Tricle (e.g. Ohya et al. 2001; Rosati et al. 2001).these muscle tissues and include transcripts that could influence absolute quantification. To provide additional assistance for the measurements of transcriptional expression and to address the situation of contamination from nonmuscle cells, we investigated myocytespecific expression of Kv4.two and Kv4.3 channels with immunohistochemistry. Powerful Kv4.3like immunoreactivity was observed in colonic myocytes, whereas Kv4.2like immunoreactivity was comparatively weaker. Kv4.two and Kv4.3like immunoreactivities have been also substantially weaker in jejunal myocytes. To additional test these observations we also characterized the current density of IA in dispersed colonic and jejunal myocytes. The stronger Kv4.3like immunoreactivity within the colon correlated with 2fold higher present density than in jejunal myocytes. There was a discrepancy amongst the levels of Kv4 transcript expression along with the levels of Kv4 protein and IA density in colonic and jejunal myocytes. We deemed the possibility that this discrepancy may be because of differential expression of KChIP proteins in these cells. KChIPs, which belong to the neuronal calcium sensor (NCS) family of proteins, are good modulators of native and heterologously expressed 41bbl Inhibitors products Kv4derived currents (An et al. 2000; Decher et al. 2001; Liss et al. 2001). These auxiliary proteins boost Kv4 existing density by rising expression on the channels inside the plasma membrane (An et al. 2000; Bahring et al. 2001). KChIPs also modify the kinetic behaviour of Kv4 channels (Beck et al. 2002). Kv4 channels underlie the Atype existing (ITO) in ventricular myocytes (Xu et al. 1999; see Nerbonne, 2000), and the pattern of KChIP2 expression has recently been shown to mirror the transmural gradient of ITO in canine and human ventricles (Rosati et al. 2001). In transgenic mice harbouring a targeted nullKChIP2 allele, heterozygotes displayed ventricular ITO that was reduced by approximately half on the existing in wildtype myocytes (Kuo et al. 2001). Homozygote nullKChIP2 mice didn’t express functional ITO. By analogy with cardiac muscle, we suggest that similar regulation of functional Kv4 channels by KChIPs may happen in gastrointestinal smooth muscles and explain the 7-Ethoxyresorufin Inhibitor disparity among transcriptional expression of Kv4 isoforms and present density in colonic and jejunal muscles. We detected transcripts encoding KChIPs in colonic and jejunal myocytes and, in agreement with our hypothesis, total KChIP transcripts were 2.6fold higher in colon than in jejunum. In these tissues KChIP1 was the dominant isoform. Our data recommend that in gastrointestinal smooth muscle tissues, functional expression of Kv4 might be regulated by the pattern of KChIP expression. A further member from the NCS protein household, frequenin (NCS1), has been shown to act as a positive modulator of Kv4 currents (Nakamura et al. 2001b). Though examination of other NCS family members in gastrointestinal smooth muscle is warranted,Journal of PhysiologyWe also designed primers for an unrelated K channelassociated protein, KChAP, the coexpression of which is also recognized to boost Kv4 existing density (Kuryshev et al. 2000, 2001). Following 35 amplification cycles, RTPCR detected KChAP transcripts in cDNA from mouse ventricle and brain, but didn’t detect KChAP transcripts in colonic or jejunal cDNA (n = three; information not shown).DISCUSSIONPreviously, we characterized an Atype existing (IA) in murine colonic myocytes that dampens excitability and may participa.
