| Connexins (Cx) are inserted into the plasma membrane as hemichannels, and normally remain closed by extracellular Ca (Cao) until partnered with hemichannels from neighboring cells to form intercellular channels in gap junctions. However, recent evidence suggests that there are potential roles for connexin hemichannels prior to their insertion into gap junctions. The studies that comprise this thesis confirm a novel physiologic role for connexin hemichannels in the processes of cellular osmoregulation and suggest that these channels may also serve as passageways for the deleterious ion fluxes that occur during myocardial ischemia. In both isolated ventricular myocytes and HeLa cells transfected with Cx43 linked to green fluorescent protein (GFP), modest hyper-osmolar stress (≥25 mOsm) or metabolic inhibition caused unpartnered Cx43 hemichannels to open in the presence of 1.8 mM Ca o. Protein immunochemistry studies revealed that Cx43 hemichannels were dephosphorylated by both hyper-osmolar and metabolic stress. In contrast, Cx26 hemichannels, which are non-phosphoproteins, did not respond to either hyper-osmolar or metabolic stress, but these responses could be rescued by linking the carboxyl (C)-terminus of Cx43 to Cx26. Mutagenesis and pharmacologic studies showed that Cx43 hemichannels opened during hyper-osmolar or metabolic stress due to increased phosphatase activity, leading to dephosphorylation of key mitogen-activated protein kinase (MAPK) phosphorylation sites in the C-terminus. By downregulating MAPK activity, inhibition of protein kinase C or protein tyrosine kinases also caused Cx43 hemichannels to open. We speculate that Cx43 hemichannels evolved as a component of MAPK-regulated osmoregulation; during metabolic stress, this response becomes maladaptive, potentially contributing to ionic disturbances causing arrhythmias and tissue injury in the setting of acute ischemia. |
