Winter flounder 3

This study evaluated the relative importance of the N arragansett Bay estuary (RI and MA, USA), and associated tidal rivers and coastal lagoons, as nurseries for juvenile winter flounder, Pseudopleuronectes americanus, and summer flounder, Paralichthys dentatus. Winter flounder (WF) and summer flounder (SF) abundance and growth were measured from May to October (2009-2013) and served as indicators for the use and quality of shallow-water habitats (water depth < 1.5-3.0 m). These bioindicators were then analyzed with respect to physiochemical conditions to determine the mechanisms underlying intra-specific habitat selection. WF and SF abundances were greatest in late May and June (maximum monthly mean = 4.9 and 0.55 flounder/m2 for WF and SF, respectively), and were significantly higher in the tidal rivers relative to the bay and lagoons. Habitat-related patterns in WF and SF abundance were primarily governed by their preferences for oligohaline (0.1-5 ppt) and mesohaline (6-18 ppt) waters, but also their respective avoidance of hypoxic conditions (< 4 mg DO/L) and warm water temperatures (> 25 °C). Flounder habitat usage was also positively related to sediment organic content, which may be due to these substrates having sufficiently high prey densities. WF growth rates (mean = 0.25 ± 0.14 mm/d) were negatively correlated with the abundance of conspecifics, whereas SF growth (mean = 1.39 ± 0.46 mm/d) was positively related to temperature and salinity. Also, contrary to expectations, flounder occupied habitats that offered no ostensible advantage in intra-specific growth rates. WF and SF exposed to low salinities in certain rivers likely experienced increased osmoregulatory costs, thereby reducing energy for somatic growth. Low-salinity habitats, however, may benefit flounder by providing refugia from predation or reduced competition with other estuarine fishes and macro-invertebrates. Examining WF and SF abundance and growth across each species' broader geographic distribution revealed that southern New England habitats may constitute functionally significant nurseries. These results also indicated that juvenile SF have a geographic range extending further north than previously recognized.

The sodium dicarboxylate cotransporter located at the basolateral side supplies renal proximal tubule cells with Krebs cycle intermediates and maintains the driving force for the exchange of organic anions like PAH against alpha-ketoglutarate through the organic anion transporter-1. Recently, we cloned sodium dicarboxylate cotransporter-3 from winter flounder kidney (fNaDC-3). To understand the regulation of fNaDC-3, we preincubated fNaDC-3-expressing oocytes with PMA, a PKC activator. PMA dose and time dependently inhibited fNaDC-3-mediated succinate uptake. Simultaneous preincubation of fNaDC-3-expressing oocytes with 50 nM PMA and either staurosporine or RO 31-8220 for 30 min attenuated PKC-mediated inhibition of succinate uptake. Site-directed mutagenesis of the five putative PKC sites (S7, T167, S174, T188, and S396) resulted in no change in PKC-mediated inhibition of the transporter. In electrophysiological studies performed at -60 mV, the K0.5 for succinate was not significantly affected (56 +/- 13 vs. 42 +/- 19 microM), but DeltaImax was reduced from -139 +/- 49 to -20 +/- 8 nA by PMA (50 nM, 30 min). Immunofluorescence analysis of fNaDC-3-expressing oocytes revealed that PMA leads to an endocytosis of fNaDC-3 protein. In conclusion, fNaDC-3 expressed in oocytes is downregulated by PMA through endocytosis. PKC consensus sites appear not to be important for this process.

In the present study, we determined the functional role of 15 positively charged amino acid residues at or within 1 of the predicted 11 transmembrane helixes of the flounder renal sodium-dicarboxylate cotransporter fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K), and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [14C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, Km and DeltaI(max) values comparable to wild-type (WT) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed but showed small transport rates and succinate-induced currents. K114I, located within transmembrane helix 4, showed [14C]succinate uptake similar to WT but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [14C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than WT, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K114I, K114Q) than, those evoked by WT. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium-dicarboxylate cotransport.