Zn 2+ activates KCNQ1 and KCNN4 K + channels in T84 human colonic epithelial cells
Mongiardo, Krystin M.
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Oral administration of Zn 2+ has been shown to reduce secretory diarrhea in children, but the mechanisms of action of this anti-secretory agent are unknown. We examined the effects of Zn 2+ on K + channels in secretory epithelial tissues of the intestine. In these tissues, the basolateral membrane K + channel KCNQ1 (KvLQT1) associates with the KCNE3 ancillary subunit to form a voltage-insensitive K + channel. Another known basolateral K + channel present in this cell type is KCNN4 (hIK1). These channels play a critical role in anion secretion by establishing an electrical driving force for anion exit through the Cl − channel, CFTR. Human colonic T84 cells were used to study the effects of Zn 2+ in an intact intestinal secretory tissue. We used 8-(4-chlorophenylthio)-cAMP (50 μM) to activate short circuit current (I sc ) across T84 monolayers. In these tissues, Zn 2+ (2 mM), in the serosal bath, transiently increased I sc followed by inhibition of the current. Clotrimazole, TRAM-34, amantadine, 293B, and charybdotoxin, inhibitors of KCNQ1 and KCNN4, were used to show that both channels are involved in the Zn 2+ activation of I sc . To directly demonstrate the significance of basolateral membrane K + channels in this response, the apical membrane of T84 monolayers was permeabilized using amphotericin B (100 μM) in the mucosal bath, and transepithelial K + currents were measured in the presence of an apical-to-basolateral chamber K + concentration gradient. Zn 2+ transiently increased K + current in the presence or absence of cpt-cAMP stimulation. Clotrimazole, TRAM-34, and amantadine treatment reduced this current. Whole-cell voltage-clamp measurements and fura-2 Ca 2+ imaging experiments were performed on single, non-polarized T84 cells. Whole-cell voltage-clamp measurements of membrane K + current show that Zn 2+ activates K + currents, eliciting two types of a response; a transient spike in current or a transient rise in current followed by oscillations in current. Fura-2 experiments demonstrated that Zn 2+ can initiate a rise in intracellular Ca 2+ levels and Ca 2+ oscillations. In addition, we used the Zn 2+ chelator, N,N,N,N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), to show that Zn 2+ can enter isolated cells. We also showed that the activation of I sc by Zn 2+ could be blocked by exposure of monolayers to TPEN. Similarly, exposure of tissues to the Ca 2+ chelator, bapta-AM, reduced the current. These results show that Zn 2+ transiently activates active Cl − secretion in T84 intestinal secretory tissue followed by a steady-state inhibition. The sites of Zn 2+ action are the cAMP-activated KCNQ1 K + channel and the Ca 2+ -activated KCNN4 K + channel. The mechanism is likely to be through direct effect on the channels at an intracellular site.