Neuronal activity leads to an influx of Na+ that needs to be rapidly cleared. The sodium-potassium ATPase (Na,K-ATPase) exports three Na+ ions and imports two K+ ions at the expense of one ATP molecule. Na,K-ATPase turnover accounts for the majority of energy used by the brain. To prevent an energy crisis, the energy expense for Na+ clearance must provide an optimal effect. Here we report that in rat primary hippocampal neurons, the clearance of Na+ ions is more efficient if Na,K-ATPase is laterally mobile in the membrane than if it is clustered. Using fluorescence recovery after photobleaching and single particle tracking analysis, we show that the ubiquitous α1 and the neuron-specific α3 catalytic subunits as well as the supportive β1 subunit of Na,K-ATPase are highly mobile in the plasma membrane. We show that cross-linking of the β1 subunit with polyclonal antibodies or exposure to Modulator of Na,K-ATPase (MONaKA), a secreted protein which binds to the extracellular domain of the β subunit, clusters the α3 subunit in the membrane and restricts its mobility. We demonstrate that clustering, caused by cross-linking or by exposure to MONaKA, reduces the efficiency in restoring intracellular Na+. These results demonstrate that extracellular interactions with Na,K-ATPase regulate the Na+ extrusion efficiency with consequences for neuronal energy balance.

NMDA receptors play a crucial role in regulating synaptic plasticity and memory. Activation of NMDA receptors changes intracellular concentrations of Na(+) and K(+), which are subsequently restored by Na/K-ATPase. We used immunochemical and biochemical methods to elucidate the potential mechanisms of interaction between these two proteins. We observed that NMDA receptor and Na/K-ATPase interact with each other and this interaction was shown for both isoforms of α subunit (α1 and α3) of Na/K-ATPase expressed in neurons. Using Western blotting, we showed that long-term exposure of the primary culture of cerebellar neurons to nanomolar concentrations of ouabain (a cardiotonic steroid, a specific ligand of Na/K-ATPase) leads to a decrease in the levels of NMDA receptors which is likely mediated by the α3 subunit of Na/K-ATPase. We also observed a decrease in enzymatic activity of the α1 subunit of Na/K-ATPase caused by NMDA receptor activation. This effect is mediated by an increase in intracellular Ca(2+). Thus, Na/K-ATPase and NMDA receptor can interact functionally by forming a macromolecular complex which can be important for restoring ionic balance after neuronal excitation. Furthermore, this interaction suggests that NMDA receptor function can be regulated by endogenous cardiotonic steroids which recently have been found in cerebrospinal fluid or by pharmacological drugs affecting Na/K-ATPase function.

Our group has studied the origin of Ca2+ oscillations triggered by the cardiotonic steroid ouabain, a highly specific ligand of Na+‐K+‐ATPase. It has been shown that these low frequency oscillations protect the cells from apoptosis by activating the NF κB/p65 survival subunit and inhibiting the intrinsic mitochondrial apoptotic pathway by reversing an imbalance between the apoptotic factor Bax and the antiapoptotic factor Bcl‐xL.The aim of this study is to reveal the identity and role of the intracellular compartments that are involved in ouabain‐triggered Ca2+ oscillations.Studies were first performed on COS7 cells which expressed genetic Ca2+ indicators targeted to mitochondria and cytosol. Following application of low doses of ouabain, we found that each cytosolic calcium wave was followed by a delayed mitochondrial calcium transient. The recovery of Ca2+ to normal levels was slower in mitochondria compared to cytosol, while the initial rate of increase was found to be similar in both compartments. Next, using an endoplasmic reticulum (ER)‐targeted genetic Ca2+ indicator, we observed rhythmic and simultaneous Ca2+ transients in both mitochondria and ER after ouabain application.We propose that Ca2+ shuttling between the ER and mitochondria may have a pivotal role in the propagation of ouabain triggered slow Ca2+ oscillations.Grant Funding Source: Swedish Research Council, Family Erling‐Persson Foundation, Märta and Gunnar V Philipson

The rapid changes in intracellular Na+ concentration due to excitatory synaptic activity by AMPA receptors are offset by the Na,K‐ATPase which is responsible for restoring the membrane potential and for maintaining a low intracellular level of sodium. Many recent studies describe the dynamics of AMPA receptor diffusion which should consequently modulate the influx of Na+. Though regulation of AMPA receptors and Na,K‐ATPase are likely coordinated, a direct link has never been investigated. In this study we use live cell imaging methods to examine population and single molecule movement of the Na,K‐ATPase a3 isoform and explore a functional regulation associated with AMPA receptor activity. Using Super Ecliptic pHluorin‐tagged al3 and FRAP we measured population mobility in cultured hippocampal neurons. We found that a majority of the Na,K‐ATPase molecules in the dendritic membrane are in lateral diffusion. With quantum dot labeling, we measured the single molecule diffusion in the extrasynaptic and synaptic membranes. After AMPA receptor stimulation the mobility of a3 was significantly increased in both the extrasynaptic and synaptic membranes. Using TTX to inhibit neuronal activity we observed a decreased mobility in the extrasynaptic and synaptic membranes. The alterations seen in response to changes in synaptic activity demonstrate that a3 is functionally regulated in the dendritic membrane.

