Identification of two pH-dependent, Zn²⁺-induced electrogenic transport pathways in human intestinal Caco-2 epithelia

We have previously reported that an electrogenic transport process is associated with apical exposure to Zn2+ in Caco-2 epithelia (Scott et al. 2002). This process is pH dependent, but the identities of the apical and basolateral current carriers are yet to be determined. While the apical membrane Fe2+ carrier in enterocytes is reported to be the H+-coupled transporter, DMT-1 (Gunshin et al. 1997), controversy exists over the route for apical Zn2+ entry. This study set out to characterise the Zn2+-induced short-circuit current (ISC) in Caco-2 epithelia. ISC determinations were made on voltage-clamped Caco-2 epithelia (passages 38-45), grown on permeable supports (Anotec, Nunc). Cells were bathed with isotonic mannitol/Hepes buffer (37 °C; pH 7.4). At the onset of the experiment, apical pH was adjusted to 6.0. Zinc-histidine (1:5 molar ratio) was applied apically. At apical pH 6.0, addition of 100 µM Zn2+ resulted in a peak inward ISC of 0.25 ± 0.05 µA cm-2 (5) (mean ± S.E.M. (n)). Cumulative additions of Zn2+ produced a saturable ISC response with KM = 286 ± 20 µM (5) and Vmax = 1.29 ± 0.12 µA cm-2. Reducing buffer temperature to 4 °C abolished the ISC response (P < 0.001; Student's unpaired t test). When apical pH was changed from 6.0 to 6.8, the ISC decreased (P < 0.05) to negligible levels. However, when apical pH was raised to 7.4, the ISC was outwardly directed, with KM = 305 ± 63 µM (6) and Vmax = 3.17 ± 0.38 µA cm-2 (6). This outward ISC was abolished (P < 0.001) at 4 °C. These data identify two distinct Zn2+-induced electrogenic transport pathways in Caco-2 epithelia. Both processes are temperature dependent and saturable. They are both pH dependent, but at pH 6.0, the evoked ISC is inward, whereas at pH 7.4 the ISC is outward. At pH 6.8 neither the inward or outward ISC is observed. The inward ISC is consistent with H+-coupled Zn2+ transport similar to Fe2+ uptake via DMT-1 (Gunshin et al. 1997), although an alternative apical membrane Zn2+ transporter (hZIP4) has recently been identified (Wang et al. 2002). The outward ISC at pH 7.4 has no immediately evident physiological function. However, it may be reporting Zn2+ transport via hZTL-1, an apical membrane transporter that operates optimally at pH 7.4 (Cragg et al. 2002).