Identifying ligands of the C-terminal domain of cardiac expressed connexin 40 and assessing its involvement in cardiac conduction disease
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2007.
Connexins (Cx) are major proteins of gap junctions, dynamic pores mediating the relay of ions and metabolites between cells. Cxs 40, 43 and 45 are the predominant cardiac isoforms and their distinct distribution raises questions about their functional differences. Their cytoplasmic (C)-terminal domains are involved in protein-protein interactions. Furthermore, mutations in the myotonic dystrophy protein kinase (DMPK)-causative gene are associated with disruptions in cardiac conduction similar to that described for Cx knock-out mice. DMPK is a Cx43 ligand, raising the possibility that defects in Cx40 ligands may be involved in the development of cardiac conduction disturbances. We hypothesised that delineation of the protein ligands of the C-termini of Cx40 and of Cx45 (parallel study conducted by N Nxumalo) would help elucidate their functional roles. Yeast-two-hybrid methodology was used to identify putative Cx40 ligands. Primers were designed to amplify the C-terminus-encoding domain of the human Cx40 gene (Cx40), the DNA product was cloned into the pGBKT7 vector which was used to screen a cardiac cDNA library in Saccharomyces cerevisiae. Successive selection stages reduced the number of putative Cx40 ligand-containing colonies (preys) from 324 to 33. The DNA sequences of the 33 ligands were subjected to BLAST-searches and internet database literature searches to assign identity and function and to exclude false positive ligands based on subcellular location and function. Eleven plausible ligands were identified: cysteine-rich protein 2 (CRP2), beta-actin (ACTB), creatine kinase, muscle type (CKM), myosin, heavy polypeptide 7 (MYH7), mucolipin1 (MCOLN1), voltage-dependent anion channel 2 (VDAC2), aldehyde dehydrogenase 2 (ALDH2), DEAH box polypeptide 30 (DHX30), NADH dehydrogenase, 6, (NDUFA6), prosaposin (PSAP) and filamin A (FLNA). Cxs 40 and 45 showed differences in the classes of proteins with which they interacted; the majority of putative Cx40 interactors were cytoplasmic proteins, while Cx45 interactors were mitochondrial proteins. These results suggest that Cxs 40 and 45 are not only functionally different, but may also have different cellular distributions. Further analyses of these protein interactions will shed light on the independent roles of Cxs 40 and 45.