Neurons communicate via synapses; the strength of each synapse is defined both pre and postsynaptically. Presynaptically, the strength of the synapse is defined by which neurotransmitter is being released and how much. On the postsynaptic membrane a corresponding receptor will receive the transmitter. Receptor abundance and availability determines the strength of the synaptic connection. Protein function is as tightly linked to structure as it is to location. Due to the fluidic nature of the plasma membrane, any membrane protein will be highly mobile unless it is being anchored or confined within a compartment by an intracellular protein or cytoskeletal complex. High mobility facilitates interactions between proteins and ensures proper localization through free energy minimization without the need of directed transport. The dynamic regulation of protein mobility is fundamental in defining the function of the protein. The overall abundance and availability of postsynaptic proteins are dependent on many processes such as exocytosis/endocytosis, activation/inactivation, and lateral diffusion. The aim of this thesis was to study how postsynaptic proteins can be regulated in the dendritic membrane by availability and mobility. Dopamine is an important modulatory neurotransmitter that is involved in cognition, memory, motoric functions and reward-mechanisms. Calcyon is an accessory protein that has been suggested to modulate dopamine receptor signaling. We show that calcyon is a neuron-specific vesicular protein with a high intracellular mobility. Furthermore we show that calcyon forms vesicular clusters located just beneath the plasma membrane. We propose a role for calcyon in the trafficking of proteins that are important for synaptic plasticity, to and from the dendritic plasma membrane. Dopamine receptors are divided into two groups, the D1-like and D2-like group, each with distinct downstream signaling pathways. We show that the two isoforms of the D1-like group, the D1 and D5 receptors have distinctly different subcellular localization in striatal neurons and interact differently with the NMDA receptors. We propose that the two isoforms, due to differences in localization and interactions with other receptors, have distinct roles in neuronal dopaminergic signaling. Most G-protein coupled receptors are transported to the plasma membrane of the soma and are then transported via lateral diffusion to the site of action. We studied several GPCRs, involved in mood regulation and behavior, to elucidate whether GPCRs share a common mode of transport. We show that the 5-HT1B receptor, in contrast to other GPCRs, is transported in vesicles in the lumen of the dendrites. We show that the vesicles release the receptors to the membrane close to inhibitory synapses, followed by subsequent lateral diffusion and confinement in inhibitory as well as excitatory synapses. We propose that this special mode of transport serves as an additional mode of regulation, which enables fine-tuning of serotonergic signaling. The Na,K-ATPase is an essential ion transporting protein that is found in all cells where it is responsible for the generation of the plasma membrane ion gradient that is the driving force for many important cellular processes. Different isoforms of the catalytic, ion-pumping, α subunit are expressed in different cell types. We show that the neuron-specific α3 isoform is responsible for the sodium clearance in dendrites following synaptic signaling, which is essential for proper neuronal function. Furthermore we show that the α3 subunit is highly mobile in the postsynaptic membrane and that it is confined in excitatory synapses. We show that mobility is modulated by neuronal activity; excitatory stimulation results in an increased mobility in both the synaptic as well as the extrasynaptic region.

Most neurons co‐express two catalytic isoforms of Na,K‐ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here we used electrophysiology and sodium imaging to study the role of α3 in neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside ouabain resulted in a modest increase in the intracellular sodium concentration ([Na+]i), accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+]i, similar to what could be expected following suprathreshold neuronal activity, inhibition of α3 almost completely abolished the capacity to restore [Na+]i in dendrites. Recordings of Na,K‐ATPase‐specific current supported the notion that α3 is the predominant isoform, responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A (PKA) consensus site, which is structurally associated with a Na+ binding site. Activation of PKA significantly attenuated both the α3 dependent current and restoration of dendritic [Na+]i, indicating that α3 is a target for phosphorylation and may participate in short‐term regulation of neuronal function.

