Secretory diarrhoea reflects the activation of electrogenic Cl– secretion within the small intestine and/or colon. This involves basolateral K+ channels, which maintain a favourable electrochemical gradient for Cl– exit across the apical (lumenal) membrane of intestinal epithelial cells. KCNQ1 (KvLQT1) and KCNE3 (MiRP2) have been identified in the T84 human adenocarcinoma-derived cell line by northern analysis of total RNA, and co-localised in mouse colon by in situ hybridisation (Schroeder et al. 2000). In addition, KCNN4 (also termed hIKCa, hSK4, hKCa4) has been identified in human colonic mucosa by reverse transcriptase-polymerase chain reaction (RT-PCR) (Warth et al. 1999), and patch-clamp studies have suggested that the functional channel is located in the basolateral membrane of human colonic crypt cells (Sandle et al. 1994). The aim of the present study was to determine whether hKCNQ1, hKCNE3 and hKCNN4 RNAs are present in human colonic crypts.
With ethics committee approval and written, informed consent, multiple biopsies were obtained during colonoscopy from the distal colon of patients who were free of mucosal disease. Intact crypts were isolated by Ca2+ chelation (Sandle et al. 1994). Crypts were pelleted (4000 r.p.m. for 1 min), and washed in DEPC-treated phosphate-buffered saline. Total RNA was extracted by a small-scale modification of the acid-phenol method (Chomczynski & Sacchi, 1987). One microgram of total RNA was reverse transcribed with 200 U Superscript according to the manufacturer’s instructions (Gibco). RT products were then subjected to two rounds of PCR with primers specific to hKCNQ1, hKCNE3 and hKCNN4. PCR products were analysed by gel electrophoresis, stained with ethidium bromide, and visualised by UV illumination. Amplification of endogenous β-actin mRNA served as a positive control. To identify potential contaminating signals from genomic DNA, reverse transcriptions were also performed in the absence of Superscript. In addition, hKCNQ1 and hKCNN4 forward and reverse primers were designed to anneal to regions which spanned one or more introns.
Human colonic crypts isolated by Ca2+ chelation consist of epithelial cells and are free of contamination by smooth muscle cells, fibroblasts, vascular tissue, or other cells in the lamina propria. RT-PCR identified hKCNQ1, hKCNE3 and hKCNN4 mRNAs in total RNA extracted from the colonic crypt isolate (RT-PCR was performed in duplicate on total RNA isolated from three different crypt isolates). PCRs yielded single products which were of the expected sizes, and their identities were confirmed by automated fluorescence sequencing (Lark Technologies). RT performed in the absence of Superscript gave no PCR products.
We have shown that hKCNQ1, and its accessory subunit hKCNE3, are present in total RNA extracted from human colonic crypt cells. Human KCNN4 mRNA was also present. Given the types of cell contained in the crypt preparation, our results show that these channels are localised to the human colonic epithelium.
This work was supported by the MRC.
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