Hyperpolarisation-activated Nucleotide-gated Channels Research Articles

Decreased expression of hyperpolarisation-activated cyclic nucleotide-gated channel 3 in Hirschsprung's disease.

To determine if hyperpolarisation-activated nucleotide-gated (HCN) channels exist in human colon, and to investigate the expression of HCN channels in Hirschsprung's disease. We investigated HCN1, HCN2, HCN3 and HCN4 protein expression in pull-through specimens from patients with Hirschsprung's disease (HSCR, n = 10) using the proximal-most ganglionic segment and distal-most aganglionic segment, as well as in healthy control specimens obtained at the time of sigmoid colostomy closure in children who had undergone anorectoplasty for imperforate anus (n = 10). Fluorescent immunohistochemistry was performed to assess protein distribution, which was then visualized using confocal microscopy. No HCN1 channel expression was observed in any of the tissues studied. Both HCN2 and HCN4 proteins were found to be equally expressed in the aganglionic and ganglionic bowel in HSCR and controls. HCN3 channel expression was found to be markedly decreased in the aganglionic colon vs ganglionic colon and controls. HCN2-4 channels were seen to be expressed within neurons of the myenteric and submucosal plexus of the ganglionic bowel and normal controls, and also co-localised to interstitial cells of Cajal in all tissues studied. We demonstrate HCN channel expression in human colon for the first time. Reduced HCN3 expression in aganglionic bowel suggests its potential role in HSCR pathophysiology.

Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system

Hyperpolarisation-activated non-specific cation currents ( I h currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the I h current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having I h currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with I h currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the I h and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent I h, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.