The Na(+)/K(+)-ATPase1 alpha subunit 3 (ATP1α(3)) is one of many essential components that maintain the sodium and potassium gradients across the plasma membrane in animal cells. Mutations in the ATP1A3 gene cause rapid-onset of dystonia parkinsonism (RDP), a rare movement disorder characterized by sudden onset of dystonic spasms and slowness of movement. To achieve a better understanding of the pathophysiology of the disease, we used immunohistochemical approaches to describe the regional and cellular distribution of ATP1α(3) in the adult mouse brain. Our results show that localization of ATP1α(3) is restricted to neurons, and it is expressed mostly in projections (fibers and punctuates), but cell body expression is also observed. We found high expression of ATP1α(3) in GABAergic neurons in all nuclei of the basal ganglia (striatum, globus pallidus, subthalamic nucleus, and substantia nigra), which is a key circuitry in the fine movement control. Several thalamic nuclei structures harboring connections to and from the cortex expressed high levels of the ATP1α(3) isoform. Other structures with high expression of ATP1α(3) included cerebellum, red nucleus, and several areas of the pons (reticulotegmental nucleus of pons). We also found high expression of ATP1α(3) in projections and cell bodies in hippocampus; most of these ATP1α(3)-positive cell bodies showed colocalization to GABAergic neurons. ATP1α(3) expression was not significant in the dopaminergic cells of substantia nigra. In conclusion, and based on our data, ATP1α(3) is widely expressed in neuronal populations but mainly in GABAergic neurons in areas and nuclei related to movement control, in agreement with RDP symptoms.
Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na+/K+-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2+/G301R) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2G301R/G301R E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2+/G301R male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2+/G301R behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.
Type III sodium-dependent phosphate (NaP i ) cotransporters, Pit1 and Pit2, have been assigned housekeeping P i transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaP i cotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu 55 and Glu 575 , with glutamine or with lysine severely impaired or knocked out, respectively, P i transport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaP i cotransporters, in fungal NaP i cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaP i cotransport. Their putative transmembranic positions together with the presented data are consistent with Glu 55 and Glu 575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 P i transport function. Inorganic phosphate (P i )1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as P i uptake across the plasma membrane in animal cells have been identified (1-3), namely the sodium-dependent phosphate (NaP i ) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4)) and Pit2 (human Pit2 formerly GLVR2 (5)). Both proteins are characterized as type III NaP i cotransporters (6) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7). Furthermore, low extracellular P i levels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1,8). These observations strongly suggest that the major cellular P i demand in mammalian cells is handled by type III NaP i cotransporters (1). However, recent results also point at type III transporters as playing specific roles in chondroblastic and osteoblastic mineralization (9, 10) as well as being critically involved in vascular calcification under hyperphosphatemic conditions, which are often present in diabetic patients and individuals with renal failure (11). The mechanisms underlying the bone-forming roles of type III NaP i transporters are presently not known. Recent results, however, showed that high extracellular P i levels can induce expression of the gene for osteopontin and that the induction is dependent on Na ϩ -dependent P i uptake across the p...
The mammalian members of the inorganic phosphate (P i ) transporter (PiT) family, the type III sodium-dependent phosphate (NaP i ) transporters PiT1 and PiT2, have been assigned housekeeping P i transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na + ) or proton (H + ) gradients to transport P i . Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N-and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N-and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P i uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP i transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na + -dependent P i transport function of human PiT2. The conservation of the aspartates among proteins using either Na + -or H + -gradients for P i transport suggests that they are involved in H + -dependent P i transport as well. Current results favor a membrane topology model in which the N-and C-terminal PiT family signature sequences are positioned in intra-and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.Abbreviations 10A1, an MLV isolate related to AM-MLV; AM-MLV, amphotropic MLV; CHO, Chinese hamster ovary; FeLV-B, feline leukemia virus subgroup B; GALV, gibbon ape leukemia virus; MLV, murine leukemia virus; NaP i , sodium-dependent phosphate; PiT, inorganic phosphate transporter; RADAR, rapid automatic detection and alignment of repeats.
BackgroundThe inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na+- or H+-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na+-dependent Pi (NaPi) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins.ResultsWe show that the human PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic for all PiT family members.A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR254-V483), was found to be a fully functional Pi transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL183-V483) did also support Pi transport albeit at very low levels.ConclusionsThe results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.
. Characterization of transport mechanisms and determinants critical for Na ϩ -dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.
The Na+/K+-ATPases maintain Na+ and K+ electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na+/K+-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3+/D801Y) was generated. The α3+/D801Y mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3+/D801Y mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na+/K+-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.
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