A New Non-Canonical Role for an Alternatively Translated Ion Channel Protein

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Ion channels have a plethora of functions than, frequently, can not be understood by their ability to insert in the plasma membrane and conduct ionic current. The Connexin43 (Cx43) gap junction channel, whose parent gene is GJA1, has been implicated in having a role in cell cycle progression, metastatic transformation, wound healing, development, migration, epithelial-mesenchymal transition, and metabolism. GJA1 has a single coding exon, and therefore it can not be subject to splicing and alternative transcription. However we have identified, and it has been confirmed by several laboratories, that GJA1 mRNA is subject to alternative translation in which initiation of ribosomal translation occurs not at the first AUG (Methionine) start codon, but at downstream Methionines. The result is that six additional progressive N-terminal truncation proteins (all containing the distal C- terminus) can be endogenously generated from the same mRNA molecule, which we identify by protein size (GJA1-43k for the full length protein, then GJA1-32k, GJA1-29k, GJA1-26k, GJA1-20k, GJA1-11k, and GJA1-7k). For the last three years, my group has explored the role of these smaller isoforms. This proposal is based on the application of a surprising, and frankly startling, finding regarding GJA1-20k which is usually the most highly translated of the smaller isoforms. We found that GJA1-20k has a strong tropism for mitochondria, so strong that it could function as a mitochondrial marker. Additional preliminary data indicate that GJA1-20k is protective against the stress of myocardial ischemia. Our central hypothesis is that following oxidative stress and ischemic injury, alternatively translated endogenous GJA1-20k is increased and targets to mitochondria as a myocardial survival signal. Furthermore, we will test whether exogenous GJA1-20k can reduce myocardial injury due to ischemic insult. Three specific aims are proposed. Aim #1- What is the expression, localization, and function of GJA1-20k during myocardial injury? Aim #2- Does exogenous GJA1-20k improves cardiomyocyte survival following oxidative stress? Aim #3- Does gene transfer of GJA1-20k reduce infarct size in a mouse model of myocardial ischemia? When the work in the three aims is complete, we expect to have identified critical pathways of GJA1-20k regulation during stress and have introduced a new therapeutic peptide for ischemic injury. Furthermore, we will develop the paradigm by which alternative translation is an important aspect of cardiac electrophysiology and should be applied to multiple channels as a means for understanding the mechanism of their non-canonical function.