Expression of the inwardly rectifying potassium ion channel, Kir2.2, and its modulating receptor, NK-1 R, in rat brain
thesisposted on 15.12.2014, 10:31 by Anthony Harold. Stonehouse
Potassium current flowing through channels of the Kir2.0 subfamily of inward rectifier channels contributes to resting membrane potential in many cell types. Suppression of inward rectifier (Kir) activity by neurokinin neurotransmitters increases neuronal excitability. Despite functional studies reporting modulation of whole-cell Kir current in the locus coeruleus (LC) of the medulla, little information is available describing the specific molecules involved.;Isoform specific polyclonal antibodies were raised, characterised and utilised, revealing differential distribution patterns. (a) Kir2.2 protein was observed in the cerebellum and medulla: a similar distribution was obtained by in situ hybridisation with Kir2.2 riboprobes. (b) Kir2.1, Kir2.2 and Kir2.3 were expressed in the hippocampus. Detailed observations of tissue sections revealed immunostaining patterns in macroglia, endothelia, ependyma and vascular smooth muscle cells for Kir2.1 and Kir2.2.;The NK-1 receptor was localised to plasma membranes of neurons, neuronal processes and oligodendroglia. Kir2.0 channels appeared to be distributed on plasma membranes and within the nucleus. This intracellular distribution was mimicked in transiently transfected Chinese hamster ovary cells expressing Kir2.0 isoforms.;Channel and receptor co-localisation supported the hypothesis that neuronal excitability in locus coeruleus is controlled by substance P-initiated suppression of Kir2.0 channel activity via protein phosphorylation. Tyrosine phosphorylation of Kir2.2 was investigated after administration of SP, in SN56 cell cultures. This produced a 62 kDa band, detectable by anti-Kir2.2 and anti-phosphotyrosine antibodies indicating that Kir2.2 protein could be phosphorylated via events initiated by SP.