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 expressing prestin are reported here. Indirect measurements, using imaging techniques, show that transfected HEK-293 or CHO-Ki 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 Km=24 nm. 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 co-transported 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.

Figure 1. Immunofluorescence of transfected cells

A, HEK-293 cell expressing the green fluorescent protein (GFP)-tagged prestin protein. As prestin is a membrane protein, the GFP tag is predominantly localised at the plasma membrane. B, HEK-293 cell expressing GFP alone; GFP was localised in the cytoplasm and not in the membrane. Scale bar, 10 μm.

Figure 2. Diameter, area and fluorescence change during sugar application on HEK-293 cells expressing rat prestin

Measurements of cell diameter (A) or area change (B) using two different techniques (see Methods). The bar indicates the sugar application. C, change in the GFP fluorescence of a region of interest contained entirely within the cytoplasmic region (as indicated) during application of 30 mm fructose (○) but not 30 mm glucose (•). Traces have been normalised to the maximum GFP fluorescence intensity at the beginning of each recording. D, same data as in C but with linear subtraction for photobleaching.

Figure 5. Sugar transport and non-linear capacitance measured in CHO-K1 cells expressing rat prestin

A, CHO-K1 cell expressing the GFP-tagged prestin protein where a membrane translocation is evident. B, the two cell types, when transfected, responded indistinguishably: iso-osmotic replacement of glucose with 30 mm fructose does not differ between HEK-293 and CHO-K1 cells. C, non-linear capacitance measured with a phase-tracking technique in response to a voltage ramp from −150 to +50 mV. Line is the fit of eqn (3) to data with values for the fit parameters of −90 mV (V_1/2) and 39 mV (α). Capacitance measured relative to the linear capacitance C_lin.

Figure 3. Sugar uptake by HEK-293 cells expressing rat prestin

A, the GFP tag did not interfere with fructose transport. B, glucose, fructose and mannose uptake by HEK-293 cells expressing prestin, measured by change in area. Area change after 150 s of isosmotic replacement of 30 mm glucose by the same solution, fructose at increasing concentration (5, 20, 30 and 50 mm) or 30 mm mannose is shown. The last two columns show the effect of two compounds added to 30 mm fructose: 5 mm salicylate (a blocker of OHC electromotility) and 1 mm DIDS (a blocker of the chloride-bicarbonate exchanger).

Figure 4. Kinetics of fructose uptake by HEK-293 cells transfected with rat prestin

A, changes in diameter following sugar application at different concentrations. The solid horizontal bar indicates the timing of the isosmotic replacement of glucose by fructose. Fructose was applied at 5 mm (n = 12), 20 mm (n = 9), 30 mm (n = 20) and 50 mm (n = 12). The dashed line superimposed on the traces is a linear fit through the rising phase of the volume response. B, initial transport rate estimated by a linear fit between t = 0 s and t = 20 s after the beginning of the application. Data were fitted by eqn (3) with V_max = 0.04 % s⁻¹ and K_m = 24 mm.

Figure 6. Water movement in HEK-293 cells transfected with rat prestin

Cell swelling caused by isosmotic 30 mm glucose/fructose replacement (○) and 30 mosmol kg⁻¹ hypo-osmotic glucose solution application (•). With prolonged hypo-osmotic glucose solution exposure (dotted line), the cell reached a steady state. Cell shrinking was caused by hyperosmotic fructose solution application (▪). The cell was equilibrated in 20 mm fructose solution and then exposed to 10 mosmol kg⁻¹ hyperosmotic solution containing 30 mm fructose. Although the cell volume decreased, the water permeability (L_p) was the same as in isosmotic fructose replacement. In all cases, the superimposed straight lines are a linear fit through the rising phase of the volume response and the horizontal bar indicates the timing of the application (200 s).

Figure 7. Sugar uptake by HEK-293 cells expressing the pendrin protein

A, HEK-293 cell expressing the GFP-tagged pendrin protein. B, change in cell area following isosmotic replacement in the bathing solution of 30 mm glucose by 30 mm fructose or 30 mm mannose. Scale bar, 10 μm.