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A Lymphedema‐Inducing Mutation of Cx47, R259C, Alters Growth Pattern and Cx43 Processing in Rat Insulinoma Cells
Author(s) -
Kanady John,
Pontifex Tasha,
Taylor SamanthaSu,
Simon Alexander,
Burt Janis
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.726.11
Subject(s) - immunocytochemistry , biology , transfection , microbiology and biotechnology , connexin , mutation , cell culture , insulinoma , western blot , gap junction , intracellular , genetics , gene , pancreas , biochemistry , endocrinology
Connexins (Cx), proteins that mediate direct intercellular communication, are essential for normal vascular development and function. Mutations in Cx47 have been reported to underlie cases of lymphedema in humans, however the cellular and molecular mechanisms responsible for dysfunction have been largely unexplored. This study examines an orthologous mutation in mouse Cx47 to one of these lymphedema‐causing mutations, an arginine to cysteine substitution (R259C) in a highly‐conserved region of the second extracellular loop. We transfected connexin‐deficient rat insulinoma (Rin) cells with wild‐type (WT) Cx47 or Cx47R259C either alone or in combination with WT Cx43. Evaluation of cell growth was performed using light microscopy, protein expression through Western blot and immunocytochemistry, and electrical coupling via dual whole‐cell voltage clamp. Expression of Cx47 in Rin cells resulted in plexiform cell growth. Cx47R259C and Cx43/Cx47R259C coexpressing Rin cells also displayed plexiform growth patterns, but exhibited more elongated gaps between cell groups. Surprisingly, in Cx43/Cx47R259C cells a multiple banding pattern for Cx43 was detected via Western blot and full length Cx43 was conspicuously absent. Immunocytochemistry of Cx43/Cx47R259C cells revealed a diffuse cytoplasmic staining pattern for Cx43 and an absence of readily identifiable gap junction plaques. Based on initial electrophysiological recordings, Cx47‐expressing Rin cells have a low incidence of electrical coupling. Together, these preliminary data suggest that Cx47 influences cell growth, and subtle alterations in pattern occur with mutation of Cx47 at R259C. Furthermore, Cx47R259C affects cellular processing of Cx43, which may be a potential molecular mechanism by which this orthologous mutation leads to lymphatic defects in humans. Support or Funding Information This work was supported by the following grants: National Institutes of Health (R21HL122443) and Heart, Lung, and Blood Training Grant (T32HL007249)