1 A specific and essential role for Na,K-ATPase α3 in neurons co-expressing α1 and α3* Background: Neurons express two Na,K-ATPase isoforms, the ubiquitous α1 and neuron-specific α3. Result: α3 is important for control of membrane potential and is fully responsible for restoration of large [Na + ] i increases. Conclusion: α1 and α3 are required for basal neuronal function, but α3 controls restoration of [Na + ] i following sustained discharge. Significance: Conditions associated with defect α3 function are likely aggravated by suprathreshold neuronal activity. SUMMARY Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here we used electrophysiological and Na + imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na + concentration ([Na + ] i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na + ] i , similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na + ] i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na + ] i is elevated in the neuron, α3 is the predominant isoform, responsible for rapid extrusion of Na +. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A (PKA) consensus site, which is structurally associated with a Na + binding site. Following activation of PKA both the α3 dependent current and restoration of dendritic [Na + ] i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short-term regulation of neuronal function. The maintenance of a steep Na + gradient across the plasma membrane is essential for the function and survival of all eukaryotic cells. The ubiquitous integral plasma membrane protein, Na,K-ATPase, which actively exports three Na + ions and imports two K + ions for each ATP hydrolyzed, is mainly responsible for maintenance of the transmembrane Na + gradient and is …

Postsynaptic receptor trafficking plays an essential role in tuning neurotransmission and signal plasticity and has emerged as a potential therapeutic target in neuropsychiatric disease. Using a novel application of fluorescence recovery after photobleaching in rat hippocampal neurons, we examined transport from the soma to dendrites of seven G-protein-coupled receptors (GPCRs) implicated in mood disorders. Most GPCRs were delivered to dendrites via lateral diffusion, but one GPCR, the serotonin 1B receptor (5-HT1B), was delivered to the dendrites in secretory vesicles. Within the dendrites, 5-HT1B were stored in a reservoir of accessible vesicles that were recruited to preferential sites in plasma membrane, as observed with superecliptic pHluorin labeling. After membrane recruitment, 5-HT1B transport via lateral diffusion and temporal confinement to inhibitory and excitatory synapses was monitored by single particle tracking. These results suggest an alternative mechanism for control of neuronal activity via a GPCR that has been implicated in mood regulation.

The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards formation of the kidney's functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that the sodium-potassium-adenosine triphosphatase (Na, K-ATPase) ligand ouabain triggers, via the Na, K-ATPase/ inositol 1,4,5-trisphosphate receptor signalosome, a calcium-nuclear factor-kappa B (NF-κB) signal that protects kidney development from adverse effects of malnutrition. Serum deprivation resulted in severe retardation of nephron formation and robust increase in apoptotic rate, but in ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Depletion of intracellular calcium stores and inhibition of NF-κB activity abolished the rescuing effect of ouabain. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given low-protein diets and treated with ouabain or vehicle throughout pregnancy.

Fetal malnutrition and other factors contributing to renal growth retardation result in increased apoptosis and reduction in nephron endowment. Our group has described a novel signaling pathway, where direct interaction between Na,K‐ATPase (NKA) and IP3R triggers slow calcium oscillations and activation of NFkappaB (PNAS 2001, JBC 2003, 2006). This signaling pathway is activated by ouabain, a specific ligand of NKA. Downstream effects include protection from apoptosis and stimulation of cell proliferation (JASN 2006). Here we report activation of this signaling pathway rescues nephrogenesis in growth factor deprived (gfd) embryonic rat kidney. Kidneys from E14 rats were cultured under normal or gfd conditions and studied after 2–3 days in vitro. Exposure to ouabain triggered calcium oscillation and activated NF‐kappaB. Growth factor deprivation retarded ureter branching and formation of new glomeruli and increased apoptotic index. Exposure to nM ouabain prevented these effects. The protective effects of ouabain were abolished by depletion of intracellular calcium stores and by inhibition of NF‐kappaB, underlining the importance of the NKA‐IP3R signaling pathway. The expression of Wt1 and Pax2, which are activated by NF‐kappaB, was increased in ouabain exposed gfd kidneys. Thus we have identified a novel mechanism by which kidney development can be protected under adverse intrauterine circumstances.

Calcyon is a brain-specific protein, implicated in clathrin-mediated endocytosis. In this descriptive study we show that calcyon is exclusively expressed in neurons, and localized in moving vesicles. The movement of calcyon-containing vesicles was dependent on temperature and on intact microtubules, in addition these vesicles were colocalized with a marker for endocytosed plasma membrane proteins, suggesting that calcyon vesicles follow the endocytic recycling pathway. We also show using evanescent wave microscopy that there is a pool of ready releasable calcyon vesiclesaccumulated beneath the plasma membrane. We conclude that the mobility and storage properties of calcyon-containing vesicles imply that they play a role in brain plasticity.