(A) Identification of hSGLT3-mRNA in human skeletal muscle and human small intestine by RT-PCR. Several different pairs of primers were used: lanes 2–4, primer HSA40/primer HSA39 (segment size: 903 bp); lanes 5–7, primer HSA38/primer HSA39 (segment size: 344 bp); lanes 9–11, primer HSA38/primer HSA37 (segment size: 1,250 bp); lanes 12–14, primer HSA42/primer HSA 37 (segment size: 251 bp). The primer pairs are separated by introns of the genomic DNA. Lanes 1 and 15, 100-bp ladder (MBI Fermentas); lane 8, 1-kbp ladder (MBI Fermentas); lanes 2, 5, 9, and 12, negative controls (–) (water instead of RNA in RT-PCR); lanes 3, 6, 10, and 13, skeletal muscle (M); lanes 4, 7, 11, and 14, small intestine (I). The PCR products were resolved on agarose gel, blotted, and hybridized with hSGLT3-specific oligonucleotides ([γ-³²P]ATP) to verify the identification of hSGLT3. Amplificates HSA40/HSA39 and HSA38/HSA39 were hybridized with HSA27 (nucleotides 982–1,006, forward). Amplificates HSA38/HSA37 and HSA42/HSA37 were hybridized with HSA30 (nucleotides 1,878 to 1,855, reverse) (data not shown). (B) Immunochemical staining of Western blots with affinity-purified antibody against amino acids 576–595 from hSGLT3. Plasma membranes from the small intestine, skeletal muscle, and Xenopus oocytes were isolated by differential centrifugation (). The oocytes were injected with 20 ng of hSGLT3-cRNA (lane 4), 20 ng of hSGLT1-cRNA (lane 5), or water (lane 6). In the Western blots, 20 μg of protein was applied per lane. The hSGLT3 antibody recognized a single protein band (≈60 kDa) in membranes isolated from whole small intestine (lane 1) and skeletal muscle (lane 3), and from oocytes injected with hSGLT3 cRNA (lane 4), but not from oocytes injected with cRNA from hSGLT1 (lane 5). Lane 2, membranes from small intestine again, but this time the antibody against SGLT3 was blocked by 1 h (37°C) incubation with 0.1 mg/ml antigenic peptide.

(A) Laser-scanning confocal micrographs of immunostaining by the anti-hSGLT3 antibody (red, 1 and 4) and anti-acetylcholine receptor β-subunit (green, 2 and 5) in the intestine. The images were superimposed to show that the antibodies stain the same structure (yellow, 3 and 6). Reaction products were found below villus crypts in the plexus submucosus (1–3 cross section with crypt in the upper right), and in the plexus myoentericus (4–6, longitudinal section). (Scale bar = 10 μm.) (B) Cartoon of a cross-section of small intestine showing the major structures and location of the myenteric neurons.

(A) The reaction of hSGLT3 antibody in a human skeletal muscle biopsy. The nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Immunostaining by the anti-hSGLT3 antibody (red, 1 and 4) and the anti-acetylcholine receptor β-subunit antibody (green, 2 and 5) in skeletal muscle. 3 and 6 show that both antibodies stain the same structure. (Scale bar = 10 μm in 3 and 2 μm in 6.)

Sugar-induced changes in membrane potential in a single hSGLT3-expressing oocyte. (A) In Na⁺buffer at pH 7.5, 5 mM glucose or αMDG depolarized the membrane by ≈4 mV, whereas 5 mM galactose had no effect. Phlorizin, a high-affinity competitive inhibitor of Na⁺-glucose cotransport, at a concentration of 250 μM, inhibited the membrane depolarization induced by 5 mM glucose. (B) Voltage depolarization in response to glucose. The data were fitted to calculate the K_0.5 (≈20 mM glucose) and the maximal depolarization (23 mV) by using the equation I = I_max·[S]/(K_0.5 + [S]) (see text for details). In six experiments, K_0.5 = 60 ± 10 mM and ΔV_max = 26 ± 9 mV.

Effect of membrane potential and pH on sugar-induced current in an hSGLT3-expressing oocyte. The figure shows the currents induced by 25 mM αMDG in Na⁺ buffer at neutral and acidic pHs and in buffer without Na⁺ at pH 5 at voltages ranging from +50 to –90 mV.

Sugar uptake is not coupled to cation uptake through SGLT3. Sugar-induced current (A) and glucose uptake (B) were simultaneously measured in the same hSGLT3-expressing oocytes (hSGLT3) and in noninjected oocytes (control). The membrane was voltage-clamped at –70 mV, and the experiment was carried out in Na⁺ buffer at pH 5. (A) In control oocytes, 2 mM glucose did not induce any current, whereas in hSGLT3-expressing oocytes, a sugar-dependent current equivalent to univalent positive charge of 81 ± 7 pmol per 5 min was recorded. (B) Glucose uptakes were recorded in the same oocytes used to measure the currents in A. The glucose uptake in the hSGLT3 oocytes was identical to that in control oocytes (P > 0.5). The data are from five hSGLT3-expressing and five control oocytes. (C) Ratio of positive ion uptake and sugar uptake in pSGLT3- and hSGLT1-expressing oocytes. These experiments were carried out as described for hSGLT3 (A and B). In hSGLT1-expressing oocytes, the ratio in Na⁺ at neutral pH is 2.1 ± 0.04 (n = 4) and at acidic pH in H⁺ is 2 ± 0.1 (n = 7) (taken from ref. ). pSGLT3-expressing oocytes show the same ratio of positive charge/sugar uptake at neutral pH: 2.1 ± 0.1 (n = 12), but at acidic pH the ratio increases to 4.7 ± 0.2 (n = 6).