Abstract
We have cloned a Na+-dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na+-dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na+-dependent transport of succinate with a Kt of 2 microM. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na+ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li+ inhibits NaDC3 in the presence of Na+. Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8. 0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6. 5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent, high affinity dicarboxylate transporter.
Highlights
Placenta plays an obligatory role in providing the developing fetus with essential nutrients and metabolic fuels [1, 2]
A high affinity sodium/dicarboxylate transporter, similar to the one expressed in the placental brush border membrane, is present in the renal basolateral membrane [9, 10] and in the liver canalicular membrane [11, 12]
Rat NaDC1 exhibits a Kt value of 25–30 M for succinate [15, 16], and the observed significant divergence in the amino acid sequence of rat NaDC1 in comparison with rabbit NaDC1 and human NaDC1 is likely to be related to the difference in the substrate affinity between rat NaDC1 and rabbit/human NaDC1
Summary
Materials—SuperScript Plasmid System for cDNA cloning and Lipofectin were purchased from Life Technologies, Inc. A commercially available membrane blot containing size-fractionated mRNA (2 g each) from brain, heart, kidney, stomach, small intestine, and skeletal muscle of rat was used for this purpose. The expected size of the PCR product is 1124 bp This sequence in rNaDC3 cDNA possesses a single restriction site each for DraIII and NcoI. When the influence of Naϩ on succinate transport was being investigated, the buffers containing 140 mM NaCl or 140 mM N-methyl-D-glucamine (NMDG) chloride were mixed to give uptake buffers of desired Naϩ composition. When the influence of pH on transport was investigated, transport buffers of different pH values were prepared by varying the concentration of Tris, Hepes, and Mes. Data Analysis—Uptake measurements were made in duplicate and each experiment was repeated two or three times with separate transfections. Kinetic analyses were carried out by nonlinear as well as linear regression methods using the commercially available computer programs Fig.P, version 6.0 (Cambridge, UK) or Sigma Plot (Chicago, IL)
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