Molecular cloning and characterization of an L-epinephrine transporter from sympathetic ganglia of the bullfrog, Rana catesbiana

Abstract

Chemical signaling by dopamine (DA) and L-norepinephrine (L-NE) at synapses is terminated by uptake via specialized presynaptic transport proteins encoded by the DA transporter (DAT) and L-NE transporter (NET) genes, respectively. In some vertebrate neurons, particularly the sympathetic neurons of amphibians, L-NE is converted to L-epinephrine (L-Epi, adrenaline) and released as the primary neurotransmitter. Although evidence exists for a molecularly distinct L-Epi transporter (ET) in the vertebrate brain and peripheral nervous system, a transporter specialized for extracellular L-Epi clearance has yet to be identified. To pursue this issue, we cloned transporter cDNAs from bullfrog (Rana catesbiana) paravertebral sympathetic ganglia and characterized functional properties via heterologous expression in non-neuronal cells. A cDNA of 2514 bp (fET) was identified for which the cognate 3.1 kb mRNA is highly enriched in frog sympathetic ganglia. Sequence analysis of the fET cDNA reveals an open reading frame coding for a protein of 630 amino acids. Inferred fET protein sequence bears 75, 66, and 48% amino acid identity with human NET, DAT, and the 5-hydroxytryptamine transporter (SERT), respectively. Transfection of fET confers Na+- and Cl--dependent catecholamine uptake in HeLa cells. Uptake of [3H]-L-NE by fET is inhibited by catecholamines in a stereospecific manner. L-Epi and DA inhibit fET-mediated [3H]-L-NE uptake more potently than they inhibit [3H]-L-NE uptake by human NET (hNET), whereas L-NE exhibits equivalent potency between the two carriers. Moreover, fET exhibits a greater maximal velocity (Vmax) for the terminal products of catecholamine biosynthesis (L-Epi > L-NE >> DA), unlike hNET, in which a Vmax rank order of L-NE > DA > L-Epi is observed. fET-mediated transport of catecholamines is sensitive to cocaine and tricyclic antidepressants, with antagonist potencies significantly correlated with hNET inhibitor sensitivity. Amino acid conservation and divergence of fET with mammalian catecholamine transporters help define residues likely to be involved in catecholamine recognition and translocation as well as blockade by selective reuptake inhibitors.

Fig. 1.

Nucleotide and deduced amino acid sequence (single letter code) of the bullfrogl-epinephrine transporter cDNA (fET). Nucleotides are numbered in the 5′ to 3′ direction, beginning at the presumptive start codon ATG. A single open reading frame of 1890 bp is present, encoding a protein of 630 amino acids. Twelve stretches of amino acid sequence predicted to encode transmembrane domains (Kyte and Doolittle, 1982) are underlined. In-frame stop codons areunderlined in the 5′ untranslated region. A putative polyadenylation signal is underlined 128 nucleotides upstream of the polymeric dA tract in the 3′ untranslated region. Canonical N-glycosylation sites, one in the N terminus, three in the large extracellular loop, and a one in the loop between TMD11 and TMD12, are indicated by clear boxes. A leucine zipper motif is indicated by double lines. Canonical phosphorylation sites are indicated by asterisks and are delineated as follows: S17 (PKA, PKC, and PKC site), S56 (PKC), T271 (PKC and PKG), S272 (PKG), S515 (PKC and PKG), Y611, (tyrosine kinase), and S629 (PKG) site. Sequence motif conserved in DATs, but not in NETs, is indicated by shaded box. The original 711 bp fragment identified by PCR of bullfrog sympathetic ganglia RNA is indicated by the nucleotide sequence from 270 to 981, between thearrows.

Fig. 2.

Alignment of amino acid sequences encoding vertebrate catecholamine transporters. Alignment was performed by using amino acid sequence of the cloned fET (this study), human NET (hNET; Pacholczyk et al., 1991), bovine NET (bNET; Lingen et al., 1994), mouse NET (mNET; L. D. Jayanthi, J. D. Fritz, M. A. Thoreson, R. D. Blakely, unpublished data), human DAT (hDAT; Giros et al., 1992), bovine DAT (bDAT; Usdin et al., 1991) and rat DAT (rDAT; Shimada et al., 1991) proteins. Residues matching fET sequences are blackened. Residues spanning putative TMDs inferred from hydrophobicity analysis are represented by a rectangular box drawnabove the sequences.

Fig. 3.

Distribution of fET mRNA in bullfrog tissues revealed by Northern and RT-PCR analyses. A, Northern analysis of bullfrog sympathetic ganglia, heart atrium, heart ventricle, human SK-N-SH neuroblastoma cells, and rat brainstem probed with the 711 bp fET RT-PCR product. Each lane contained 2 μg of poly(A⁺) RNA. Ethidium bromide staining and hybridizations with a β-actin (data not shown) cDNA probe revealed even loading and transfer across lanes. B, RT-PCR analysis of fET expression in bullfrog tissues. RT-PCR was performed as described in Materials and Methods with 2 μg of total RNA and oligonucleotides originally used to identify fET cDNA.C, Southern analysis of RT-PCR-derived products fromB probed with fET 711 bp cDNA obtained by RT-PCR. Blotting and hybridization were performed as described in Materials and Methods. Note that amplification products seen in brain RNA did not hybridize with fET cDNA probes, even with long exposure.D, RT-PCR products obtained from RNAs used inC amplified with oligonucleotides encoding highly conserved sequences of 28S ribosomal RNA.

Fig. 4.

fET-induced catecholamine transport in transfected HeLa cells. A, Time-dependent accumulation of [³H]-l-NE uptake. Cells were transfected either with pBluescript SKII⁻ vector containing the fET cDNA or pBluescript vector and assayed in normal buffer (squares) or buffer with complete isotonic substitution of Na⁺ by choline (filled circles).B, Sodium dependence of [³H]-l-NE uptake in HeLa cells expressing fET cDNA, assessed in the presence of different concentrations of extracellular Na⁺ with isotonicity maintained with LiCl.C, Chloride dependence of [³H]-l-NE uptake in HeLa cells expressing fET cDNA assessed in the presence of different concentrations of extracellular Cl⁻. Isotonicity was maintained with sodium gluconate. D, Catecholamine inhibition of [³H]-l-NE uptake in transfected HeLa cells. Nonspecific uptake was determined by parallel transfections with pBluescript SKII⁻. [³H]-l-NE uptake assays were performed as described in Materials and Methods, with increasing concentrations of l-NE, l-Epi,d-Epi, or DA added simultaneously with 20 nm[³H]-l-NE. Data are expressed as a percentage of specific l-NE uptake and presented as mean ± SEM of three to five separate experiments.

Fig. 5.

Concentration dependence of l-Epi ( A), l-NE (B), and DA (C) transport into HeLa cells transiently transfected with fET cDNA. [³H]-l-Epi, [³H]-l-NE, and [³H]-DA uptake assays were performed on separate cultures transfected in parallel as described in Materials and Methods. Data are presented as mean ± SEM of four separate experiments. Insets are Eadie–Hofstee linear transformations of saturation data.