first post-translational modifications of Cx26 (Table 1 ). These modifications included hydroxylation and/or phosphorylation in the amino-terminal domain of both connexins, -carboxyglutamate residues in the cytoplasmic loop of both isoforms, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation of the carboxyl-terminus of Cx32.

The pIs of the processed proteins were also calculated using protein sequences modified to reflect the charge changes arising from the observed post-translational modifications. Most of the acidic shift of the connexin pIs we observed by liquid-phase IEF could be accounted for by the observed post-translational modifications.

CONCLUSIONS AND SIGNIFICANCE

The functional properties of connexin channels have been regarded as primarily determined by channel isoform composition. However, channel function and biological regulation can also be profoundly and dynamically governed by the location, character, extent, and combination of different post-translational modifications. Modulation of connexin channel properties and cellular dynamics by covalent modification is largely unexplored, except for phosphorylation, yet other post-translational modification(s) are likely to be involved in channel trafficking, assembly, or function. To date, none of the other common post-translational modifications have been reported for any connexin. We propose a number of novel post-translational modifications of Cx26 and Cx32 in HeLa cells and rodent livers using MALDI-TOF-MS, which are likely to be involved in channel trafficking and assembly, or functional modulation. These post-translational modifications contribute to the measured acidic pIs of Cx26 and Cx32 (Fig. 1 and Table 1 ).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5309fje

² These authors contributed equally to this work.

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5309fjev1 fj.05-5309fjev2 fj.05-5309fjev3 20/8/1221    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Locke, D. Articles by Harris, A. L. Search for Related Content PubMed PubMed Citation Articles by Locke, D. Articles by Harris, A. L.