Chloride Flux in Prestin-Expressing Cells

Abstract

In prestin-expressing cells, intracellular Cl- ([Cl-]i) flux plays a preeminent role in promoting prestin activity, especially since the resulting Vh shift in prestin's state-probability function (inferred from nonlinear capacitance - NLC) along the Vm axis will effect a motile response. For example, with perforated patch clamp and local perfusion, changing extracellular Cl concentration from 1 mM to 140 mM using prestin's NLC as a measure of intracellular Cl indicates a several mM increase in intracellular Cl concentration. Nevertheless, the mechanism underlying Cl- flux in prestin-expressing cells is not clear.To better define Cl- flux, the CFP-YFP-based ratiometric Cl indicator (Cl-sensor, J. Neurosci. Meth., 2008, 170, 67) was modified to reduce pH sensitivity by shifting the pKa away from the physiological pH range. Using excitation ratiometric imaging of fluorescence, [Cl]i was measured in either induced- or non-induced prestin-expressing HEK cell-lines transfected with our new Cl-sensor construct. Upon changing local extracellular perfusion from 0.2 mM to 140 mM Cl buffer, fluorescence measures indicate a significant difference between induced- and non-induced prestin cell-lines.We also evaluated [Cl]i flux using a Cl-sensor fusion product of prestin at the C-terminal to better gauge flux near prestin's intracellular binding site. Comparison of [Cl]i flux in HEK-293T cells transfected with a normal prestin-Cl-sensor or a prestin construct with a C-terminal deletion that eliminated NLC (P709-Cl-sensor) showed no statistical difference in flux. These data suggest that prestin's NLC is separable from Cl- movement induced by prestin, as we previously suggested (Bai et al., BJ, 3179, 2009). Further experiments are underway, including voltage dependence of flux by simultaneously monitoring fluorescence ratio signals and NLC using perforated patch.(Supported by NIDCD DC00273 and NIDCD DC 008130)