Molecular cloning, characterisation and function expression of cyclic nucleotide-gated ion channel genes expressed in sino-atrial node region of heart.
thesisposted on 2015-11-19, 09:08 authored by Sukhinder Paul Singh. Hundal
Pacemaker cells of the mammalian sino-atrial node (SAN) contain a hyperpolarization-activated, non-specific cationic current, If which is an important component involved in the initiation and neurotransmitter-mediated control of cardiac rhythm. cAMP can directly modulate If by a mechanism independent of phosphorylation, demonstrating that cyclic nucleotide-sensitive ion channel genes are expressed within cardiac pacemaker cells. Through a combination of library screening methods based on cross-hybridising cyclic nucleotide-gated channel probes and a PCR 'fingerprint' employing primers designed to sequences encoding an ion channel cyclic nucleotide-binding domain, partial cDNA clones were isolated from a prepared sino-atrial node regional-specific cDNA library, which were either homologues of previously identified ion channels shown to be expressed in sensory tissues or putative new channel clones. Isolate rscNGC 1 following retrieval of a full coding region by anchor-PCR, demonstrated 90.4% sequence identity to the a-subunit of the rod photoreceptor cGMP-gated channel. The PCR 'fingerprint' identified a SAN homologue of the olfactory neuron cAMP-gated channel within library aliquots. This was the first demonstration that two distinct cyclic nucleotide-gated ion channel genes were expressed in SAN region of heart. Heterologous expression of rscNGC 1 following micro-injection of capped cRNA in Xenopus oocytes, gave rise to cGMP-stimulated channel activity exhibiting electrophysiological properties similar to the characterised a-subunit of the rod photoreceptor cGMP-gated channel. A reconstituted second messenger-pathway mediating endogenous receptor coupling to heterologously expressed cAMP-gated ion channels - shown to be present within native nodal tissue - was attempted within MEL cells. However, the absence of endogenous receptors positively-coupled to adenylyl cyclase within MEL cells, and the inability to functional characterise cAMP-stimulated cationic conductances via electrophysiological methods, prevented such studies. Thus demonstrating the inappropriateness of the MEL cell, as a heterologous system for studying receptor-mediated second messenger coupling to cNG channels. Although cyclic nucleotide-gated ion channels are obligatorily coupled to intracellular signalling agonists commonly found in heart, they have yet to be described in functional terms within SAN or any other cardiac subregion. It is postulated that they may have a role in vasculature - underlying mechanisms of smooth muscle relaxation.
Date of award1994-01-01
Awarding institutionUniversity of Leicester