Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC.

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antibodies targeting pS124-NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment-specific markers localized pS124-NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124-NCC co-localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich-Wistar rats or vasopressin-deficient Brattleboro rats with the vasopressin type 2 receptor-specific agonist dDAVP significantly increased pS124-NCC abundance, with no changes in total NCC plasma membrane abundance. pS124-NCC levels also increased in abundance in rats after stimulation of the renin-angiotensin-aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, (36)Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane-associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline-inducible MDCKII-NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC.

Water transport between CNS compartments: contributions of aquaporins and cotransporters.

Large water fluxes continuously take place between the different compartments of the brain as well as between the brain parenchyma and the blood or cerebrospinal fluid. This water flux is tightly regulated but may be disturbed under pathological conditions that lead to brain edema formation or hydrocephalus. The molecular pathways by which water molecules cross the cell membranes of the brain are not well-understood, although the discovery of aquaporin 4 (AQP4) in the brain improved our understanding of some of these transport processes, particularly under pathological conditions. In the present review we introduce another family of transport proteins as water transporters, namely the cotransporters and the glucose uniport GLUT1. In direct contrast to the aquaporins, these proteins have an inherent ability to transport water against an osmotic gradient. Some of them may also function as water pores in analogy to the aquaporins. The putative role of cotransport proteins and uniports for the water flux into the glial cells, through the choroid plexus and across the endothelial cells of the blood-brain-barrier will be discussed and compared to the contribution of the aquaporins.

Vasopressin-dependent short-term regulation of aquaporin 4 expressed in Xenopus oocytes.

Aquaporin 4 (AQP4) is abundantly expressed in the perivascular glial endfeet in the central nervous system (CNS), where it is involved in the exchange of fluids between blood and brain. At this location, AQP4 contributes to the formation and/or the absorption of the brain edema that may arise following pathologies such as brain injuries, brain tumours, and cerebral ischemia. As vasopressin and its G-protein-coupled receptor (V1(a)R) have been shown to affect the outcome of brain edema, we have investigated the regulatory interaction between AQP4 and V1(a)R by heterologous expression in Xenopus laevis oocytes. The water permeability of AQP4/V1(a)R-expressing oocytes was reduced in a vasopressin-dependent manner, as a result of V1(a)R-dependent internalization of AQP4. Vasopressin-dependent internalization was not observed in AQP9/V1(a)R-expressing oocytes. The regulatory interaction between AQP4 and V1(a)R involves protein kinase C (PKC) activation and is reduced upon mutation of Ser(180) on AQP4 to an alanine. Thus, the present study demonstrates at the molecular level a functional link between the vasopressin receptor V1(a)R and AQP4. This functional interaction between AQP4 and V1(a)R may prove to be a potential therapeutic target in the prevention and treatment of brain edema.

Periodic high-conductance states in spinal neurons during scratch-like network activity in adult turtles.

Intense synaptic activity may alter the response properties of neurons in highly interconnected networks. Here we investigate whether the excitability and the intrinsic response properties of neurons in the spinal cord are affected by the increased synaptic conductance during functional network activity. Scratch episodes were induced by mechanical stimulation in the isolated carapace-spinal cord preparation from the adult turtle. Intracellular recordings revealed a dramatic increase in synaptic activity in interneurons and motoneurons during scratch activity. Superimposed slow depolarizing waves were phase-related to the rhythmic bouts of spike activity in the hip flexor nerve. The increase in synaptic conductance in interneurons and motoneurons varied with the scratch rhythm. During individual episodes, the conductance shifted smoothly with the scratch rhythm from near-resting levels to levels two to four times higher. In slice experiments, we found that even moderate increases in the conductance of motoneurons suppressed the slow afterhyperpolarization and the plateau potentials. We conclude that the excitability and the intrinsic response properties of spinal neurons are periodically quenched by high synaptic conductance during functional network activity.

Water transport in the brain: role of cotransporters.

It is generally accepted that cotransporters transport water in addition to their normal substrates, although the precise mechanism is debated; both active and passive modes of transport have been suggested. The magnitude of the water flux mediated by cotransporters may well be significant: both the number of cotransporters per cell and the unit water permeability are high. For example, the Na(+)-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic ions are transported by K(+)-Cl(-) and Na(+)-K(+)-Cl(-) cotransporters, neurotransmitters are reabsorbed from the synaptic cleft by Na(+)-dependent cotransporters located on glial cells and neurons, and metabolites such as lactate are removed from the extracellular space by means of H(+)-lactate cotransporters. We have previously determined water transport capacities for these cotransporters in model systems (Xenopus oocytes, cell cultures, and in vitro preparations), and will discuss their role in water homeostasis of the astroglial cell under both normo- and pathophysiologal situations. Astroglia is a polarized cell with EAAT localized at the end facing the neuropil while the end abutting the circulation is rich in AQP4. The water transport properties of EAAT suggest a new model for volume homeostasis of the extracellular space during neural activity.

Engineered Zn(2+) switches in the gamma-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents.

Two high affinity Zn(2+) binding sites were engineered in the otherwise Zn(2+)-insensitive rat gamma-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn(2+) binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [3H]GABA uptake by Zn(2+) (IC(50) = 35 and 44 microM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn(2+) was a potent inhibitor of the GABA-induced current (IC(50) = 21 microM for T349H/E370H and 51 microM for T349H/Q374C), albeit maximum inhibition was only approximately 40% in T349H/E370H versus approximately 90% in T349H/Q374C. In the wild type, Zn(2+) did not affect the Na(+)-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na(+) binding. However, in both mutants Zn(2+) caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn(2+) is inhibiting transporter function by stabilizing the outward-facing Na(+)-bound state. Translocation of lithium by the transporter does not require GABA binding and analysis of this uncoupled Li(+) conductance revealed a potent inhibition by Zn(2+) in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes of the transporter.

Water transport by the human Na+-coupled glutamate cotransporter expressed in Xenopus oocytes.

The water transport properties of the human Na+-coupled glutamate cotransporter (EAAT1) were investigated. The protein was expressed in Xenopus laevis oocytes and electrogenic glutamate transport was recorded by two-electrode voltage clamp, while the concurrent water transport was monitored as oocyte volume changes. Water transport by EAAT1 was bimodal. Water was cotransported along with glutamate and Na+ by a mechanism within the protein. The transporter also sustained passive water transport in response to osmotic challenges. The two modes could be separated and could proceed in parallel. The cotransport modality was characterized in solutions of low Cl- concentration. Addition of glutamate promptly initiated an influx of 436 +/- 55 water molecules per unit charge, irrespective of the clamp potential. The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein primarily from the downhill fluxes of Na+ to the uphill fluxes of water. Experiments using the cation-selective ionophore gramicidin showed no unstirred layer effects. Na+ currents in the ionophore did not lead to any significant initial water movements. In the absence of glutamate, EAAT1 contributed a passive water permeability (Lp) of (11.3 +/- 2.0) x 10(-6) cm s(-1) (osmol l(-1))(-1). In the presence of glutamate, Lp was about 50 % higher for both high and low Cl- concentrations. The physiological role of EAAT1 as a molecular water pump is discussed in relation to cellular volume homeostasis in the nervous system.