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Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance
Tokiyoshi Ayabe1, Heike Wulff, Dalila Darmoul
1Department of Pathology, College of Medicine, University of California, Irvine, California 92697-4800, USA.
Abstract:
Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.
Insights
The calcium-activated potassium channel mIKCa1 regulates Paneth cell secretion of alpha-defensins, crucial for intestinal mucosal defense against bacteria. This finding highlights mIKCa1
Area of Science:
- Immunology
- Cell Biology
- Gastroenterology
Background:
- Paneth cells in the small intestine secrete alpha-defensins, which are microbicidal peptides essential for innate immunity.
- These defensins are released in response to bacterial stimuli, playing a key role in mucosal defense.
Purpose of the Study:
- To investigate the role of the calcium-activated potassium channel mIKCa1 in modulating Paneth cell secretion.
- To determine if mIKCa1 is specifically expressed in Paneth cells and involved in intestinal defense mechanisms.
Main Methods:
- Isolation and sequencing of mIKCa1 cDNA from mouse small intestinal crypts.
- Reverse transcriptase-PCR to assess mIKCa1 expression in different intestinal epithelial cell populations.
- Functional characterization of the cloned mIKCa1 channel and its inhibition by various blockers.
- Measurement of Paneth cell secretion (bactericidal activity and cryptdin protein) under stimulated conditions with and without channel inhibitors.
Main Results:
- mIKCa1 cDNA clones were identified and found to be identical to those from erythroid cells and liver, with conserved genomic organization between mouse and human.
- mIKCa1 expression was detected specifically in Paneth cells (lower crypts) but not in upper crypt cells or villus epithelium, indicating lineage-specific expression.
- The cloned mIKCa1 channel was calcium-activated and sensitive to a wide range of inhibitors, similar to its human homologue.
- Selective IKCa1 inhibitors (TRAM-34, TRAM-39) significantly inhibited Paneth cell secretion stimulated by bacteria or lipopolysaccharide, while an inactive analog (TRAM-7) did not.
Conclusions:
- The calcium-activated potassium channel mIKCa1 is a key modulator of alpha-defensin secretion by mouse Paneth cells.
- mIKCa1 plays a significant role in the intestinal epithelium's defense against ingested bacterial pathogens.
- This study reveals a novel mechanism involving ion channel function in innate mucosal immunity.