An Un-Bleachable YFP-Based Chloride Sensor

Abstract

Prestin (SLC26A5), the protein responsible for electromotility in the mammalian outer hair cell, remains incompletely characterized. We are interested in the protein's transporter/channel-like behavior. Clearly, the functional analysis of many chloride-related membrane proteins, such as channels/transporters/receptors, would benefit from a genetically encoded chloride sensor with reliable properties. Recently we developed an YFP-based chloride sensor, mCl-YFP (Sheng et al., PLoS ONE 2014, 9(6): e99095), with chloride Kd of 14 mM and pKa of 5.9, as well as 15-fold better photostability than wild-type EYFP. Using this chloride sensor, we demonstrated enhanced dynamic flux of chloride in the mM range into HEK cells expressing the fused protein of prestin and mCl-YFP. In order to avoid photobleaching interference during long exposure measurements, a new chloride sensor was developed with 80-fold better photostability than wild-type EYFP by stabilizing the chromophore structure via hydrogen-bond network. Other characteristics are similar to the original sensor we developed. We are currently testing the new sensor's sensitivity to other anions that typically exist in cells. We expect that this sensor will provide more detailed information on the transporter function of prestin, as well as other membrane proteins.