Upregulation of basolateral small conductance potassium channels (KCNQ1/KCNE3) in ulcerative colitis

Abstract

Background: Basolateral K(+) channels hyperpolarize colonocytes to ensure Na(+) (and thus water) absorption. Small conductance basolateral (KCNQ1/KCNE3) K(+) channels have never been evaluated in human colon. We therefore evaluated KCNQ1/KCNE3 channels in distal colonic crypts obtained from normal and active ulcerative colitis (UC) patients.

Methods: KCNQ1 and KCNE3 mRNA levels were determined by qPCR, and KCNQ1/KCNE3 channel activity in normal and UC crypts, and the effects of forskolin (activator of adenylate cyclase) and UC-related proinflammatory cytokines on normal crypts, studied by patch clamp recording.

Results: Whereas KCNQ1 and KCNE3 mRNA expression was similar in normal and UC crypts, single 6.8 pS channels were seen in 36% of basolateral patches in normal crypts, and to an even greater extent (74% of patches, P < 0.001) in UC crypts, with two or more channels per patch. Channel activity was 10-fold higher (P < 0.001) in UC crypts, with a greater contribution to basolateral conductance (5.85 ± 0.62 mS cm(-2)) than in controls (0.28 ± 0.04 mS cm(-2), P < 0.001). In control crypts, forskolin and thromboxane A2 stimulated channel activity 30-fold and 10-fold respectively, while PGE2, IL-1β, and LTD4 had no effect.

Conclusions: KCNQ1/KCNE3 channels make only a small contribution to basolateral conductance in normal colonic crypts, with increased channel activity in UC appearing insufficient to prevent colonic cell depolarization in this disease. This supports the proposal that defective Na(+) absorption rather than enhanced Cl(-) secretion, is the dominant pathophysiological mechanism of diarrhea in UC.

Keywords: Human colonic crypts; KCNQ1/KCNE3 channels; Patch clamp; Ulcerative colitis.

Fig. 1

(A) Agarose gel showing RT-PCR products of KCNQ1 and KCNE3 in control human sigmoid colon. L = 100 bp ladder, +ve = cDNA template present, -ve = cDNA template absent. (B) Plot of logarithmic fluorescence versus RT-PCR cycle number, showing the log-linear phase of PCR amplification. (C) Melting curve analysis of PCR products. (D) Relative levels of KCNQ1 and KCNE3 mRNA in control and UC patients.

Fig. 2

(A) SK channel currents across a cell-attached basolateral membrane patch on a crypt from control sigmoid colon at different command potentials (V_com) referenced to the pipette interior. Broken line indicates closed channel current and downward deflections indicate channel openings. (B) Linear I/V_com relationship of the SK channel shown in A.

Fig. 3

SK channel currents across cell-attached basolateral membrane patches on crypts from (A) control and (B) UC sigmoid colon, at a command potential (V_com) of −100 mV (referenced to pipette interior). Broken line indicates closed channel current and downward deflections indicate channel openings.

Fig. 4

SK channel currents across cell-attached basolateral membrane patches on crypts from normal sigmoid colon, in the basal state and after the addition of 10 μM forskolin (A) or (B) 1 μM TXA₂. Recordings were obtained at a command potential (V_com) of −100 mV (referenced to pipette interior), broken lines indicating closed channel currents and downward deflections indicating channel openings.