Basic Science Paper
Decreased expression of Kv7 channels in Hirchsprung's disease

https://doi.org/10.1016/j.jpedsurg.2016.12.028Get rights and content

Abstract

Purpose

Voltage-dependent K+ channels (Kv channels) participate in electrical rhythmicity and smooth muscle responses and are regulated by excitatory and inhibitory neurotransmitters. Kv channels also participate in the interstitial cell of Cajal (ICC) and smooth muscle cell (SMC) responses to neural inputs. The Kv family consists of 12 subfamilies, Kv1-Kv12, with five members of the Kv7 family identified to date: Kv7.1-Kv7.5. A recent study identified the potassium channel Kv7.5 as having a role in the excitability of ICC-IM in the mouse colon. We therefore designed this study to test the hypothesis that Kv7 channels are present in the normal human colon and are reduced in Hirschprung's disease (HSCR).

Material and methods

HSCR tissue specimens were collected at the time of pull-through surgery (n = 10), while normal control tissue specimens were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). Kv7.3-Kv7.5 immunohistochemistry was performed and visualized using confocal microscopy to assess their distribution. Western blot analysis was undertaken to determine Kv7.3-Kv7.5 protein quantification.

Results

Kv7.3 and Kv7.4-immunoreactivity was co-localized with neuron and ICC markers, while Kv7.5 was found to be expressed on both ICCs and SMCs. Western blot analysis revealed similar levels of Kv7.3 and Kv7.5 expression in the normal colon and HSCR colon, while Kv7.4 proteins were found to be markedly decreased in ganglionic specimens and decreased further in aganglionic specimens.

Conclusion

A deficiency of Kv7.4 channels in the ganglionic and aganglionic bowel may place a role in colonic dysmotility in HSCR.

Section snippets

Tissue samples

This study was approved by the Ethics Medical Research Committee, Our Lady's Children's Hospital, Dublin, Ireland (Ref. GEN/292/12) and tissue samples were obtained with informed parental consent. HSCR specimens from 10 patients who underwent pull-through surgery were studied. These specimens were divided into aganglionic and ganglionic samples. HD patients were aged 6 ± 3 months old. We compared the most distal aganglionic segments with the most proximal ganglionic segments. Normal control colon

Immunofluorescence staining and confocal microscopy

Immunofluorescence double-staining revealed that Kv7.3 and Kv7.4 were expressed in Hu-positive neurons located within both the myenteric and submucosal plexuses of the normal human colon, as well as in ICCs in the both the myenteric plexus and in the smooth muscle layer (Fig. 1). Kv7.5 was found to be expressed on both ICCs and SMCs in the normal human colon (Fig. 2). A decreased expression of Kv7.4 was observed within both the ganglionic and aganglionic tissue specimens, while Kv7.3 and Kv7.5

Discussion

K+ channels play important roles in excitable cells, neurons and SMCs, in which they are involved in several physiological processes, such as action potential firing, neurotransmitter release and smooth muscle contractility [5]. The cell membrane potential hyperpolarizes whenever K+ channels open, as K+ flows out of the cell due to the large transmembrane electrochemical gradient, and cell excitability decreases. In the smooth muscle, this results in a reduction of contractility, that is mainly

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