Te in keeping the phasic pattern of electrical activity observed in intact colon tissue preparations (Koh et al. 1999b). Subsequent investigation identified 19 pS DBCO-PEG4-DBCO medchemexpress channels in colonic myocytes with voltagedependent and regulatory properties constant with macroscopic Atype currents (Amberg et al. 2001). Kinetic and molecular analysis of colonic IA suggested that Kv4 asubunits, as opposed to other Kv loved ones members (e.g. Kv1.four), could encode IA (Koh et al. 1999b). Within the present study we sought to establish the relative contribution of Kv4 isoforms to Atype currents inside the murine colonic cells. Applying a variety of procedures we conclude that the Atype currents are most likely to become as a result of Kv4 expression, and analyses of transcription and protein expression suggest that Kv4.three is definitely the predominant isoform. Our data also recommend that expression of KChIP1 in gastrointestinal myocytes may perhaps regulate the current density of Atype currents. We used quantitative realtime PCR to establish the relative expression levels of transcripts encoding every single Kv4 isoform in mouse proximal colon. For comparative purposes, we also determined relative expression of Kv4 isoforms in jejunal smooth muscles. We’ve previously demonstrated smooth muscle cellspecific expression of Kv4 transcripts employing qualitative RTPCR on isolated colonic myocytes (Koh et al. 1999b). In this study we showed that transcripts encoding Kv4.3 had been 3fold extra abundant than Kv4.1 transcripts and 2fold a lot more abundant than Kv4.two transcripts in colonic and jejunal smooth muscle. Kv4.3 seems to be alternatively spliced in some tissues (e.g. Ohya et al. 2001); we only detected the extended form in colonic and jejunal muscle tissues. This observation is consistent using a prior report describing tissuespecific expression of Kv4.3 splice variants (Ohya et al. 1997). There were no substantial differences within the levels of Kv4 transcripts in colon and jejunum. A caveat to this conclusion is the fact that RNA from colonic and jejunal muscles with mucosa and submucosa removed was utilized for the quantitative analysis of Kv4 expression. Cell varieties besides myocytes, like interstitial cells of Cajal and enteric neurons, are present inJ. Fmoc-NH-PEG4-CH2COOH Technical Information Physiol. 544.Kv4 channels in murine colonJournal of Physiologydifferences, namely recovery from inactivation and elevated present density, between heterologously expressed Kv4 channels and native colonic IA are much more constant with all the actions of KChIP than these of frequenin (An et al. 2000; Nakamura et al. 2001a,b). Similarly, expression of other modulatory subunits such as minKrelated peptide 1(MiRP1; Zhang, M. et al. 2001) and Kvb (Yang et al. 2001) should be examined, although the importance of these proteins may well be tentatively discounted for related reasons to frequenin. Expression of yet another constructive effector of Kv4 channels, KChAP (Kuryshev et al. 2000, 2001), was not evident in colonic and jejunal muscle tissues. The pharmacological characterization of colonic IA presented in this study provides additional supportive proof linking Kv4 channels to this present. We examined the sensitivity of IA for the antiarrhythmic flecainide. Atype currents formed by Kv4 channels are far more sensitive to inhibition by flecainide (IC50 20 mM) than those formed by Kv1 channels (IC50 50 mM; Grissmer et al. 1994; Yamagishi et al. 1995; Yeola Snyders, 1997; Rolf et al. 2000). Colonic and jejunal IA were sensitive to low micromolar concentrations of flecainide, with IC50 values of 11 and 24 mM, respective.
