Sugar Transport by Mammalian Members of the SLC26 Superfamily of Anion‐Bicarbonate Exchangers

Abstract

The mammalian cochlea contains a population of outer hair cells (OHCs) whose electromotility depends on an assembly of 'motor' molecules in the basolateral membrane of the cell. Named 'prestin', the molecule is a member of the SLC26 anion transporter superfamily. We show both directly and indirectly that SLC26A5, rat prestin, takes up hexoses when expressed in several cell lines. Direct measurements of labelled fructose transport into COS-7 cells expessing prestin are reported here. Indirect measurements, using imaging techniques, show that transfected HEK-293 or CHO-K1 cells undergo reversible volume changes when exposed to isosmotic glucose-fructose exchange. The observations are consistent with the sugar transport. A similar transport was observed using a C-terminal green fluorescent protein (GFP)-tagged pendrin (SLC26A4) construct. Cells transfected with GFP alone did not respond to sugars. The data are consistent with fructose being transported by prestin with an apparent K m = 24 mM. From the voltage-dependent capacitance of transfected cells, we estimate that 250 000 prestin molecules were present and hence that the single transport rate is not more than 3000 fructose molecules s _1. Comparison of the transfected cell swelling rates induced by fructose and by osmotic steps indicates that water was cotransported with sugar. We suggest that the structure of SLC26 family members allows them to act as neutral substrate transporters and may explain observed properties of cochlear hair cells. Journal of Physiology METHODS Cell culture and transfectionHuman embryonic kidney (HEK)-293, COS-7 and Chinese hamster ovary (CHO)-K1 cells were grown at 37°C and in 5 % CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum. Cells were split weekly when confluent. They were transfected directly in a 75 cm 3 flask (Falcon) using LipofectAmine (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's recommendations. A plasmid coding for the six transmembrane KCNQ4 potassium channel (a gift from A. Tinker, Department of Clinical Pharmacology, UCL, UK) was used as a control for expression of a membrane protein. Plasmids containing rat prestin (a gift from B. Fakler, Department of Physiology II, University of Freiburg, Germany) and human pendrin (a gift from R. Trembath, Department of Genetics, University of Leicester, UK) cDNAs were both driven by cytomegalovirus promoter. Prestin cDNA was ligated into the eukaryotic expression plasmid vector pBK-CMV (Stratagene) with the GFP-mut3 coding sequence fused to the 3‚ end of the prestin sequence (see Ludwig et al. 2001).

Tau Aggregation and Toxicity in a Cell Culture Model of Tauopathy

“…Second, the current model employs tau concentrations of ϳ5.6 pg/cell. Assuming a typical HEK-293 cell volume of ϳ4 pl (66,67), these levels correspond to intracellular tau concentrations (ϳ25 M) well above estimates of physiological tau concentrations in cells (68) and also adult brain homogenates (3). In either case, however, the amount of free tau is unknown.…”

Hypotonic swelling of salicylate-treated cochlear outer hair cells

“…Belyantseva et al (2000) obtained values of P f = 9.7 · 10 À3 cm/s (adult rats) and P f = 11.1 · 10 À3 (adult guinea pigs) with puff pipettes of radius R = 0.5-1 lm placed at a distance of H = 25-30 lm applying an osmotic challenge of DC = À5 mOsm for 20 s. Appendix A.1 re-evaluated data by Morimoto et al (2002) in the range 1.5-2 · 10 À3 cm/s for untreated and salicylatetreated OHCs with R = 2.5 lm, H = 10 lm and DC = À45 mOsm for 20-25 s. Following the discovery that the motor mechanism underlying OHC electromotility involves the membrane protein prestin which is a member of the SLC26 family of anion transporter proteins (Zheng et al, 2000), Chambard and Ashmore (2003) obtained P f = 2 · 10 À4 cm/s from prestin-transfected HEK-293 cells with R = 0.5-1 lm, H = 100-200 lm and DC = À5 mOsm for 200 s.…”

Functional Characterization of Wild-Type and a Mutated Form of SLC26A4 Identified in a Patient with Pendred Syndrome

“…Several different studies were made using the expression of the SLC26A4 protein in non-polarized human-embryonic-kidney (HEK) cells [20,36,37], claiming, that the SLC26A4 protein is not expressed in those cells [4,20]. Here we show, that the SLC26A4 protein is expressed in non polarized HEK293-Phoenix as well as in non-polarized WRO cells [13], and therefore we can assume that these cells also possess the environment needed for SLC26A4 function (despite the fact that the cells we use are not polarized).…”