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

doi:10.1016/j.neuroscience.2004.08.027

Neuroscience

Volume 129, Issue 3, 2004, Pages 603-614

https://doi.org/10.1016/j.neuroscience.2004.08.027 Get rights and content

Abstract

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.

Section snippets

Tissue preparation and animals

The experiments utilized adult guinea-pigs of 250–300 g, adult Sprague–Dawley rats of 240–280 g and 5–7 week Balb/c mice, of either sex. Tissue samples were taken from animals that were killed by a blow to the head and cutting the carotid arteries and the spinal cord. All animal procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. The experiments comply with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (//www.health.gov.au/nhmrc

Distribution of HCN isoforms in the ENS

Immunoreactivity (IR) for three HCN isoforms was found to be differentially distributed in the ENS (Fig. 1), however no IR for HCN3 was observed. IR for HCN1 occurred on the membranes of myenteric neurons with strong IR on the surfaces of DII cell somas and their processes in rat ileum (Fig. 1D) and mouse distal colon. HCN1-IR was not detectable in guinea-pig enteric neurons. IR for HCN2 was found in all myenteric neurons, including DII neurons and small, non-DII neurons in the guinea-pig

Discussion

The results of the present investigations, summarized in Table 4, indicate that HCN1, 2 and 4 channel proteins are present in enteric neurons. However, the HCN3 channel protein is probably not present in the neurons. HCN3 IR was not found using immunohistochemistry in enteric neurons of guinea-pig, rat or mouse, although the antibodies were tested and found to be effective in localizing channels in the retina. Furthermore, HCN3 protein was not detected by Western analysis of lysates from

Conclusion

In conclusion, HCN IR occurred in AH/DII neurons, where an I_h current is readily demonstrable. Most AH neurons in the guinea-pig myenteric ganglia co-expressed HCN2 and HCN4 channel isoforms. The kinetics of the current indicate that there may be modifying factors that accelerate current onset in enteric AH neurons. The equivalent neurons in mouse and rat co-expressed HCN1 and HCN2 channel isoforms. HCN channel IR also occurred in S neurons, few of which exhibit an I_h current. The significance

Acknowledgments

This research was supported by the National Health and Medical Research Council of Australia and GlaxoSmithKline. We thank Dr. Erica Fletcher for retinal tissue and advice on HCN distribution in the retina, and Dr. Miles Epstein for the provision of the anti-Hu antiserum, ANA2.

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

Abstract

Section snippets

Tissue preparation and animals

Distribution of HCN isoforms in the ENS

Discussion

Conclusion

Acknowledgments

Neuron

Prog Neurobiol

J Auton Nerv Syst

J Biol Chem

Neuroscience

Electrophysiologic and molecular properties of cultured enteric glia

J Neurosci Res

Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the guinea-pig

Neuroscience

Projections and chemistry of Dogiel type II neurons in the mouse colon

Cell Tissue Res

Fine structure of the myenteric plexus in the guinea-pig ileum

J Anat

Cation current activated by hyperpolarization (IH) in guinea-pig enteric neurons

Am J Physiol

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Am J Physiol

Morphology of horseradish peroxidase HRP-injected glial cells in the myenteric plexus of the guinea-pig

Cell Tissue Res

Molecular diversity of pacemaker ion channels

Annu Rev Physiol

Immunoreactivity of Hu proteins facilitates identification of myenteric neurones in guinea-pig small intestine

Neurogast Motil

Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon

J Physiol (Lond)

A family of hyperpolarization-activated mammalian cation channels

Nature

Cation current activated by hyperpolarization (I_H) in guinea-pig enteric neurons