Proceedings of The Physiological Society

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, PCB113

Poster Communications

YFP detection of chloride transport by the outer hair cell protein prestin

L. Cruddas2, A. Krasnow1,2, T. West1, M. Gomez2, N. Daudet2, J. Ashmore1,2

1. Neuroscience, Physiology & Pharmacology, UCL, London, United Kingdom. 2. UCL Ear Institute, UCL, London, United Kingdom.

Prestin is a protein expressed abundantly in the lateral membrane of the cochlear outer hair cells (OHCs) and is a critical component in the mechanism of sound amplification in the mammalian inner ear. It is a member of the SLC26 superfamily of anion transporter related proteins but in OHCs, it acts as a voltage-dependent area motor. Its transport function has proved more difficult to show and it has been termed an incomplete transporter. We have recently shown that in an expression system prestin acts a weak electrogenic Cl : HCO3 exchanger (Mistrik et al., 2012) by using a prestin-pHluorin construct to measure the initial rate of recovery from a CO2 - induced intracellular acidification close to the membrane. We show here, using a prestin-YFP construct, that chloride is counter-transported by prestin. To detect chloride movement near the membrane, CHO cells were transfected with prestin linked on its cytoplasmic surface to a particular variant of YFP, enhanced to detect chloride (EYFP: 148H, 152I, 163V; a kind gift of F. Pereira). EYFP fluorescence is quenched by elevated chloride. Expression of EYFP appeared as a bright ring localised to the surface membrane and only cells which had a high level of expression were imaged using an excitation wavelength of 488 nm and emission and collection at 510-530 nm. Cells were continuously superfused and external solutions changed as appropriate. When cells were exposed to high (25 mM) external HCO3- and low (4 mM) Cl- containing solution (gluconate replacing Cl-), cells responded with an increase of fluorescence, indicative of a reduction of intracellular Cl-. As control, the experiments were repeated with cells transfected with the same EYFP fused to a membrane-targeting domain. In response to the same external solution switch, no change in membrane EYFP fluorescence was observed, indicating that that there is a requirement for prestin in order for cells to exhibit significant Cl- transport.By observing permeabilised cells , we estimated IC50 = 51 mM for fluorescence quench of EYFP in CHO cells. Using whole cell recording with different pipette chloride concentrations we estimated the internal Cl-. Prestin density /cell was obtained by measuring non-linear capacitance in transfected cells (Ashmore, 2008), and hence we determined the turnover number for prestin to be approximately 800 s-1, two orders of magnitude less than for the Cl:HCO3 exchanger in red blood cells. Prestin is therefore an inefficient but not an incomplete transporter.

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