Tu1389 Increased Basolateral Small Conductance Potassium Channel (KCNQ1/ KCNE3) Activity in Ulcerative Colitis

Abstract

Introduction and Aims: Basolateral K+ channels keep colonic cells in a hyperpolarized state to ensure transcellular ionic movements. Intermediate conductance K+ (IK) channels dominate the basolateral membrane conductance in human colonic cells, but their expression and activity are markedly decreased in ulcerative colitis (UC), thus contributing to defective Na+ and water absorption across inflamed mucosa (J Pathol 2012;226:463-470). This study is the first to characterize basolateral small conductance (KCNQ1/KCNE3) K+ channels in normal human colon, and explore their activity in UC. Methods: Intact crypts were isolated from distal colonic biopsies taken while investigating patients with functional diarrhea (normals) and patients with UC not receiving steroids. Expression of KCNQ1 and KCNE3 mRNAs was determined by qPCR. Patch clamp recording was used to study (i) basolateral KCNQ1/KCNE3 channel activity in normal and UC crypt cells and (ii) in normal crypts cells, the effects of forskolin (an activator of adenylate cyclase) and inflammatory mediators present at high concentrations in UC. Results: qPCR product bands were of the expected size (KCNQ1 160 bp, KCNE3 119 bp, β-actin 148 bp). Product identity was confirmed by automated sequencing and melting curve analysis, and KCNQ1 and KCNE3 mRNA expression was similar in normal and UC patients. In normal crypts (n=34), usually single channels (conductance 6.8 ± 0.5 pS) were seen in 36% of patches, whereas in UC crypts (n=9), two or more channels were seen in 74% of patches, the difference in channel prevalence between the two groups being significant (P<0.001). Overall channel activity was 10-fold greater in UC than control patients (P<0.001). These channels constituted a greater calculated basolateral conductance (GSK) in UC (5.85 ± 0.62 mS.cm-2) than in control patients (0.28 ± 0.04 mS.cm-2, P<0.001), but this increase in GSK did not compensate for the decrease in basolateral membrane conductance (from 22 mS.cm-2 to 5 mS.cm-2) previously attributed to decreased IK channel expression/activity in UC (J Pathol 2012;226:463-470). In control crypts, 10μM forskolin and 1μM thromboxane A2 stimulated channel activity 30-fold (n=8, P<0.005) and 10-fold (n=5, P<0.05) respectively, while 100nM PGE2, 10nM IL-1β and 1μM LTD4 had no effect. Conclusions: Crypt cells in normal human colon express cAMP-activated KCNQ1/ KCNE3 channels which make a far smaller contribution to overall basolateral conductance than IK channels. In active UC, KCNQ1/KCNE3 channel activity is significantly increased, but this fails to compensate for the depolarizing effect of decreased IK channel expression/ activity. This may explain, at least in part, why defective electrogenic Na+ absorption rather than increased electrogenic Cl secretion is the dominant pathophysiological mechanism of diarrhea in active UC (J Pathol 2012;226:463-470).