Fig. 8. Expression properties of ZAC^Thr128 and ZAC^Ala128 in tsA201 and HEK293 cells.

A. Functional properties of HA-ZAC^Thr128 and HA-ZAC^Ala128 expressed in oocytes injected with 1.84 ng cRNA of both subunits. Left: Averaged current amplitudes evoked by Zn²⁺ (10 mM) in ZAC^Thr128-, ZAC^Ala128-, HA-ZAC^Thr128- and HA-ZAC^Ala128-expressing oocytes (mean ± S.E.M., n = 4–8). Middle: Averaged concentration-response relationships displayed by Zn²⁺ at HA-ZAC^Thr128 and HA-ZAC^Ala128 (means ± S.E.M., n = 6–8). Right: Averaged current amplitudes evoked by H⁺ (pH 4.0) in HA-ZAC^Thr128- and HA-ZAC^Ala128-expressing oocytes (means ± S.E.M., n = 6–8). B. Total and cell surface expression levels of HA-ZAC^Thr128 and HA-ZAC^Ala128 transiently expressed in tsA201 cells in the ELISA experiments. All transfections used a total of 4 μg cDNA (all in the pCIneo vector) per 6-cm dish, where the quantities of specific cDNAs indicated in the figure were supplemented with “empty” pCIneo up to 4 μg. Total and cell surface expression was determined in permeabilized (Triton-X-treated) and non-permeabilized cells, respectively. Data (in %, normalised to the absorbance for the reference “HA-ZAC^Thr128 (4.0)”-transfected cells) are given as mean ± S.E.M. and are based on a total of three experiments (n = 3). A line marker indicates the 100% level. C. Co-immunoprecipitation of ZAC^Thr128 and ZAC^Ala128. HEK293 cells were transfected with myc-ZAC^Thr128 alone, myc-ZAC^Thr128/HA-ZAC^Thr128, myc-ZAC^Thr128/HA-ZAC^Ala128, or myc-ZAC^Ala128/HA-ZAC^Ala128. ZAC proteins were immunoprecipitated using myc-beads. Lysates and immunoprecipitated samples were resolved by SDS-PAGE. The myc-, HA- and actin-expression levels were measured by immunoblot.