IK_Ca channels in epithelial and non-epithelial cells are exquisitely sensitive to Ca²⁺ despite the absence of Ca²⁺-binding sites. Intracellular Ca²⁺ regulates IK_Ca activity by complexing with calmodulin, which is bound to the cytosolic carboxyl tail (Fanger et al. 1999; Khanna et al. 1999). Although calmodulin antagonists (e.g. W-7) might be expected to decrease IK_Ca activity, their effects are by no means clear, W-7 either having no effect (Fanger et al. 1999) or decreasing IK_Ca channel activity (Khanna et al. 1999) in human T-lymphocytes. The aim of the present study was to use W-7 to evaluate the role of calmodulin in the regulation of basolateral IK_Ca channels in human colonic crypt cells.

With ethics committee approval and informed consent, multiple biopsies were obtained from the distal colon of patients undergoing colonoscopy, who were free of mucosal disease. Intact crypts were isolated by Ca²⁺ chelation (Sandle et al. 1994) and used for patch clamp studies of the basolateral membrane in the cell-attached configuration (NaCl solution containing variable Ca²⁺ in bath and KCl containing 1.2 mM Ca²⁺ in pipette, holding voltage -40 mV). Data are shown as mean (± S.E.M.) single channel open probability (P_o). Differences between mean values were assessed by Student’s unpaired t test.

In the absence of W-7, decreasing bath [Ca²⁺] from 1.2 mM to 100 mM produced a reversible decrease in IK_Ca channel activity, whereas with 25 mM W-7 in the bath solution there was a high level of channel activity which was unaffected by changes in bath [Ca²⁺] (Table 1). These results suggest that W-7 ‘clamps’ IK_Ca activity at a high level set by the high initial bath [Ca²⁺]. To explore this possibility, experiments were done with a low initial bath [Ca²⁺], in which increasing bath [Ca²⁺] from 100 mM to 1.2 mM produced a reversible increase in IK_Ca channel activity, whereas the presence of 25 mM W-7 ensured a high level of IK_Ca activity irrespective of bath [Ca²⁺] (Table 2).

We conclude that W-7 enhances the activity of basolateral IK_Ca channels in human colonic crypt cells. This novel finding is inconsistent with the role of W-7 as a calmodulin antagonist, and raises the possibility that W-7 may increase intracellular Ca²⁺ concentration irrespective of extracellular Ca²⁺ concentration and/or enhance the sensitivity of human colonic IK_Ca channels to Ca²⁺.

This work was supported by the MRC and The Wellcome Trust.

All procedures accord with current local guidelines and the Declaration of Helsinki.