TRAM 39

TRAM-39, 2-(2-chlorophenyl)-2, 2-diphenyl acetonitrile, is a potent, synthetic, selective inhibitor of intermediate-conductance Ca2+-activated K+channels. TRAM-39 selectively block K-Ca3 but do not inhibit other Ca2+-activated K+channels.[1,3]

Intermediate conductance Ca2-activated K channels are the product of the IKCa1 genes and are important in regulating the membrane potential of colonic epithelial cells, and lymphocytes. The IKCa1 gene is also important in volume regulation of red blood cells. IKCa1 modulates the paneth cell alpha-defensin secretion and involve in the mucosal defense of the intestinal epithelium against ingested bacterial pathogens.[1,2]

TRAM-39 inhibits the Kca3,so as to Inhibit paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide. Endothelium-derived hyperpolarizing factor (EDHF ) responses are brought about by the activation of endothelial SKCa and IKCa.[1,3]

A combination of TRAM-39 and apamin abolished endothelium-derived hyperpolarizing factor (EDHF) mediated relaxation of the rat isolated mediated relaxation. The combination of TRAM-39 and apamin evoked a small, transient depolarization of endothelial cells.[2]

References:
[1] Ayabe T1, Wulff H, Darmoul D, etal. , Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel. J Biol Chem. 2002 Feb 1;277(5):3793-800.
[2]Hinton JM, Langton PD.  Inhibition of EDHF by two new combinations of K+-channel inhibitors in rat isolated mesenteric arteries. Br J Pharmacol. 2003 Mar; 138(6):1031-5.
[3]Burnham MP, Johnson IT, Weston AH.   Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats. Br J Pharmacol. 2006 Jun;148(4):434-41.

Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats.[Pubmed:16682967]

Br J Pharmacol. 2006 Jun;148(4):434-41.

We have examined the relative contributions of small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium-derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to -69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre-diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed.

Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel.[Pubmed:11724775]

J Biol Chem. 2002 Feb 1;277(5):3793-800.

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.

Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.[Pubmed:10884437]

Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8151-6.

The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K(+) channel, IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -CN group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K(d) of 20-25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [(3)H]thymidine incorporation of preactivated lymphocytes with EC(50)-values of 100 nM-1 microM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.