Cloning and functional co-expression of cardiovascular receptors and ion channels.
thesisposted on 19.11.2015, 09:07 by Charlotte Fenton. Ratcliffe
There is an expanding family of cyclic nucleotide-gated cation channels (CNGCs) with expression of family members reported in rod and cone photoreceptor cells, olfactory epithelium, heart, kidney, sperm and aorta. Although functions have been assigned to CNGCs in sensory cells, the function of such channels in non-sensory cells is unknown. A PCR-based screen showed that a CNGC is expressed throughout bovine heart tissue and also in bovine aorta, a bovine aorta endothelial cell line and a human umbilical vein endothelial cell line. Sequence data from these amplified products showed that the CNGC expressed in bovine heart and vasculature is highly related to the bovine rod photoreceptor channel. This was also the case for PCR-generated clones spanning the entire coding sequence of the CNGC from porcine coronary artery smooth muscle tissue. Cyclic GMP is an important messenger in vascular smooth muscle relaxation and therefore this CNGC may play a key role in this process. The second messenger pathways which may be involved in the gating of cardiovascular CNGCs have also been studied by attempting to heterologously co-express a cGMP-generating receptor, ANP-RA, with a CNGC in HEK293 cells. The inward rectifier K+ channel subunits Kir 3.1 and Kir 3.4 have been heterologously co-expressed in MEL cells using a mammalian expression vector which incorporates the ?-globin LCR and promoter allowing high levels of gene expression in a 'position independent' manner. Electrophysiological analysis of these co-expressing cell lines shows that Kir 3.1 and Kir 3.4 form a heteromultimeric ion channel complex which displays the features of the native G-protein activated atrial muscarinic K+ channel, KACh, and it is probable that these two inward rectifier channel subunits are major components of KACh.