Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

Abstract

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s⁻¹) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.

Figures

Fig. 1.

Radioactive tracer flux experiments in human Na+/d-glucose cotransporter 2 (hSGLT2) and hSGLT1. [14C]-α-methyl-d-glucopyranoside (αMDG, 50 μM) uptake was measured in hSGLT2-, hSGLT1-, and untransfected human embryonic kidney 293T (HEK293T) cells at 37°C. Uptake is expressed as quantity of tracer (pmol) per minute per well; n = 3 wells per bar; error bars are ± SE. Cells transiently expressing either hSGLT2 or hSGLT1 showed significant (P < 0.05) αMDG uptake (−Pz) above background; when SGLT inhibitor phlorizin (Pz, 100 μM) was added, uptake was reduced to background levels (+Pz), as seen in untransfected cells [not significant (N.S.), P > 0.05].

Fig. 2.

Steady-state hSGLT2 Na+/d-glucose cotransport is electrogenic and membrane potential (Vm) and temperature dependent. A: whole cell recordings at 37°C and a Vm of −60 mV in HEK293T cells expressing hSGLT2 revealed inward current upon addition of glucose to the extracellular Na+ solution. Upon returning to Na+-only, current was restored to baseline. In Na+-free buffer (i.e., 150 mM cholineCl), there was no glucose-induced current (Fig. 3B). B: current-voltage (I-V) relationships of the glucose-induced current (□, 22°C; ■, 37°C).

Fig. 3.

Current records in response to step jumps in membrane voltage. Cells were held at the holding membrane potential (Vh = −60 mV). With application of the test voltage pulse, current relaxation showed an initial capacitive spike due to membrane bilayer capacitance followed by decay (It,On) to the steady-state level (Iss). Upon return to Vh, the transient current was in the opposite direction and consisted of the capacitive spike followed by a decay (It,Off) to the baseline holding current value (Ihold). Test voltage pulse duration was 30 ms, starting at +10 mV and ending at −110 mV, in 20-mV decrements. A–C: transient and steady-state current records from a single HEK293T cell expressing hSGLT2 at 37°C, in Na+ alone (A) and Na+ and saturating d-glucose (50 mM) (B), and Na+-d-glucose difference current with the steady-state currents removed (C). D–F: current records for a single HEK293T cell expressing hSGLT1 at 37°C in Na+ alone (D), Na+ and saturating d-glucose (100 mM) (E), and Na+-d-glucose difference current (F). In view of the settling time of the voltage clamp, the transient currents in C and F are shown 1.2 ms after the voltage pulse.

Fig. 4.

Kinetic properties. A: steady-state kinetics of d-glucose transport by hSGLT2 at 37°C and Vm = −60 mV. Glucose-induced currents are plotted against extracellular glucose concentration, and the curves represent the data fit to the Hill equation (Eq. 2) with the Hill coefficient, nh, constrained to 1. Maximal glucose-induced current (Imaxd-glucose) indicates the level of expression in a given cell, whereas the glucose concentration at half-maximal current (K0.5d-glucose) reflects the transporter's apparent affinity for glucose. B: steady-state kinetics of Na+ activation of hSGLT2, 37°C and Vm = −60 mV. Inward, 100 mM glucose-induced current is plotted as a function of extracellular Na+ concentration and the data are fitted to the Hill equation, maximal current (Imax), nh, and apparent Na+ affinity (K0.5Na) values. The statistics for maximal current (ImaxNa) and apparent Na+ affinity (K0.5Na) in B (as in A for sugar) are SEs of the fit. For both substrates, K0.5 values were averaged from multiple cells and compared with those from hSGLT1 (Table 1).

Fig. 5.

d-Glucose vs. d-galactose as substrates of hSGLT2. Inward current resulting from 100 mM d-glucose vs. 100 mM d-galactose was compared in a single HEK293T cell expressing hSGLT2 at 37°C. K0.5d-glucose and K0.5d-galactose values are presented, based on dose-response studies of both substrates; the K0.5d-galactose value was estimated ≥100 mM.

Fig. 6.

Determination of Na+:glucose coupling ratio for hSGLT2. Reversal potentials (Vr) for hSGLT2 at 37°C with fixed Na+ and αMDG gradients [75 mM extracellular Na+ concentration ([Na+]o), 50 mM intracellular [Na+] ([Na+]i), 30 mM [αMDG]i, and 2, 4, 9, or 16 mM [αMDG]o]. With [Na+]o, [Na+]i, and [αMDG]i constant, Vr is plotted vs. (RT/F) × ln([αMDG]o), where R is gas constant, T is absolute temperature, and F is Faraday's constant. The inverse of the slope is the Na+:glucose coupling ratio, n (Eqs. 4 and 5). As shown in the plot, n = 1.0 ± 0.02 (± error of the fit).

Fig. 7.

Time course of phlorizin inhibition of hSGLT2 and 1. Continuous current records of the sugar current (100 mM glucose, 60 pA for hSGLT2; 2 mM glucose, 400 pA for hSGLT1) as phlorizin (1 μM) was added or removed from the external solution, in hSGLT2 (A) and hSGLT1 (B). t1/2,On and t1/2,Off are the half-times for the current to reach steady state with phlorizin addition and removal. Upon washout of phlorizin (in the presence of glucose), the current returned to approximately the same level of Iglucose. t1/2,On was estimated to be within the half-time of the solution change: ≈2 s (A) and ≈0.5 s (B). As a result, t1/2,On for phlorizin binding could not be directly determined. For both A and B, t1/2,Off was much slower than the solution change half-time. Also, t1/2,Off by definition does not depend on bath concentrations of either d-glucose or phlorizin. t1/2,Off was used to calculate the kOff (Eq. 7), which along with the Ki was used to determine the theoretical kOn (Eq. 8). In each experiment the t1/2 for solution changes was determined from the current responses upon replacing the external Na+ with choline+.