Mechanism of potassium channel activation by zinc
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Oral administration of Zn 2+ is known to lessen both the severity and duration of secretory diarrhea, but the mechanisms of action of Zn 2+ are still unknown. I studied the effects of Zn 2+ on Cl- secretion in human colonic T84 epithelial cells. In these tissues, there are two known basolateral membrane K + channels, KCNQ1 (Kv7.1) and KCNN4 (KCa3.1), which are involved in regulating Cl- secretion by establishing an electrical driving force for Cl- exit through CFTR. In T84 monolayers, after stimulation of short circuit current (I sc ) by 8-(4-chlorophenylthio)-cAMP (50µM), Zn 2+ (2mM) applied to the serosal bath caused a biphasic transient activation of I sc followed by inhibition. The biphasic activation phase of I sc contained fast peak and slow peak components. Both the activation and the inhibition of I sc by Zn 2+ was dose-dependent between 100µM and 2mM Zn 2+ . The addition of U73122 (10µM), a phospholipase C (PLC) inhibitor, to the basolateral side of tissues caused a decrease in the Zn 2+ -activated second, slower peak in I sc . The addition of catalase (1000U/mL), which catalyzes the conversion of H 2 O 2 into H 2 O and O 2 , to both the apical and basolateral side of the tissue potentiated activation of both peaks in I sc during Zn 2+ exposure. Neither U73122 nor catalase had any effect on the inhibition of I sc by Zn 2+ . Cd 2+ (500µM and 2 mM), a divalent cation in the same periodic group as Zn 2+ , also caused a transient activation of I sc followed by inhibition, in a similar manner to Zn 2+ . Whole-cell voltage-clamp measurements were made on single T84 cells. Zinc activated K+ currents (IK) in a transient or sustained manner, and the effects were dose-dependent between 80µM and 2mM Zn 2+ . The addition of dithiotriothol (DTT, 2 mM), a compound that reduces disulfide bonds, also activated I K . Hydrogen peroxide (5mM) also activated IK. The addition of 293B, an inhibitor of KCNQ1, reduced the Zn 2+ -activated I K . The addition of clotrimazole and TRAM-34 (10µM), inhibitors of KCNN4, inhibited the Zn 2+ -activated IK. Overall, these results show that Zn 2+ activates KCNQ1 and KCNN4 K + channels, and suggest the Zn 2+ -induced slow peak in I sc is the result of activation of KCNN4, while activation of the fast peak in I sc is the result of activation of KCNQ1. In addition, Zn 2+ is likely to activate these channels by acting on cysteine residues. Finally, the mechanisms by which Zn 2+ inhibits I sc are not revealed by these studies, but it is likely that Zn 2+ directly inhibits CFTR after crossing the basolateral cell membrane.