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Biochem Biophys Res Commun
2020 Feb 05;5222:539-544. doi: 10.1016/j.bbrc.2019.11.137.
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Molecular characterization of the orphan transporter SLC16A9, an extracellular pH- and Na+-sensitive creatine transporter.
Futagi Y
,
Narumi K
,
Furugen A
,
Kobayashi M
,
Iseki K
.
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Human monocarboxylate transporters (hMCTs) mediate the transport of monocarboxylates across plasma membranes. One such transporter, hMCT9, has been shown to be related to serum uric acid levels and the risk of renal overload gout. However, the functional characteristics of hMCT9 remain unknown. The aim of this study was to investigate the expression and localization of hMCT9 using a Xenopus laevis oocyte heterologous expression system and characterize its transport properties. Kinetic analysis of hMCT9-mediated creatine uptake revealed that uptake consisted of two components, with apparent Km values of 237 mm (low-affinity) and 23.7 mm (high-affinity), respectively. The transport activity of hMCT9 was dependent on the extracellular pH and activity sharply increased with increasing pH. Under Na+-free conditions, hMCT9-mediated creatine uptake was reduced by one-half, indicating that hMCT9 is a Na+-sensitive transporter. Moreover, carbonyl cyanide 3-chlorophenylhydrazone (a protonophore) inhibited hMCT9 activity, whereas valinomycin (a K+-ionophore) did not inhibit the transporter. These results suggest that hMCT9 is susceptible to changes in H+ gradients. A cis-inhibition assay of hMCT9-and hMCT12-mediated creatine transport revealed that cyclocreatine, creatine, guanidineacetate, and 3-guanidinopropionate are recognized by the transporter, and 4-guanidinobutyrate and guanidinoethyl sulfonate selectively inhibited hMCT9 activity. These findings demonstrate that hMCT9 is an extracellular pH- and Na+-sensitive creatine transporter.
Fig. 1. Membrane localization of hMCT9 and hMCT12 in Xenopus oocytes. Localization of hMCT9 (A) and hMCT12 (B) in the oocyte membrane. hMCT9 cRNA-injected and hMCT12 cRNA-injected oocytes were incubated with antibodies against hMCT9 and hMCT12. Water-injected oocytes were used as negative controls.
Fig. 2. hMCT9-and hMCT12-mediated creatine uptake by oocytes.(A, B) Time course of [14C]creatine uptake through hMCT9 (A) and hMCT12 (B). hMCT9 cRNA-injected and hMCT12 cRNA-injected oocytes were incubated for the indicated time periods. The data were fitted to a two-parameter single exponential equation as described in the Materials and methods section. (C, D) Concentration dependence of [14C]creatine uptake through hMCT9 (C) and hMCT12 (D). Creatine uptake was assayed at the indicated concentrations. The apparent Km was determined using non-linear fitting of the Michaelis–Menten equation, as described in the Materials and methods. Inset: Eadie–Hofstee plot of the data, where the creatine uptake rate (V) was plotted against V/[Creatine] (V/S). Transporter-specific uptake was calculated by subtracting the uptake by water-injected oocytes from the uptake by cRNA-injected oocytes. Values are the means ± S.E. from 3 independent experiments, each with 3–5 replicates.
Fig. 3. Influence of ion composition on creatine transport. (A, B) Effects of extracellular pH and Na+ on [14C]creatine uptake through hMCT9 (A) and hMCT12 (B). Uptake by hMCT9 cRNA-injected and hMCT12 cRNA-injected oocytes was assayed for 60 min (hMCT9) or 10 min (hMCT12) using standard buffers containing 100 mm NaCl or choline chloride at the indicated pH values. (C, D) Effects of CCCP and valinomycin on [14C]creatine uptake through hMCT9 (C) and hMCT12 (D). The requirement of an H+ gradient or plasma membrane potential for creatine uptake was assayed for 60 min (hMCT9) or 10 min (hMCT12) in the absence or presence of 50 μm CCCP or 10 μm valinomycin. a, p < 0.05; c, p < 0.005; d, p < 0.001 versus the control. Transporter-specific uptake was calculated by subtracting the uptake in water-injected oocytes from the uptake in cRNA-injected oocytes. Values represent the means ± S.E. from 3 independent experiments, each performed in 3–5 replicates. Choline chloride, choline Cl; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.
Fig. 4. Inhibitory effects of various compounds on creatine uptake through hMCT9 and hMCT12. hMCT9 cRNA-injected and hMCT12 cRNA-injected oocytes were incubated for 60 min (hMCT9) or 10 min (hMCT12) in standard buffers containing 10 μm [14C]creatine with the test inhibitors (10 mm). a, p < 0.05; b, p < 0.01; c, p < 0.005; d, p < 0.001 versus the control (hMCT9), and B, p < 0.01; D, p < 0.001 versus the control (hMCT12). Transporter-specific uptake was calculated by subtracting the uptake by water-injected oocytes from the uptake by cRNA-injected oocytes. Values presented are the means ± S.E. of 3 independent experiments, each performed with 3–5 replicates.
