A novel plate reader based assay for inhibitor studies of slc4a10 protein and other acid/base transporters

The function of members of the slc4a family of HCO 3 − transporters is commonly determined using fluorescence‐microscopy of tissue loaded with pH i sensitive dyes. The cells are subjected to an acidification induced by NH 4 Cl and the Na + dependent pH i recovery is determined in the presence of CO 2 /HCO 3 − . The purpose of the present study was to implement the method in a plate‐reader based assay to increase the throughput of the method by measuring an entire cell population, and performing multiple experiments in parallel, thus improving stability and reliability of data. A 3T3 NIH‐fibroblast cell line was stably transfected with the Na + /HCO 3 − transporter encoded by slc4a10 . pH i of cells was measured in 8 wells of 96‐well plates in an Enspire® platereader using the fluorescent pH i indicator BCECF. Control experiments included DIDS, an inhibitor of bicarbonate transporters. The Na + dependent pH i recovery in slc4a10 transfected cells was 17.8±5.35 ×10 −4 pH units/s (n=12). 200 μM DIDS inhibited the recovery by 65% (6.16±3.35 ×10 −4 pH units/s, n=12). The endogenous Na + /H + exchange in the cells was similarly reduced to 66% by 10 μM EIPA in the absence of CO 2 /HCO 3 − . In conclusion, this method is a novel and valuable tool for high throughput screening of pharmacological agents inhibiting the acid/base transporters.