Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.

Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na(+), Cl(-), and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na(+):1 Cl(-):1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na(+), Cl(-), and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na(+), Cl(-), or GABA. The shifts in V rev for a tenfold change in the external Na(+), Cl(-), and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na(+), Cl(-), and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na(+):1 Cl(-):1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease.

Turnover rate of the gamma-aminobutyric acid transporter GAT1.

We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the gamma-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The two-electrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA (I(NaCl)(GABA)), macroscopic charge movements (Q (NaCl)) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q (NaCl) and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 +/- 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 +/- 1 elementary charges per GAT1 monomer. This information and the relationship between I(NaCl)(GABA) and Q (NaCl) were used to estimate a GAT1 unitary turnover rate of 15 +/- 2 s(-1) (21 degrees C, -50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79-93 s(-1) (37 degrees C, -50 to -90 mV).

Na(+)/monocarboxylate transport (SMCT) protein expression correlates with survival in colon cancer: molecular characterization of SMCT.

We report an extensive characterization of the Na(+)/monocarboxylate transporter (SMCT), a plasma membrane protein that mediates active transport of monocarboxylates such as propionate and nicotinate, and we show that SMCT may play a role in colorectal cancer diagnosis. SMCT, the product of the SLC5A8 gene, is 70% similar to the Na(+)/I(-) symporter, the protein that mediates active I(-) uptake in the basolateral surface of thyrocytes and other cells. SMCT was reported in the apical surface of thyrocytes and formerly proposed also to transport I(-) and was called the apical I(-) transporter. However, it is now clear that SMCT does not transport I(-). Here we demonstrate a high-affinity Na(+)-dependent monocarboxylate transport system in thyroid cells, which is likely to be SMCT. We show that, whereas thyroidal Na(+)/I(-) symporter expression is thyroid-stimulating hormone (TSH)-dependent and basolateral, SMCT expression is TSH-independent and apical not only in the thyroid but also in kidney and colon epithelial cells and in polarized Madin-Darby canine kidney cells. We determine the kinetic parameters of SMCT activity and show its inhibition by ibuprofen (K(i) = 73 +/- 9 microM) in Xenopus laevis oocytes. SMCT was proposed to be a tumor suppressor in colon cancer. Significantly, we show that higher expression of SMCT in colon samples from 113 colorectal cancer patients correlates with longer disease-free survival, suggesting that SMCT expression may be a favorable indicator of colorectal cancer prognosis.

Calcineurin activation influences muscle phenotype in a muscle-specific fashion.

BACKGROUND: The calcium activated protein phosphatase 2B, also known as calcineurin, has been implicated as a cell signaling molecule involved with transduction of physiological signals (free cytosolic Ca2+) into molecular signals that influence the expression of phenotype-specific genes in skeletal muscle. In the present study we address the role of calcineurin in mediating adaptations in myosin heavy chain (MHC) isoform expression and muscle mass using 3-month old wild-type (WT) and transgenic mice displaying high-level expression of a constitutively active form of calcineurin (MCK-CN* mice). RESULTS: Slow muscles, e.g., soleus, were significantly larger (by ~24%), whereas fast muscles, e.g., medial gastrocnemius (MG) and tibialis anterior were significantly smaller (by ~26 and ~16%, respectively) in MCK-CN* mice compared to WT. The masses of mixed phenotype muscles, such as the plantaris and the extensor digitorum longus, were not significantly changed from WT. The soleus, plantaris, MG and diaphragm displayed shifts toward slower MHC isoforms, e.g., soleus from WT mice contained ~52% MHC-I, ~39% MHC-IIa, and ~9% MHC-IIx, whereas MCK-CN* mice had ~67% MHC-I, ~26% MHC-IIa, and ~7% MHC-IIx. The specific isoforms that were either up or down-regulated were muscle-specific. For instance, the proportion of MHC-IIa was decreased in the soleus and diaphragm, but increased in the plantaris and MG of MCK-CN* mice. Also, the proportion of MHC-IIx was unchanged in the soleus, decreased in the diaphragm and increased in the plantaris and MG of MCK-CN* relative to WT mice. Fast to slow shifts in fiber type proportions were evident for the plantaris, but not the soleus. Fast, but not slow, plantaris fibers of MCK-CN* mice had higher oxidative and lower glycolytic properties than WT. CONCLUSION: These data suggest that calcineurin activation can influence muscle phenotype and that the specific influence of calcineurin activation on the phenotypic and mass characteristics of a muscle is dependent upon the original phenotypic state of the muscle.